2020--Analysis of polyphenolics

C H A P T E R
3
Analysis of polyphenolics
Kamal Niaza, Fazlullah Khanb
a
Department of Pharmacology and Toxicology, Faculty of Bio-Sciences, Cholistan University
of Veterinary and Animal Sciences (CUVAS), Bahawalpur, Pakistan bDepartment of Toxicology
and Pharmacology, Faculty of Pharmacy, The Institute of Pharmaceutical Sciences (TIPS), Tehran
University of Medical Sciences, Tehran, Iran
Introduction
Plants produce enormous amount of health-promoting compounds, including polyphenols, dietary fibers, vitamins, and minerals (Filannino et al., 2018). Basically, food items of
plant origin provide a high nutritive value, low caloric density, and low-energy constituents
(e.g., minerals, vitamins, dietary fibers) as well as being rich sources of bioactive phytochemicals such as carotenoids, sterols, polyphenols (Fig. 3.1), and glycosylates (Manach et al.,
2017). Dietary polyphenols like flavonoids are strong antioxidants that act through
interacting with reactive oxygen species (ROS) producing reactive compounds. For example,
curcumin is a dietary polyphenol with strong antioxidant activity that have been used against
a number of aging-related diseases like Alzeimer’s disease (AD) for more than a century.
Curcumin acts via chelating reactive metal ions like Fe2+, thus diminishes their oxidative
power and the resulting OS due to free radicals. Ginkgo biloba extract has also been used
to treat aging-related diseases. Ginkgo biloba extract contains various polyphenols, such
as flavonoids, and terpenes, which interact with superoxide anion, hydroxyl, and peroxyl free
radicals to quench the radical chain reactions in mitochondrial respiratory chain function (Liu
et al., 2007). The important effect of tea polyphenols on cognitive function of elderly people is
attributed to its flavonoid (catechin) content (Song et al., 2012). Various studies have
Recent Advances in Natural Products Analysis
https://doi.org/10.1016/B978-0-12-816455-6.00003-2
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# 2020 Elsevier Inc. All rights reserved.
40
3. Analysis of polyphenolics
Phenolic acids
Hydroxybenzoic
acids
Gallic acid
Ellagic acid
Vanillic acid
Syringe acid
Protocatechulic acid
Salicylic acid
Other phenolics
Flavonoids
Flavones
Luteolin
Apigenin
Flavonols
Quercetin
Rutin
Isohanmetin
Myricetin
Kaempferol
Flavan-3-ols
Catechin
Gallocatechin
Epicatechin
Isoflavones
Genestein
Daidzin
Glycitein
Equol
Flavanones
Narinagin
Hesperidin
Hydroxycinnamic
acids
Caffeic acid
Caftaric acid
Chlorogenic acid
Cinnamic acid
Coumaric acid
Ferulic acid
Curcumin
Stilbenes
Resveratrol
Piceatannol
Lignans
Pinoresinol
Lariciresinol
Matairesinol
Secoisolaricire
Sinol
Sesamol
Enterodiol
Enterolaction
Tannins
Anthocyanidins and
anthocyanins
Hydrolyzable
tannins
Nonhydrolyzable/
condensed tannins/
proanthocyanidins
Tannic acid
Galloetannins
Ellagitannins
Procyanidin B2
Procyanidin A2
Xanthones
Lignins
Malvidin
Cyanidin
Delphinidin
Peonidin
Chromones
Anthraquinones
FIG. 3.1 Classification of polyphenols.
confirmed that vegetable and fruit juices available commercially also have high level of
antioxidant polyphenolic compounds (Proteggente et al., 2002). This might be due to the
high-pressure mechanical extraction during processing of vegetable and fruit juices that
drives out a considerable amount of the antioxidant compounds from the peels and pulp
in addition to the main fruit and vegetable fluids.
According to various in vitro, in vivo, and clinical studies, a stronger neuroprotective activity than antioxidant vitamins has been reported in polyphenols from grape, apple, and
II. Phenolics
References
41
citrus fruit juices (Dai et al., 2006). Other antioxidant molecules like β-carotene, dietary polyphenols (flavonoids), and Se also reduce OS in neurons. The antioxidant, antiinflammatory,
and neuroprotective effect of vitamin D has also been widely reported (Alles et al., 2012).
Due to the high nutritive values of plants and antioxidant potential, various polyphenolic
compounds such as flavonoids, glycosidic derivatives of flavonoids, isoflavonoids, lignans
and flavonolignans, stilbenoids, tannins, curcuminoids, coumarins and phloroglucinols,
xanthones, and anthrones have been discussed in this book chapter, addressing their phytochemistry and classification, main representatives, biological activities, techniques of
extraction and purification, techniques of identification and quantification, levels founds
in foods, effects of food processing and pharmaceutical applications.
References
Alles, B., Samieri, C., Feart, C., Jutand, M.A., Laurin, D., Barberger-Gateau, P., 2012. Dietary patterns: a novel approach to examine the link between nutrition and cognitive function in older individuals. Nutr. Res. Rev.
25 (2), 207–222. https://doi.org/10.1017/s0954422412000133.
Dai, Q., Borenstein, A.R., Wu, Y., Jackson, J.C., Larson, E.B., 2006. Fruit and vegetable juices and Alzheimer’s disease:
the Kame Project. Am. J. Med. 119 (9), 751–759.
Filannino, P., Di Cagno, R., Gobbetti, M., 2018. Metabolic and functional paths of lactic acid bacteria in plant foods: get
out of the labyrinth. Curr. Opin. Biotechnol. 49, 64–72.
Liu, X., Dong, M., Chen, X., Jiang, M., Lv, X., Yan, G., 2007. Antioxidant activity and phenolics of an endophytic
Xylaria sp. from Ginkgo biloba. Food Chem. 105 (2), 548–554.
Manach, C., Milenkovic, D., Van de Wiele, T., et al., 2017. Addressing the inter-individual variation in response to
consumption of plant food bioactives: towards a better understanding of their role in healthy aging and
cardiometabolic risk reduction. Mol. Nutr. Food Res. 61 (6) 1600557.
Proteggente, A.R., Pannala, A.S., Paganga, G., Buren, L.V., Wagner, E., Wiseman, S., Put, F.V., Dacombe, C., RiceEvans, C.A., 2002. The antioxidant activity of regularly consumed fruit and vegetables reflects their phenolic
and vitamin C composition. Free Radic. Res. 36 (2), 217–233.
Song, J., Xu, H., Liu, F., Feng, L., 2012. Tea and cognitive health in late life: current evidence and future directions.
J. Nutr. Health Aging 16 (1), 31–34.
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3. Analysis of polyphenolics
S U B C H A P T E R
3.1
Flavonoids (flavones, flavonols, flavanones, flavanonols,
flavanols or flavan-3-ols, isoflavones, anthocyanins,
chalcones/coumestans)
Mohammed Bulea, Fazlullah Khanb,c, Kamal Niazd
a
Department of Pharmacy, College of Medicine and Health Sciences, Ambo University, Ambo,
Ethiopia bInternational Campus, Tehran University of Medical Sciences (IC-TUMS), Tehran,
Iran cDepartment of Toxicology and Pharmacology, Faculty of Pharmacy, The Institute of
Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran
d
Department of Pharmacology and Toxicology, Faculty of Bio-Sciences, Cholistan University of
Veterinary and Animal Sciences (CUVAS), Bahawalpur, Pakistan
3.1.1 Phytochemistry of the flavonoids
Polyphenols are a group of secondary plant metabolites comprising more than 8000 compounds identified till date. Based on the number of phenolic rings, they are categorized into
different classes, namely, are flavonoids, lignans, and stilbenoids. On the other hand, flavonoids have seven subclasses, which represents 60% of dietary polyphenols, including flavones, flavonols, flavanones, isoflavones, flavanols, anthocyanins, and chalcones (Legeay
et al., 2015). Flavonoids are biological active polyphenols with potential antioxidant and immunomodulatory bioactivities. Various epidemiological and animal model studies indicated
that dietary flavonoids can reduce the incidence of age-related diseases (Ciz et al., 2012).
Structurally flavonoids are biosynthesized from aromatic rings of the phenyl- and
malonyl-coenzyme A. Their classification thus depends on the position of different functional
groups on ring C and the position of ring B as shown in Fig. 3.1.1. In ring C of flavanones and
flavanols, the bond between second and the third atoms is a single bond where as it is an aromatic or a double bond in the case of flavones, flavonols, isoflavones, and anthocyanidins.
With the exception of isoflavones, the attachment of ring B to the ring C is carried out at position 2 (De Martino et al., 2012).
Flavonoids mainly occur as glycosides instead of aglycones. Chalcones are the precursors
of flavonoids composed of two benzene rings connected by a three-carbon α,β-unsaturated
carbonyl structure (Lago et al., 2014). On the other hand, dihydrochalcones, which are a small
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43
3.1.1 Phytochemistry of the flavonoids
3¢
4¢
2¢
9
7
A
6
B
1
8
HO
O
2
1¢
5¢
6¢
C
3
10
5
OH
4
OH
OH
OH
Flavan-3-ols
OH
HO
Flavonol
Flavones
HO
O
O
HO
O
OH
OH
OH
OH
O
OH
OH
O
Anthocyanidins
Isoflavones
OH
O
HO
HO
O+
Flavanones
HO
O
OH
OH
OH
FIG. 3.1.1
O
OH
O
The general structure of some of the main flavonoids.
group of flavonoids, have an open C-ring structure. The dihydrochalcones have a limited
dietary significance and they are abundantly found in apples (Malus domestica) and rooibos
tea (Del Rio et al., 2013). The other groups of flavonoids with a double bond at C-2 are the
flavones, which include chrysin, apigenin, rutin, and the isoflavones genistin, genistein,
daidzin, and daidzein (Lago et al., 2014). Apart from lacking C-3 oxygen flavones, apigenin,
luteolin, wogonin, and baicalein bears structural resemblance to flavonols.
The content of flavones in celery (Apium graveolens) and parsley (Petroselinum hortense)
is reported considerably high. Besides, other herbs of the citrus family are rich in
polymethoxylated flavones like nobiletin and tangeretin (Del Rio et al., 2013).
Isoflavones are a group of phytoestrogens that have structural and/or functional similarity
to 17-estradiol. Therefore, they are known to have potent estrogenic or antiestrogenic actions.
This is a group of flavonoids with the B-ring attached at C-3 instead of C-2. They exist abundantly in legumes with significant quantities of daidzein and genistein. In soybean,
isoflavones occur mainly as 7-O-(600 -O-malonyl) glucosides, 7-O-(600 -acetyl) glucosides,
7-O-glucosides, and aglycones (de la Parra et al., 2012; Del Rio et al., 2013). The gut microflora
plays a central role in the absorption and metabolism of isoflavones. For instance, the glycosidic isoflavone daidzin needs to be metabolized into equol by the intestinal bacteria before
final absorption (de la Parra et al., 2012).
The oxidation at the C-3 position of the flavonoid structure gives flavonols such as
kaempferol, isorhamnetin, myricetin, and quercetin. Flavonols are found abundantly in plants
mainly as glycosides with conjugate bonds at 5, 7, 30 , 40 , and 50 positions in their structure.
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3. Analysis of polyphenolics
Yellow and red onions (Allium cepa) are potential sources of flavonols with significantly high
amount of quercetin-40 -O-glucoside and quercetin-3,40 -O-diglucoside (Del Rio et al., 2013).
Flavanones are flavonoids formed as a result of closure of the C-ring that produces
chromone unit and they have a C-4 carbonyl group. The most common flavanones include
naringin, naringenin, taxifolin, eriodictyol, and hesperidin (Lago et al., 2014). Flavanones occur in abundant amount in flavedo of citrus fruits as hydroxyl, glycosylated, and O-methylated
derivatives where the most common one is hesperetin-7-O-rutinoside (hesperidin) (Del Rio
et al., 2013). The flavanone naringin is a glycoside containing of naringenin, an aglycone,
and neohesperidose on the hydroxyl group at C-7. Naringin has a bitter taste because of its
glucose moiety, but its 1,3-diphenylpropan-1-one derivative produced by treating naringin
with a strong base is about 300–1800 times sweeter than sugar (Bharti et al., 2014).
The flavan-3-ols such as catechin, epicatechin, and epigallocatechin gallate, and the
anthocyanidins include apigenidin and cyanidin are formed through the reduction of the flavonols by reductases (Lago et al., 2014). The flavan-3-ols are subclass of flavonoids, which consists of the monomers, oligomers, and polymeric proanthocyanidins (condensed tannins). At
positions C-2 and C-3 of the monomeric flavan-3-ols, there are two chiral centers that result in
four isomeric structures. Two of these isomeric flavan-3-ols, (+)-catechin and ()-epicatechin,
are abundant, whereas the others are found rarely in nature. For instance, flavan-3-ol monomers such as ()-epigallocatechin, ()-epigallocatechin-3-O-gallate, and ()-epicatechin3-O-gallate are available in green tea (Camellia sinensis) in very high quantity (Del Rio et al.,
2013). Moreover, there are over 500 anthocyanin derivatives identified in plants with a varying
degree of hydroxylation and/or methoxylation. Anthocyanins possess radical scavenging
property in relation to the hydroxyl backbone in their structure (Kovinich et al., 2014).
Anthocyanidins are well known for their characteristic colors in fruits and flowers.
The formation of various colors ranging from blue and purple to orange and red is due to
the presence of conjugates produced as a result of a reaction between anthocyanidin
aglycones (aglycones are pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and
malvidin) and organic acids (Del Rio et al., 2013). These compounds mainly occur in epidermal
cell layer of vegetative tissues at some developmental stage and serve as photoprotectants and
attractants for pollinators or seed-dispersing organisms. An anthocyanidin is a stable
anthocyanin-containing aglycone at C-3 position, while the additions of other moieties like acyl
and hydroxycinnamic acid to the backbone yield complex anthocyanins (Kovinich et al., 2014).
The structural activity relationship (SAR) of flavonoids depends, in general, on the nature
of substitution on rings B and C. The SAR explains their radical-scavenging, metal-chelating,
and other biological activities in relation to various moieties on the main structures. Particularly, an ortho-dihydroxyl substitution on ring B (catechol group) confers higher activity because of the electron delocalization that stabilizes the carboxyl moiety, which also serves as a
binding site for trace metals. Furthermore, the antioxidant activity is enhanced as the number
of OH groups increases especially at positions 3, 4, and 5 of the ring B. In addition, presence of
a conjugated 4-oxo or a 3-OH group along with a double bond between C-2 and C-3 position
of the ring C further enhances the radical-scavenging activity of flavonoids. In contrast, the
methoxy derivatives of flavonoids formed by substitution of OH groups of ring B have lower
radical-scavenging potential (Balasundram et al., 2006; Lago et al., 2014). In general, the following are the biological activities that determine the activity of flavonoids (Balasundram
et al., 2006).
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3.1.2 Biological activities of flavonoids
45
3.1.2 Biological activities of flavonoids
Flavonoids are a class of polyphenols with low molecular weight and they have an important role in the cell wall synthesis (Ahmed et al., 2014). They have a wide range of pharmacological activities, including anticancer, and antidiabetic activities, neuroprotective role and
reduce the risk of cardiovascular diseases (CVDs) (Hanrahan et al., 2011). The term
“phytopharmaceutical” is coined to plant secondary metabolites possessing biological activities. Naringin is one of the “phytopharmaceutical” among many flavonoids, which have
hypolipidemic, antiatherosclerotic, antidiabetic, neuroprotective, hepatoprotective, and anticancer properties (Bharti et al., 2014). In regard to their antidiabetic activity, flavonoids had
shown to act via different intracellular signaling mechanisms such as regulating insulin secretion, insulin signaling, carbohydrate digestion, and glucose uptake in insulin-sensitive tissues
(Ahmed et al., 2014). Various epidemiological studies support the potential of flavonoids to
reduce the risk of CVDs. Especially their activity toward reducing oxidation of low-density lipoproteins (LDL) is attributed to prevent endothelial dysfunction, platelet aggregation and adhesion, and smooth muscle cell migration and proliferation (Legeay et al., 2015). For instance,
there is a growing interest in the role of chocolate and one of its bioactive metabolites, such as
flavan-3-ol, has efficacy in the prevention and management of CVDs due to improving metabolic syndrome risk factors. A large number of observational studies have demonstrated a significant association between cocoa use, which minimized risk of CVDs and related mortality.
Moreover, numerous other in vitro studies have exhibited the potential of flavan-3-ols activity
toward reduction of CVDs risk via their effect on angiotensin-converting enzyme (ACE) activity, inflammation, platelet function, endothelial function, and glucose transport, whereas their
effectiveness is still unclear in in vivo model (Bule et al., 2018; Hooper et al., 2012). In addition,
experimental studies on flavonoids suggested that their potential antiepileptic activity is modulated via γ-aminobutyric acid type A (GABAA)-Cl-channel complex due to their structural
resemblance to benzodiazepines. Such results from herbal remedies containing flavonoids
with neuroactive properties play a profound evidence for their potential action against
GABAA receptor-mediated pathologic conditions. Hence, flavone derivatives are considered
interesting principal compounds in the discovery of selective and potent benzodiazepine-like
drugs (Diniz et al., 2015). The neuroprotective effect of flavonoids was initially suggested to be
through an indirect action as a result of their antioxidant and free-radical scavenging activities.
However, it was later discovered that flavonoids exert a direct pharmacological action on enzymes, receptors, and signaling pathways. Currently, much attention is given to the biological
and physiochemical activities of flavonoids against neurodegeneration associated with
Parkinson’s and Alzheimer’s disease and improve cognitive function in central nervous system (CNS) (Hanrahan et al., 2011).
The antimicrobial potential of flavonoids is a widely reported biological activity especially
in plants containing flavones and flavonones. Structurally diverse isolates of the root bark
of Morus like morusin, kuwanon C, sanggenon B and D were shown to have potent and
wide spectrum activity against a range of microbes (Edziri et al., 2012). Flavonones,
pinocembrin and cryptocaryone, from the leaves of Cryptocarya chinensis demonstrated significant activity against M. tuberculosis in in vitro model. Besides, biflavonoids from Garcinia
livingstonei leaves such as amentoflavone and 40 monomethoxy amentoflavone have also
shown potential antibacterial activity against Mycobacterium smegmatis (Santhosh and
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3. Analysis of polyphenolics
OH
HO
O
O
O
HO
HO
O
OH
OH
OH
O
OH
O
O
OH
O
OH
OH
O
Naringin
FIG. 3.1.2
OH
O
Apigenin
OH
Chrysin
Chemical structure of bioactive flavonoids naringin, apigenin, and chrysin.
Suriyanarayanan, 2014). The other prominent biological activity that flavonoids possess is inhibitory effect on cancer cell growth in in vitro and in vivo study. Apigenin is a flavonoid with
a strong radical scavenging activity and antiinflammatory property. Moreover, a number of
studies on apigenin action against several cancer cell lines demonstrated that it vigorously
inhibits tumor cell invasion, metastasis, mitogen-activated protein kinases (MAPKs), and
downstream oncogenes. On the other hand, chrysin, which is structurally similar to apigenin,
is a potent inhibitor of aromatase of human immunodeficiency virus (HIV) in latent infection
model. It was also reported that chrysin with slight modification has antiinflammatory and
antioxidant activities in addition to its cancer chemopreventive potential in in vitro model
(Khoo et al., 2010) (Fig. 3.1.2).
3.1.3 Current and potential industrial applications of flavonoids
Currently, over 9000 different flavonoids are available with a varying substitution pattern
on their ring C. These flavonoids include various subclasses such as chalcones, flavones,
flavonols, flavanones, anthocyanins, and isoflavonoids. Most of these flavonoids have considerable commercial importance. For instance, the flavonoid resveratrol (a stilbene), which is
used for its animal longevity effect, is now gaining a greater market as nutritional supplement
in products. Besides, traditional Chinese medicines comprise ingredients many of which are
flavonoid derivatives such as the isoflavonoid, puerarin, and the flavone betalain (Wang
et al., 2011). On the other hand, the metabolic engineering of flavonoid pathways, which
became outstanding research area in the past few decades, begins since 1987. Particularly,
in the area of ornamental plant breeding, engineering of flavonoids plays a major role in
generating uncommon variety of flower colors such as blue and yellow flowering cultivars.
In this regard, the molecular methods in combination with classical methods have been used
in metabolic engineering to cultivate flowers and obtain novel colors while keeping other
desirable original traits of the plant. Furthermore, flavonoids are indicated as screening
pigments against UV-B, phytoalexins (antipathogenic microorganisms), antifeedants,
pollination agent attractants, and plant growth promoters (Forkmann and Martens, 2001).
II. Phenolics
3.1.4 Possible interactions of flavonoids
47
The other interesting feature of anthocyanins is the color change they generate when in contact with colorless substances, which are not copigments such as, surfactants, emulsions, gels,
proteins, DNA, cotton, wool, and hair. Anthocyanins are becoming an alternative to synthetic
colorants and produce novel dyes (cationic dyes) for hair coloring. In addition, they have also
been used as dyes in the textile, cosmetic, for attractive presentation of vegetables and preservation, as well as for photoprotection against sunlight. They are also known for their reversible color modification character under different physicochemical environment, including
solvatochromism, thermochromism, and photochromism (Brouillard et al., 2010).
Grape-derived nutraceutical products are quite popular and common agents as nutritional
products with a potential health benefit for the human body. What makes grape a special
source of exceptional nutraceutical products is its composition of unique polyphenols, including flavonoids, anthocyanins, proanthocyanins, and stilbenes (Georgiev et al., 2014). Nowadays, polyphenolic antioxidants are considered vital ingredients as food additives and/or as
important ingredients that impart extra health benefits to the food item they are added. In this
regard, grape seed extract (GSE) is extensively utilized as additive to improve the antioxidant
capacity of bread in addition to reduce the level of Nε-(carboxymethyl)-lysine (CML). In general, GSE-fortified bakery products are promising as functional foods having relatively lower
CML-related health risks but stronger antioxidant activity (Peng et al., 2010). On the other
hand, wheat species such as emmer wheat, which have significantly high flavonoid content
and strong antioxidant capacity, can be utilized as novel grains with rich natural antioxidants
in bakery products. Lutein content of einkorn samples is also considered as a potential source
of lutein for producing bakery products to improve dietary carotenoid intake (Dziki et al.,
2014). In recent years, grape-fruit-derived nutritional products and food additives have
dominated the market world widely. Most of these products are obtained as by-products
of pomace or grape juice processing, including grape skin or seed extracts, grape skin powder, dry seed powder, pomace powder, and anthocyanin colorants. Swiss company “Mibelle
biochemistry” recently commercialized the biomass derived from grape cell suspension
(V. vinifera L. “Gamay Freaux” var) for use in skin care and cosmetic products. This highlights
the commercialization of modern high-end plant biotechnology products, where this is the
first of its kind for the anthocyanin-rich grape products. As a matter of fact, anthocyanins
are valuable ingredients in skin care products since they have strong UV protectants and
antiaging activity (Georgiev et al., 2014).
3.1.4 Possible interactions of flavonoids
Nutritional supplements containing flavonoid glycosides or aglycones in milk thistle
(Silybum marianus) and red clover extracts (Trifolium pratense) are widely utilized because
of their beneficial health effects and good safety profile. However, the plasma concentration
of flavonoids after taking these herbal supplements is considerably high. For instance, chrysin
supplement is taken at a dose of 1–4 capsules/day, while the recommended daily dose of
quercetin supplements is 620 mg (one capsule/day). Hence, the concentration of flavonoids
is possibly very high immediately after taking these supplements and might cause potential
drug interactions (Morris and Zhang, 2006).
II. Phenolics
48
3. Analysis of polyphenolics
Despite the influence of structural features on absorption and metabolism, about 90%–95%
of dietary polyphenols reaches the colon intact without being absorbed in the small intestine.
Flavonoids are hardly absorbed in the small intestine but metabolized highly in the large
intestine (Duenas et al., 2015). Although understanding the bioavailability of flavonoids is
important to determine their health effects, the bioavailability of this class of compounds
is barely studied and their metabolism is not fully understood. A number of species of
colon microflora were able to hydrolyze flavonoid glycosides to their related aglycones,
though their enzyme could possibly lead to total breakdown of the flavonoid as well
(Georgiev et al., 2014). In addition, the physiological importance of isoflavone glucosides
was also suggested. Very few glucosides such as genistin and daidzin are partly absorbed
directly from the small intestine intact (Iovine et al., 2012). Once absorbed, the flavonoid
aglycones are subjected to three types of conjugation: methylation, sulfation, and
glucuronidation. The plasma concentration of conjugation products is very high indicating
that most of the absorbed flavonoids undergo conjugation ( Jager and Saaby, 2011). In addition, flavones and flavanones demonstrated higher inhibitory activity against aromatase in
comparison to other flavonoid derivatives such as isoflavones and isoflavanones. Moreover,
the major problem associated with flavonoids and their derivatives is in their use for pleiotropic effect. The limiting factor in exploiting therapeutic applications of the flavonoid
derivatives is their potential to interact with many biological molecules. For instance, a
dietary flavonoid called luteolin is reported to have aromatase inhibitory activity even at concentrations as low as 2.44 μM. In contrast, hespartine, a dietary flavonoid, demonstrated
upregulation of aromatase at the mRNA level (Mocanu et al., 2015). Consequently, large dose
and frequent intake of flavonoids increase the risk of flavonoid-induced pharmacokinetic
interactions with drugs. Recent studies reported considerable life-threatening interactions between products containing flavonoids and conventional drugs. For example,
coadministration of silymarin with metronidazole facilitates metronidazole’s clearance
rate. On the other hand, naringin, which is mainly found in grapefruit juice, is reported to
increase the oral bioavailability of felodipine, nimodipine, cyclosporine, and saquinavir in
humans. In animal model studies, naringin increased the oral bioavailability of quinine
and quercetin demonstrated to raise the oral bioavailability of paclitaxel in rats (Morris
and Zhang, 2006). Additionally, herbal remedies such as ginkgo and St. John’s wort contain
flavonoids that were shown to affect the pharmacokinetics of some drugs. Their interaction
with cyclosporine affected pharmacokinetics of the drug, thus facilitating organ rejection
during organ transplant. Likewise, St. John’s wort administered along with digoxin reduced
the oral bioavailability of the drug by 18% through inducing P-glycoprotein (P-gp)
(Roufogalis, 2012).
3.1.5 Techniques of extraction, purification, and fractionation of flavonoids
The extraction, separation, and purification of polyphenols are a challenging process due
to their chemical complexity and similar structural features. However, recent advancements
in technologies and instrumentations have made them easier. Overall, the collected samples
II. Phenolics
3.1.5 Techniques of extraction, purification, and fractionation of flavonoids
Sample
preparation
Extraction
49
Drying, lyophilizing (freezing)
Centrifugation, filtration
Liquid-liquid, solid-liquid, solid phase extraction
Supercritical fluid extraction, sonication, soxhlet, microwave
SPE, TLC, acid-base fractionation, column chromatography
Isolation
Instrumental
analysis
GC, HPLC, LC-MS, CE, NMR
FIG. 3.1.3
Schematic diagram showing the process of extraction, fractionation, and purification strategies of flavonoids from biological fluids, plants, and food (including beverages). Abbreviations: SPE, solid-phase extraction;
TLC, thin-layer chromatography; GC, gas chromatography; HPLC, high-performance liquid chromatography; LCMS, tandem liquid chromatography-mass spectrometry; CE, capillary electrophoresis; NMR, nuclear magnetic
resonance.
to be extracted (e.g., plants, animal products, and food) undergo drying, freezing, or lyophilizing ahead of extraction to avoid degradation, which is particularly a problem during hightemperature drying. Besides, the sample should be kept in a closed and opaque container
since the composition of polyphenolic compounds is mostly affected by exposure to light
and oxygen. Once the sample is ready, different extraction methods (Fig. 3.1.3), including liquid/liquid partitioning and solid/liquid extraction, can be employed immediately after
preliminary sample pretreatments (filtration and centrifugation) (Tsao, 2010). In the process
of crude extraction of flavonoids, heating the sample-solvent mixture at about 60°C in ethanol/water mixture (3:7, v/v), which is mostly considered suitable solvent, is the first
step. After heating the sample-solvent mixture for enough period of time, the extract will
be cooled, filtered, and the excess solvent will be evaporated to obtain the final crude extract
(Shin et al., 2013). However, the crude extract needs further purification in order to obtain a
pure flavonoid. Thus, a solution of the crude extract will be prepared and poured to a column
(400 2.5 cm i.d.) packed with pretreated AB-8 resin. Once the solution is absorbed, distilled
water will be used to wash carbohydrates out of the column and then preferably 65% ethanol
is poured into the column to elute flavonoids. The collected mixture will be concentrated by
rotary evaporation apparatus and dried under vacuum. Since the obtained eluate of
flavonoids could contain many derivatives, further chromatographic procedures such as
HPLC, GC, and others can be employed to get component flavonoids (Cai et al., 2010;
Shi et al., 2016).
II. Phenolics
50
3. Analysis of polyphenolics
3.1.6 Techniques of identification and quantification of flavonoids
Usually the total flavonoid content in natural products is estimated of rutin equivalents.
In the calculation of rutin equivalents, primarily a serial dilution of solution containing different concentration of rutin ranging from 4.58 to 54.9 μg/mL is prepared, which will then
be used to draw the calibration curve. The stock solution of rutin is further diluted with
0.3 mL of 5% (w/v) NaNO2, 0.3 mL of 10% Al(NO3)3, and 4 mL of 4% (w/v) NaOH. In
the meantime, the solutions are left to stand for about 6 min and then further diluted to a
volume of 25 mL using distilled water. After a period of 15 min, the absorbance can be measured using a spectrometer. The extracted natural product sample should also be prepared
and analyzed in a similar manner. Finally, the total flavonoids content of the extracted natural product sample is calculated using the already-established standard calibration curve
(Shi et al., 2016). On the other hand, anthocyanins and yellow flavonoids analysis is done by
preparing a 10-mL solution of 1 g freeze-dried sample in 1.5 N HCl in 85% ethanol solvent.
The sample is then homogenized and extracted for 13 h at low temperature (refrigeration) in
the dark. Next, after filtering the sample, the absorbance is measured at 535 and 374 nm
for anthocyanins and yellow flavonoids, respectively. The result is then calculated using
the following equation to determine the content of anthocyanins and yellow flavonoids
(da Silva et al., 2014).
ðABS dilution factorÞ 1000
Anthocyanins content mg=100 g ¼
sample dry weight ε1%
1 cm 535
ABS ¼ absorbance reading of sample at 535 nm, ε1%
1 cm 535 ¼ the absorption coefficient for
anthocyanins.
Similarly, the content of yellow flavonoids is calculated by the same equation only by
changing the absorbance reading to 374 nm and its absorption coefficient (da Silva et al.,
2014). Other methods have also been used for the determination of total flavonoids content.
This includes the aluminum complexation method, where quercetin is generally used as standard to draw the calibration curve (Mello et al., 2010). Furthermore, Cai and colleagues have
used modified version of rutin standard curve method to determine the content of total flavonoids, which is slightly different from the procedure discussed (Cai et al., 2010).
3.1.7 Levels founds of flavonoids in plants/food-based plants
The level of flavonoids, which are the most representative of the polyphenols, in different
plant species varies typically. Along with tea, coffee, spices, and herbal medicines, fruits
and vegetables are the major dietary sources of flavonoids. In general, the estimated
daily flavonoid intake varies depending on their subclasses such as 0.1–1.2 mg (isoflavones),
0.3–1.6 mg (flavones), 5.4–27.4 mg (flavonols), 20.4–50.6 mg (flavanones), 12–189.2 mg
(flavan-3-ols), and 180–215 mg (anthocyanins) (Mocanu et al., 2015). In addition, the amount
of daily flavonoids intake might vary based on the type of plant-based food consumed. In
grapes, flavonoids mainly accumulate in seeds, but they are also found in the skin of the
II. Phenolics
3.1.8 Effects of food processing in flavonoids
51
grape berries as well. The major grape flavonoids are (+)-catechin, ()-epicatechin, and
proanthocyanidins. For instance, the flavonoid content in white grape ranges from 46%
to 56% of total phenolics content, while in red grape it is between 13% and 30%. Although
grapes contain a considerable number of flavonoids, the predominant flavonoids in grapes
are quercetin-3-O-glucoside and quercetin-3-O-glucuronide (Georgiev et al., 2014). Green
tea polyphenols, which also comprises flavonoids, have been broadly studied for their beneficial health effects. Data from HPLC analysis revealed that green tea leaf infusions have
30%–40% polyphenols of the dry weight content of which flavonoids represent about 80%
(Legeay et al., 2015). Another rich source of polyphenols is the extra virgin olive oil obtained
from the olive tree (Olea europaea L.) fruits. Although the olive tree fruits are consumed
widely, olive leaves are those which contain higher number of polyphenols. Ironically,
the most commonly consumed olives contain the phenolic compound oleuropein only
about 0.005%–0.12%, while its amount in the leaves is between 1% and 14%. Similarly,
the total flavonoids and polyphenols content of the olive leaves is found to be high.
It was determined that the total flavonoids and polyphenols content in olive leaves reaches
up to 2.058 mg GAE (gallic acid equivalents) per 100 g and 858 mg CTE (catechin equivalents) per 100 g (Vogel et al., 2014). Another class of flavonoids, anthocyanins, are also
abundantly present in fruits especially in blueberries and blackcurrants. Generally, the
total anthocyanins content of Surinam cherry and acerola (alkaloids, triterpenes, tannins,
flavonoids, and anthraquinones) is very high as compared to other berries such as
red grapes (137.8 mg/100 g d.b.), strawberries (236 mg/100 g d.b.), red raspberries
(647.9mg/100g d.b.), cherries (616.2mg/100 g d.b.), and blackberries (2954.2 mg/100 g d.b.).
In contrast, the total anthocyanins content in apple and peach is lower, which is estimated
to be between 8.2–84.8 and 0.8–3.1 mg/100 g d.b., respectively (da Silva et al., 2014). According
to the USDA’s flavonoids database, in the United States, the main sources of anthocyanins
for adults include berries (20%), wine (16%), grapes (11%), red/purple vegetables (8%),
100% noncitrus juice (6%), yogurt (6%), and other food sources (33%). The mostly utilized
berries with higher content on anthocyanins are bilberries, blueberries, blackberries,
blackcurrants, chokeberries, strawberries, and elderberries. Nowadays the increasing demand
of coloring agents in the food industry has increased the popularity of anthocyanins as an
alternative to synthetic colorants (Wallace and Giusti, 2015).
3.1.8 Effects of food processing in flavonoids
Nowadays, consumers’ interest has shifted toward a diet richer in fruits and vegetables
due to their health benefits. However, multiple factors such as cost, seasons of the year, and
time restraints limit the access to fresh fruits and vegetables on a daily basis (Schmidt et al.,
2005). Grape is one of the most consumed fruits due to its importance in the making of wine,
grape juice, and other food products. Grape flavonoids are major constituents among the
phenolic compounds and they are responsible for its antioxidant activity (Georgiev et al.,
2014). However, these phenolics compounds such as anthocyanins and proanthocyanidins
are unstable under different storage and processing conditions, which in turn influences
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52
3. Analysis of polyphenolics
their biological activity. In today’s market, canned, dried, or frozen have become preferable
because of convenience and long-life span. Despite this, the processing methods used
to maintain freshness and physical integrity mostly affect the level and composition of
bioactive components of the product. Heat, pH, oxygen, and other storage conditions
have clear impact on anthocyanins stability. This has been remarkably observed in blueberries and other small fruits, in which their color is a major determinant of their quality
and pigment stability is affected under different processing conditions (Schmidt
et al., 2005).
UV irradiation is now a well-established nonthermal, physical method employed in preserving food (cold pasteurization). It is more advantageous than thermal processing since it
uses an ambient temperature and thus causes insignificant changes in the color, test, flavor,
nutrients, and other quality characteristics of foods. However, some studies indicated that
based on the technological criteria and type of food processed, the irradiation can alter the
antioxidant compounds composition of the food. A study that used g-irradiation demonstrated that the antioxidant potential of soybean was enhanced after irradiation due to increase the levels of genistein (an isoflavone) and diadzein degradation products. In
another study on nutmeg oil, g-irradiation resulted in increased amount of phenolic acids,
which are degradation products of tannins (Alothman et al., 2009a). The ultraviolet light
(UV-C) (200–280 nm) has been used to disinfect water, surfaces, and packaging in foodprocessing industries. The germicidal effect of utilizing UV irradiation has demonstrated
promising results as compared to thermal food processing. Studies showed UV-C irradiation
improves the total polyphenols profile of banana, guava, honey, and pineapple. By increasing
the period of treatment, the overall content of polyphenols and flavonoids is boosted, despite
the decrease in vitamin C content. Moreover, it has a preventive action against various
physiological and pathological conditions in addition to improving the nutritional value of
fresh-cut fruits (Alothman et al., 2009b). Pomegranate (Punica granatum Linn.) fruit has a high
concentration of polyphenols such as anthocyanins, catechins, and ellagic tannins. In this
fruit, the anthocyanins provide the bright red color, which is one of the main parameters that
determine the quality and consumers acceptance of pomegranate juice. As compared to heat
treatment, UV irradiation of pomegranate juice enhances its anthocyanins and polymeric
color. Furthermore, other qualities of the fruit such as the total phenolics, antioxidant activity,
and physicochemical characteristics remained the same under UV treatment (Pala and
Toklucu, 2011).
3.1.9 Trends and concluding remarks
In the past few decades, human, animal, and cell-culture studies demonstrated that flavonoids have a great potential for animal and human health. Flavonoids are polyphenols
abundantly available in our diet, including in vegetables, fruits, wine, and tea (George
et al., 2017). Experimental evidences suggested that the risk of osteoporosis-related bone
fracture and bone density decrease can be attenuated by dietary approach that results in
improving bone mineral density (BMD), bone strength, and bone microstructure. Dietary
II. Phenolics
3.1.9 Trends and concluding remarks
53
polyphenols such as tea flavonoids, particularly black tea and green tea flavonoids, have
been shown to mitigate bone loss and to reduce risk of fracture. Antiosteoporosis effect
of flavonoids from green tea and black tea has been demonstrated in animal models with
estrogen deficiency (due to ovariectomy, OVX) (Shen and Chyu, 2016). Similarly, blueberry
flavonoids, which have attracted researchers’ attention due to their strong antioxidant potential, halt whole-body bone loss with no change in fracture-prone sites (Weaver et al.,
2012). Another important source of flavonoids is Rhizoma drynariae, which is currently a post
marketing Chinese medicine by a name Qianggu capsule, and has shown that its total
flavonoids include naringin, naringenin, and neoeriocitrin. The Rhizoma drynariae flavonoids have strong osteoprotective activity mainly acting through regulating signaling pathways, including OPG (osteoprotegerin)/RANK (nuclear factor kappa B)/RANKL (RANK
ligand), CTSK (cathepsin K) cysteine protease, Wnt/β-catenin, and consequently
preventing osteoporosis (Zhang et al., 2017). Moreover, consumption of soy isoflavones,
which bears structural similarity to estrogen, is also suggested to mitigate the risk of hip
fractures in Asians (Weaver et al., 2012).
Likewise, increase in consumption of dietary isoflavones has been suggested to reduce
the incidence of estrogen-related cancers and vascular diseases (Batra and Sharma, 2013).
Additionally, various other flavonoid derivatives available in the regular diet have been related to the chemoprevention and treatment of cancer. Specially, the consumption of dietary
flavonoids has been associated with the reduced risk of smoking-related cancer (Raffa et al.,
2017). Knekt et al. reported that higher intake of quercetin reduces the risk of lung cancer
in men and consumption of myricetin decreases the incidence of prostate cancer (Batra
and Sharma, 2013). However, in comparison to quercetin and baicalein, their glycoside
derivatives rutin and baicalin demonstrated no inhibitory effect against colon cancer
(Sak, 2014). As anticancer agents, flavonoids act through various mechanisms, including
cell-cycle arrest by modulating several cell-cycle regulatory proteins (Raffa et al., 2017).
Being the source of bioactive flavonoids, the positive associations of healthy dietary
approach (optimal intake of fruits and vegetables) with decreased risk of cancer and
better bone health can bring about a new suggestion toward consuming a particular type
of fruits and vegetables, although the evidence is incomplete currently (Welch and
Hardcastle, 2014).
Dietary supplements and nutraceuticals that contain important flavonoids are widely
available in the market. However, they have not been extensively utilized to benefit world
population. Being strong antioxidants in nature, flavonoids have a range of biological activities as well as wider application in food-processing industries. Moreover, their characteristic coloring ability, especially that of anthocyanins, has been of interest as dyes
in textile, food-processing, and cosmetic industries. Generally, the current trends in flavonoids research indicate there is an emerging potential to drive a lead compound or a drug
candidate that can be developed to a drug for treating infectious diseases, diabetes, cancer,
Alzheimer’s disease, and other age-related ailments. Therefore, further research is important to provide reliable data on the flavonoids and their metabolites that are active and
their respective doses, their interaction with other drugs as well as their mechanisms of
actions.
II. Phenolics
54
3. Analysis of polyphenolics
Acknowledgment
All the authors of the manuscript thank and acknowledge their respective universities and institutes.
Conflict of interest
There is no conflict of interest.
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3.2.2 Phytochemistry and classification of glycosidic derivatives of flavonoids
57
S U B C H A P T E R
3.2
Glycosidic derivatives of flavonoids
Muhammad Farrukh Nisara, Chunpeng Wanb, Zahid Manzoorc, Yasir Waqasa,
Kamal Niazc, Muhammad Mazhar Ayazd
a
Department of Physiology and Biochemistry, Faculty of Bio-Sciences, Cholistan University of
Veterinary and Animal Sciences (CUVAS), Bahawalpur, Pakistan bCollege of Agronomy, Jiangxi
Agriculture University, Nanchang, China cDepartment of Pharmacology and Toxicology, Faculty
of Bio-Sciences, Cholistan University of Veterinary and Animal Sciences (CUVAS), Bahawalpur,
Pakistan dDepartment of Parasitology, Faculty of Veterinary Science, Cholistan University of
Veterinary and Animal Sciences (CUVAS), Bahawalpur, Pakistan
3.2.1 Introduction of glycosidic derivatives of flavonoids
In plants, the phenolic compounds are among the major classes of secondary metabolites
and they depict a huge diversity in their structures, which make them important part of foods
(fruits and vegetables) and beverages. Found in various parts of plant organs either as pigments or signalizing compounds, flavonoids serve multiple roles in the human body, i.e.,
antiphotoaging or skin protection against ultraviolet A/B (UVA/UVB) radiations (Semwal
et al., 2016), antiaging (Wang et al., 2018b), antiinflammatory (Wang et al., 2018b), antiallergic,
hepatoprotective, neuroprotective, antiobesity principle, osteogenic activity, antidiabetic, antiviral, antiproliferation, vasodilation, antidiarrheal, antiprotozoal activity,
anticomplementary activity, plant-microbe interactions, pollinator guidance, male fertilization, antifungal, antimicrobial activities, among others (Cirmi et al., 2016; Jiang et al., 2005;
Proestos and Komaitis, 2006; Shirley, 1996). Flavonoids are a large and diverse group. They
are mainly present as glycosides and accumulated in plants in response to certain abiotic
stressors, e.g., UV radiations (Bußler et al., 2015), drought, low and high temperatures
(Popova and Hincha, 2016). Following sections will elaborate the details of flavonoids.
3.2.2 Phytochemistry and classification of glycosidic derivatives of flavonoids
A huge list of natural compounds and antioxidants has been reported to date, which is capable to protect organisms from free-radicals-induced oxidative stress damages. Plants are
rich in diverse class of primary and secondary metabolites (Gupta and Gupta, 2016), mainly
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58
3. Analysis of polyphenolics
FIG. 3.2.1
Basic structure of flavonoids that
consisted of C6-C3-C6 rings primarily linked
in a sequence. (A) Flavonoid C6-C3-C6 and
(B) 2-phenyl-chromone.
the polyphenolics bearing antioxidant, anticancer, antiviral, and antiinflammation properties
(Vinson and Dabbagh, 1998). In the latter case, most of the phenolic compounds such as flavonoids and terpenoids play an important part particularly as medicines to cure veterinary
and human diseases (Williams et al., 2004). Flavonoids are cosmopolitan in distribution as
secondary metabolites in almost each and every plant species. Flavonoids present a huge
group of phenolic compounds accounting for over 9000 naturally occurring distinct structures (Falcone Ferreyra et al., 2012; Geleijnse and Hollman, 2008; Perez-Cano and Castell,
2016; Wang et al., 2011), and they are classified into several major groups, viz. flavonols, flavones, isoflavones, flavanols, flavanones, proanthocyanidins, anthocyanins, and chalcones,
based on the aglycone structure (Brodowska, 2017; Harborne, 1998; Kumar et al., 2009;
Popova and Hincha, 2016) though, some authors classified flavonoids into fourteen classes
(Tebou et al., 2017). Moreover, flavonoids are a major and scattered group of plant compounds in nature and abundantly included in our diet (Brodowska, 2017; Wang et al.,
2018b). Flavonoids are mainly found as glycosylated forms or either esters that consist of
C6-C3-C6 rings primarily linked in a sequence: C6 rings are joined by 3 carbon-ring
(Fig. 3.2.1) (Isoda et al., 2014; Symonowicz and Kolanek, 2012). Basically, the flavonoids
are low-molecular-weight compounds comprising 2-phenyl-chromone nucleus. Following
the variations found in substitution patterns, flavonoids broadly fall into different subclasses,
and produce a huge number of their functional derivatives (Isoda et al., 2014). Universal and
cosmopolitan distribution of flavonoids in plant kingdom offers thousands of benefits, but the
lower bioavailability checks its nutraceutical effects significantly due to lack of proper and
authentic pharmacological evidences (Wang et al., 2018b).
Many of the residues of acetic acid/phenylalanine in the shikimic acid pathway give rise to
the majority of the flavonoids (Wang et al., 2018b). The extent of oxidation level, attachment of
rings, and annularity of rings help to classify the flavonoids (Fig. 3.2.1). In plants, coexisting
flavonols and flavones, comprise the majority of compounds in the group 2-benzo-γ-pyrone
(e.g., quercetin), and possess saturated C2]C3 bonds. On the other hand, isoflavones (e.g.,
Narcissus) make another class of 3-phenyl-chromone compounds. Chalcones are the basic
units or precursors during flavonoid biosynthetic pathway, and its C-opening isomers of
dihydroflavones help impart colors in the various parts of the plants (Wang et al., 2018b).
Moreover, aurones make another class of derivatives, i.e., 5-cornered ring of C benzofuran,
which do not follow the unique structural pattern of flavonoids. The ionic form chromene
pigments form another group, namely, anthocyanidins, and these are known to give certain
specific colors to plants in certain families. Dihydroflavonols (e.g., catechins) having the
flavan-3-ols may undergo reduction following certain environmental conditions to its final
products, i.e., flavonols. Other than these aforementioned classes of flavonoids, few minor
II. Phenolics
3.2.3 Main representatives of glycosidic derivatives of flavonoids
FIG. 3.2.2
59
Structural diversity in the flavonoids class.
classes such as biflavones, furan chromones, xanthones also exist in plant kingdom and
have shown the mimicking structural pattern. The flavonoids also included certain other
compounds, viz. biflavonoids (e.g., ginkgetin), flavonolignans (silybin), prenylflavonoids,
chalcones, proanthocyanins, and ester flavonoids (Wang et al., 2011). Glycosides, classified
into different categories, variable numbers, and connecting patterns, are predominant and
active forms of flavonoids and help create structural diversity (Fig. 3.2.2). Preferred glycosylation sites are linked with the structure of aglycones.
3.2.3 Main representatives of glycosidic derivatives of flavonoids
Bioflavonoids or flavonoids are the cyclic aromatic compounds and important components in the plants. Around 7000–9000 different flavonoids have been isolated and reported
so far (Falcone Ferreyra et al., 2012; Wang et al., 2011). Some of the major and cosmopolitan
representatives of the group have been illustrated in Fig. 3.2.3.
The occurrence and distribution of some common flavonols are enlisted in Table 3.2.1.
The leaves of the Holarrhena floribunda plant have Kaemperol-3-O-rutinoside, kaemperol-3O-glucoside, quercetin-3-O-glucoside, and inseparable mixture of quercetin-3-O-glucose/galactose has been well defined (Badmus et al., 2016). Moreover, it is further elucidated that these
flavonoids have quercetin as the basic structure, and these isolated flavonoid glycosides bearing strong antioxidant activity had been traditionally used since centuries (Badmus et al., 2016).
In maize plant, the ethyl acetate (EtOAc) fractions showed the presence of new molecules along
with three known flavonoids, i.e., tricin, salcolin A, and salcolin B ( Jung et al., 2015). The water
alcoholic extracts ( 80% methanol) of many croton species (Euphorbiaceae) have been studied
extensively recently (Furlan et al., 2015; Salatino et al., 2007; Savietto et al., 2013; Zou et al., 2010),
and the presence of O-glycosides of flavonols (quercetin, isorhamnetin, and kaempferol),
acylglycosides of quercetin and kaempferol (tiliroside), flavones, viz. apigenin, rutin, and
proanthocyanidins are the dominant part of these extracts in croton species (Furlan et al.,
2015; Zou et al., 2010). It is further endorsed that the antioxidant properties of the croton species
II. Phenolics
60
FIG. 3.2.3
3. Analysis of polyphenolics
Distribution of common flavonoids in plants.
TABLE 3.2.1
Diversity and occurrence of some common flavonoids in different plant families.
Family
Plant Species
Active compound
Reference
Actinidiaceae
Actinidia deliciosa
Luteolin/luteolin glycosides
Bhagwat et al. (2014), Harnly
et al. (2006), Lugasi and
Takács (2002), and
Sakakibara et al. (2003)
Amaranthaceae
Spinacia oleracea
Apigenin, luteolin
Arai et al. (2000), Bhagwat
et al. (2014), Chu et al. (2000),
Franke et al. (2004), Hertog
et al. (1992a), and Lugast and
Hovari (2000)
Apiaceae
Petroselinum crispum
Apigenin/apigenin
O-glycosides
Luteolin
Bhagwat et al. (2014) and
Lechtenberg et al. (2007)
Petroselinum crispum
Apigenin, luteolin
Arai et al. (2000), Bhagwat
et al. (2014), Justesen et al.
(1998), Lugast and Hovari
(2000), Mattila et al. (2000),
and Sakakibara et al. (2003)
Apium graveolens var. dulce,
A. graveolens var. Secalinum
Apigenin, luteolin
Bhagwat et al. (2014), Harnly
et al. (2006), Hertog et al.
(1992b), and Justesen et al.
(1998)
II. Phenolics
61
3.2.3 Main representatives of glycosidic derivatives of flavonoids
TABLE 3.2.1
Diversity and occurrence of some common flavonoids in different plant families—cont’d
Family
Plant Species
Active compound
Reference
Asteraceae
Chamaemelum nobile
Apigenin O-glycosides
Apigenin
Luteolin O-glycosides
Luteolin
Carnat et al. (2004)
Matricaria chamomilla
Apigenin O-glycosides
Svehlikova and Repcak
(2006)
Tanacetum vulgare
Apigenin, Luteolin
Wojdyło et al. (2007)
Lactuca sativa
Apigenin, Luteolin
Arabbi et al. (2004), Arai et al.
(2000), Bahorun et al. (2004),
Bhagwat et al. (2014), Chu
et al. (2000), DuPont et al.
(2000), Franke et al. (2004),
Harnly et al. (2006), Hertog
et al. (1992a), Huber et al.
(2009), Lugast and Hovari
(2000), and Young et al. (2005)
Cynara scolymus
Apigenin/apigenin,
glycosides
Luteolin/luteolin, glycosides
Azzini et al. (2007), Bhagwat
et al. (2014), Ferracane et al.
(2008), Lattanzio and van
Sumere (1987), Sch€
utz et al.
(2004), and Wang et al. (2003)
Chicorium intybus
Apigenin/apigenin
O-glycosides
Luteolin/luteolin
O-glycosides
Arabbi et al. (2004), Bhagwat
et al. (2014), and Heimler
et al. (2009)
Brassicaceae
Brassica napus var.
napobrassica, B. oleracea,
B. oleracea var. acephala, B. rapa
var. Chinensis
Apigenin glycosides
Luteolin
Bahorun et al. (2004),
Bhagwat et al. (2014), Chu
et al. (2000), Franke et al.
(2004), Hertog et al. (1992a),
Lugast and Hovari (2000),
Miean and Mohamed (2001),
Sakakibara et al. (2003), and
Young et al. (2005)
Cucurbitaceae
Citrullus lanatus
Luteolin
Arai et al. (2000), Bhagwat
et al. (2014), Harnly et al.
(2006), Lugasi and Takács
(2002), Owen et al. (2003), and
Sampson et al. (2002)
Cucumis melo
Luteolin
Bhagwat et al. (2014), Harnly
et al. (2006), and Lugasi and
Takács (2002)
Cucirbota sp.
Luteolin
Bhagwat et al. (2014), Lugasi
and Takács (2002), and Miean
and Mohamed (2001)
Continued
II. Phenolics
62
TABLE 3.2.1
3. Analysis of polyphenolics
Diversity and occurrence of some common flavonoids in different plant families—cont’d
Family
Plant Species
Active compound
Reference
Ericaceae
Vaccinium sp.
Luteolin
Bhagwat et al. (2014), Franke
et al. (2004), Harnly et al.
(2006), and Justesen et al.
(1998)
Fabaceae
Aspalathus linearis
Apigenin C-glycosides
Luteolin C-glycosides
Bramati et al. (2003), Breiter
et al. (2011), and Pengilly
et al. (2008)
Trigonella foenum-graecum
Apigenin
Luteolin
Wojdyło et al. (2007)
Pisum sativum
Apigenin, luteolin
Apigenin C-glycosides
Luteolin C-glycosides
Luteolin O-glycosides
Quercetin and kaempferol
Quercetin-3-O-p-coumaroyltriglucoside
Bhagwat et al. (2014), Bußler
et al. (2015), Franke et al.
(2004), Magalhaes et al.
(2017), and Miean and
Mohamed (2001)
Cicer arietinum
Luteolin C-glycosides
Magalhaes et al. (2017)
Vicia faba
Apigenin C-glycosides
Apigenin
O-neohesperidoside
Luteolin C-glycosides
Bhagwat et al. (2014) and
Magalhaes et al. (2017)
Mentha xpiperita
Apigenin O-glycosides
Luteolin O-glycosides
Areias et al. (2001) and Fecka
and Turek (2007)
Origanum vulgare
Apigenin, luteolin
Bhagwat et al. (2014)
Perilla frutescens
Apigenin O-glycosides
Luteolin O-glycosides
Meng et al. (2009)
Rosmarinus officinalis
Apigenin, luteolin/luteolin
O-glycosides, diosmetin
O-glycosides
del Baño et al. (2004) and
Wojdyło et al. (2007)
Salvia officinalis
Luteolin/luteolin
O-glycosides
Fecka and Turek (2007) and
Wojdyło et al. (2007)
Oleaceae
Olea europaea (black, green,
olive oil)
Apigenin, luteolin
Bhagwat et al. (2014), Ouni
et al. (2011), and Owen et al.
(2003)
Poaceae
Digitaria exilis
Apigenin, luteolin
Sartelet et al. (1996)
Sorghum bicolour L., (yellow
and red sorghum)
Apigenin
Luteolin
Bhagwat et al. (2014), Dykes
et al. (2011), and Wu et al.
(2016)
Triticum aestivum,
T. carthlicum, T. monococcum,
T. dicoccum, T. carthlicum,
T. turgidum, T. durum,
T. spelta, T. urartu,
T. polonicum
Apigenin C-glycosides
Wijaya and Mares (2012)
Lamiaceae
II. Phenolics
63
3.2.3 Main representatives of glycosidic derivatives of flavonoids
TABLE 3.2.1
Family
Diversity and occurrence of some common flavonoids in different plant families—cont’d
Plant Species
Active compound
Reference
Oryza sativa (Black, White,
Red, and Brown rice)
C-Glycosides of apigenin and
luteolin
Pereira-Caro et al. (2013)
Lophatherum gracileBrongn.
Luteolin 6-C-b-Dglucuronopyranosyl-(1/2)-bD-glucopyranoside,
isoorientin, swertiajaponin,
luteolin 6-C-b-Dglucuronopyranosyl-(1/2)-aL-arabinopyranoside,
isovitexin, swertisin, luteolin
7-O-b-D-glucopyranoside,
and luteolin 6-C-a-Larabinopyranoside
Fan et al. (2015a)
Polygonaceae
Fagopyrum esculentum
Apigenin C-glycosides
Luteolin C-glycosides
Bhagwat et al. (2014),
Dietrych-Szostak and
Oleszek (1999), and
Kiprovski et al. (2015)
Rutaceae
Citrus bergamia
C. deliciosa
C. medica
C. sinensis
Apigenin C-glycosides
Apigenin O-glycosides
Chrysoeriol C-glycosides
Chrysoeriol O-glycosides
Diosmetin C-glycosides
Diosmetin O-glycosides
Luteolin C-glycosides
Caristi et al. (2006),
Fernandez de Simon et al.
(1992), Gattuso et al. (2006),
Gil-Izquierdo et al. (2002),
and Mullen et al. (2007)
Fortunella japonica
Acacetin O-glycosides
Acacetin C-glycosides
Apigenin C-glycosides
Barreca et al. (2011)
Citrus deliciosa, C. sinensis
Apigenin/apigenin
Glycosides, luteolin
Bhagwat et al. (2014), Franke
et al. (2004), Harnly et al.
(2006), Hoffmann-Ribani
et al. (2009), Lugasi and
Takács (2002), and
Sakakibara et al. (2003)
Fortunella crassifolia
Apigenin glycosides
Bhagwat et al. (2014) and
Sakakibara et al. (2003)
Scrophulariaceae
Linaria japonica MIQ.
Linariin, isolinariin,
pectolinarin
Pectolinarigenin
Apigenin
Luteolin
Widyowati et al. (2016)
Solanaceae
Capsicum annuum
Luteolin/luteolin
Glycosides
Arabbi et al. (2004), Bhagwat
et al. (2014), Justesen et al.
(1998), Lugast and Hovari
(2000), and Sakakibara et al.
(2003)
Continued
II. Phenolics
64
TABLE 3.2.1
3. Analysis of polyphenolics
Diversity and occurrence of some common flavonoids in different plant families—cont’d
Family
Plant Species
Active compound
Reference
Theaceae
Camellia sinensis
Black, oolong and green
Apigenin C-glycosides
Luteolin C-glycosides
Engelhardt et al. (1993)
Vitaceae
Vitis vinifera
Apigenin, luteolin
Bevilacqua et al. (2004),
Bhagwat et al. (2014), Fang
et al. (2008), Gambelli and
Santaroni (2004), and Sun
et al. (2008)
Vitis sp.
Luteolin
Bhagwat et al. (2014), Franke
et al. (2004), Hertog et al.
(1992a), Justesen et al. (1998),
and Lugasi and Takács (2002)
Amaranthaceae
AtriplexhalimusL.
Flavonol glycosides,
syringetin 3-O-β-Drutinoside, syringetin 3-O-βD-glucopyranoside,
isorhamnetin 3-O-β-Drutinoside (narcissin),
artiplexoside A
El-Aasr et al. (2016)
Clusiaceae
Garcinia gracilis Pierre,
Apigenin-8-C-α-Lrhamnopyranosyl-(1 ! 2)-βD-glucopyranoside, 5hydroxymethyl-2furaldehyde, and vanillic
acid
Supasuteekul et al. (2016)
Bignoniaceae
Oroxylum indicum L.
Hispidulin, baicalein,
chrysin, oroxylin A
Tran et al. (2015)
are primarily due to the presence of the quercetin, phenols, and proanthocyanidins derivatives
(Furlan et al., 2015; Salatino et al., 2007).
Phytochemistry analyses of Allium tuberosum Rottler reported the presence of two
new phenylpropane glycosides (tuberonoid A and tuberonoid B) along with kaempferol 3-O-b-sophoroside, 3-O-b-D-(2-O-feruloyl)-glucosyl-7,40-di-O-b-D-glucosylkaempferol,
3-O-b-sophorosyl-7-O-b-D-(2-O-feruloyl)glucosyl kaempferol, kaempferol 3,40-di-O-b-Dglucoside (Han et al., 2015). Previously, studies of many Allium species reported that the
leaves are enriched with dietary flavonoids (Kaneta et al., 1980; Yoshida et al., 1987).
The phytochemistry of Leucaeana leucocephala showed the presence of many secondary
metabolites particularly an acylated flavanol glycoside, quercetin-3-O-(200 -transp-coumaryl)-α-rhamnopyranosyl-(1000 !600 )-β-glucopyranoside, in addition to quercetin3-O-α-rhamnopyranosyl-(1000 !200 )-β-glucopyranoside, quercetin-7-O-α-rhamnopyranosylquercetin-3-O-α-rhamnopyranoside,
quercetin-3-O-β(1000 !200 )-β-glucopyranoside,
glucopyranoside, isovitexin, vitexin, quercetin, and many others have been reported from
the Leucaeana leucocephala (Lowry et al., 1984; Mohammed et al., 2015). Opuntia ficus-indica
plants are enriched with isorhamnetin glycosides flavonoids, which are a good source of
II. Phenolics
3.2.4 Biological activities of glycosidic derivatives of flavonoids
65
traditional antiaging medicines as well as food in North American societies (AntunesRicardo et al., 2015). The flavonol glycoside, methylene-bisnicotiflorin, was extracted and
reported from ripe Pu-er tea plant, particularly 1,3-dihydroxyphenyl-2-O-sulfate, 2,3,4trihydroxybenzoic acid, and 3,30,4,40-tetrahydroxybiphenyl are also described through
spectroscopic analyses (Tao et al., 2016). Another new 31arcissi C-glycoside, apigenin 6-Cα-arabinofuranosyl 8-C-α-arabinopyranoside and a novel bibenzyl, and bulbotetusine, were
successfully extracted and reported in the tubers of Bulbophyllum retusiusculum (Yang
et al., 2016).
3.2.4 Biological activities of glycosidic derivatives of flavonoids
The history of using flavonoids to treat various ailments and diseases dates back to the ages
when the science of chemistry evolved. Flavonoids are well known since ages to be used in the
traditional as well as modern medication systems throughout the world. Moreover, the production and biosynthesis of these secondary metabolites is considered as the protective measure in plants against various pathogens and environmental threats; therefore, these
important phytochemicals are not only important for the plant species in which they occur
but also for post consumers, including animals and humans (Di Carlo et al., 1999). The polyphenolic compounds bear diverse applications, including spanning multiple fields and
industries. Although the different flavonoids are classified based on their similar chemical
structures, it is noteworthy to mention that the biological as well as chemical properties
are not the same even when they fall in the same group (Erlund, 2004). The protection being
offered by flavonoids is attributed to the capability of electrons transfer, chelation of metals,
provoke the cellular antioxidant responses, reduction of α-tocopherol radicals, and suppress
oxidases enzymes within the cells (Heim et al., 2002).
Among many vegetables, Allium tuberosum is being traditionally applied to cure abdominal pain, diarrhea, hematemesis, snakebites, and bronchial issues since centuries (Kuo
et al., 2005). Various mono- and biflavonoids have been extracted from the fruits of Juniperus
communis, including apigenin, hypolaetin 7-O-β-xylopyranoside, hypolaetin 7-O-β-Dxylopyranoside, isoscutellarein-7-O-β-xylopyranoside, amentoflavone, podocarpus flavone
A, robustaflavone, cupressuflavone, and hinokiflavone ( Jegal et al., 2016). Abrus mollis Hance
(Fabaceae) plant has a long history of traditional utilization in Asian countries, including
China, but in clinical cases it cures hepatitis and alcoholic liver diseases. These strong healing
properties of Abrus mollis are mainly due to the presence of flavonoid C-glycosides (Xiong
et al., 2015). The glycosides of quercetin, isoquercetrin, and rutin have been reported for significantly providing neuroprotection under 6-OHDA toxicity in PC-12 cells (Magalingam
et al., 2016). Cassia angustifolia M. Vahl is a medicinal herb used traditionally to cure many
illnesses (liver diseases, constipation, typhoid, cholera, etc.) in Southeast and central Asia;
however, its active ingredients are three flavonoids, e.g., rutin, quercimeritrin, and
scutellarein (Ahmed et al., 2016). Martynia annua L., a member of Martyniaccae family, has
long been traditionally used to cure sore throats, inflammatory disorders, and epilepsy,
and these therapeutic effects are mainly due to the presence of luteolin (Lodhi et al., 2016).
Moreover, it was further described in the same study that quercetin is the main constituent
in Tephrosia purpurea, which has previously been used against asthma, gonorrhea, rheumatism, and ulceration in traditional medication systems (Lodhi et al., 2016).
II. Phenolics
66
3. Analysis of polyphenolics
3.2.5 Techniques of extraction and purification of glycosidic
derivatives of flavonoids
Revolutionary changes occurred in the mass spectrometric techniques in recent decades,
which made it possible to analyze low-molecular-weight natural products bearing distinguished physicochemical properties (Gross, 2011). The versatility of mass spectrometric techniques lies in applicability of various physical phenomena for ionization of the molecules
analyzed, production and separation of the ions generated. The majority of the applications
that utilize liquid chromatography, gas chromatography, and capillary electrophoresis with
mass spectrometry (LC-MS, GC-MS, or CE-MS, respectively) along with multiple ionization
methods could potentially be approached (Gross, 2011). With the onset of the 21th century,
and advancement in equipments, a revolution in novel methodologies to extract new compounds arrived even when present in minute quantities (Table 3.2.2).
TABLE 3.2.2
List of phytochemicals extraction methods.
Extraction
methods
Plants
Flavonoids
References
Strobilanthes
crispus
Major bioactive flavonoid, i.e., (+)-catechin,
()-epicatechin, rutin, myricetin, luteolin, apigenin,
naringenin, and kaempferol
Liza et al.
(2010)
Mentha
spicata L.
Catechin, epicatechin, rutin, myricetin, luteolin,
apigenin, and naringenin
Bimakr et al.
(2011)
Camellia
sinensis L.
191.28 mg QE/100 mL of TFC
Nam et al.
(2015)
Mentha spicata
L.
Catechin, epicatechin, rutin, myricetin, luteolin,
apigenin, and naringenin
Bimakr et al.
(2011)
Radix astragali
1.292 mg/g flavonoids
Xiao et al.
(2008)
Rape bee
pollen
Quercetin, kaempferol, isorhamnetin
Tu et al. (2017)
Tailor-made deep
eutectic solvents
Flossophorae
Quercetin, kaempferol, and isorhamnetin
Nam et al.
(2015)
Heat reflux
extraction (HRE)
Radix astragali
0.934 mg/g flavonoids
Xiao et al.
(2008)
Pandanus
maryllifolius
Roxb.
Myricetin, luteolin, and quercetin
Ghasemzadeh
and Jaafar
(2014)
Radix astragali
1.234 mg/g flavonoids
Xiao et al.
(2008)
Supercritical
carbon dioxide
(SC-CO2)
Soxhlet extraction
(CSE)
Microwaveassisted extraction
(MAE)
II. Phenolics
67
3.2.5 Techniques of extraction and purification of glycosidic derivatives of flavonoids
TABLE 3.2.2
List of phytochemicals extraction methods—cont’d
Extraction
methods
Pressurized liquid
extraction (PLE)
Ultrasoundassisted extraction
(UAE)
Plants
Flavonoids
References
Saussurea
medusa Maxim.
Rutin
Gao et al. (2006)
Eucalyptus
camaldulensis
Dehn.
5.8 mg/g flavonoids
Gharekhani
et al. (2012)
Myrtus
communis L.
5.02 mg/g flavonoids, myricetin 3-O-galactoside,
myricetin galloylgalactoside, epigalocatechin, myricetin
3-O-rhamnoside, quercetin 3-O-rhamnoside
Dahmoune
et al. (2015)
Epimedium
15 Flavonoids, including hexandraside E (1), kaempferol3-O-rhamnoside (2), hexandrasideF (3), epimedin A (4),
epimedin B (5), epimedin C (6), icariin (7), epimedoside C
(8), baohuoside II (9), caohuosideC (10), baohuoside VII
(11), sagittatoside A (12),sagittatoside B (13), 200 -Orhamnosyl icariside II (14), and baohuosideI (15)
Chen et al.
(2007)
Houttuynia
cordata Thunb.
1, Rutin; 2, hyperin; 3, quercitrin; 4, quercetin
Zhang et al.
(2008)
Spinach
Patuletin 3-O-β-D-glucopyranosyl(1 ! 6)-[β-Dapiofuranosyl (1 ! 2)]-β-D-glucopyranoside, spinacetin
3-O-β-D-glucopyranosyl
(1 ! 6)-[β-D-Apiofuranosyl (1 ! 2)]-β-D-glucopyranoside,
patuletin 3-O-β-D-(200 -feruloylglucopyranosyl (1 ! 6)-β-Dapiofuranosyl (1 ! 2)]-β-D-glucopyranoside, spinacetin
3-O-β-D-(20 -p-feruloylglucopyranosyl (1 ! 6)-[β-Dapiofuranosyl
(1!2)]-β-D-glucopyranoside, spinacetin 3-O-β-Dglucopyranosyl (1 ! 6)-β-D-glucopyranoside, jaceidin
40 -glucuronide, 5,3,40 -trihydroxy-3-methoxy-6:7methylenedioxyflavone 40 -glucuronide, 5,40 -dihydroxy3,30 -dimethoxy-6:7-methylenedioxyflavone
40 -glucuronide
Howard and
Pandjaitan
(2008)
Radix astragali
0.736 mg/g flavonoids
Xiao et al.
(2008)
Eucalyptus
camaldulensis
Dehn.
5.5 mg/g flavonoids
Gharekhani
et al. (2012)
Olea europaea
Luteolin-40 -O-glucoside, apigenin-7-O-glucoside, rutin,
luteolin, quercetin, and apigenin
Wang et al.
(2018a)
Pollen typhae
Quercetin, naringenin, kaempferol, and isorhamnetin
Meng et al.
(2018)
II. Phenolics
68
3. Analysis of polyphenolics
3.2.6 Techniques of identification and quantification of glycosidic derivatives
of flavonoids
The polyphenolic compounds or flavonoids are frequently found in the plant kingdom,
and help impart a unique flavor and color to the fruit and vegetables. Most of these plant polyphenolics or flavonoids show medicinal, food, and cosmetic industrial values, encouraging
the scientists to explore more and more compounds in a precise way.
Detection of the novel flavonoid compounds formerly remained undetectable in plant
samples is recently easier by competent dereplication methods that recognize flavonoids even
when present in minute quantities (Table 3.2.3). The main techniques, thin-layer chromatography (TLC) alone or in combination with mass spectrometry (MS), exploited the new and
TABLE 3.2.3
Various phytochemicals extraction methods.
Identification and quantification
methods
Plants
Flavonoids
References
Oroxylumindicum
Flavonoid 5,6,7-trimethoxyflavone8-O-b-D-glucopyranoside
Fan et al.
(2015b)
Caragana
brachyantha
Two new flavonol glycosides,
quercetin 5-O-[α-L-rhamnopyranosyl(1!6)-β-D-glucopyranoside]-7-O-[αL-rhamnopyranoside] and quercetin
5-O-[α-L-rhamnopyranosyl-(1!6)β-D-glucopyranoside]-7-O-[α-Lrhamnopyranoside]-40 -O-[α-Lrhamnopyranoside]
Perveen
et al. (2015)
Scabiosa
prolifera L.
New flavonol glycoside, kaempferol3-O-(400 ,600 -di-E-p-coumaroyl)-β-Dgalactopyranoside
Al-Qudah
et al. (2017)
Apple juice
Quercetin, quercetin arabinose,
quercitrin, phloretin glucose,
kaempferol rhamnose, quercetin
arabinose, quercetin glucose, rutin
Fougère
et al. (2018)
Rose flower
Glycosides of kaempferol and
quercetin
Fougère
et al. (2018)
Soldanella alpine
Rutin, luteolin-7-O-glucoside,
quercetin, and luteolin
Kroslakova
et al. (2016)
OLE-QTOF-MS (online extractionquadrupole time-of-flight tandem
mass spectrometry)
Citrus paradisi
Hesperidin and neohesperidin
Tong et al.
(2019)
TLC-ESI-MS (thin-layer
chromatography with electrospray
ionization mass spectrometry)
Juniperus
communis L.
(+)-Catechin, procyanidin dimmer,
procyanidin trimmers
Smrke and
Vovk (2013)
Punica
granatum L.
()-Epicatechin, (+)-gallocatechin,
procyanidin dimers/trimmers
Smrke and
Vovk (2013)
1
13
H-NMR, C-NMR and 2D-NMR
(H, H-COSY, HSQC, HMBC)
TLC-MALDI-TOF-MS (thin-layer
chromatography matrix-assisted laser
desorptiontime-of-flight mass
spectrometry)
II. Phenolics
69
3.2.7 Levels founds of glycosidic derivatives of flavonoids in foods/plants
TABLE 3.2.3
Various phytochemicals extraction methods—cont’d
Identification and quantification
methods
GC-MS
HPLC-ESI-QTOF-MS
Plants
Flavonoids
References
Berry fruits
Anthocyanins and anthocyanidins
Cretu and
Morlock
(2014)
Salvia
lavandulifolia
Genkwanin, nepetin, isoquercitrin,
and luteolin-5-rutinoside
Sajewicz
et al. (2011)
Propolis
Polymethoxylated flavones or
flavanones
Gautam
et al. (2013)
Vitex negundo
Flavones 40 -OH,5-OH,7-di-Oglucoside, 5-hydroxy-3,6,7,30 ,40 pentamethoxy flavones
Marquez
Hernandez
et al. (2010)
Rubusidaeus L.
Various derivatives of Quercetin,
kaempferol, luteolin, and
isorhamnetin
Li et al.
(2016)
Morus alba L.
Quercetin derivatives, kaempferol
derivatives, two new quercetin
derivatives morkotin A and
morkotin C
Kwon et al.
(2019)
Prunus fruits
23 Flavonoids were confirmed, three
new identified: quercetin 3-O-(200 -Oacetyl) neohesperidoside, quercetin
3-O-(400 -O-acetyl) rutinoside, and
kaempferol 3-O-(400 -O-acetyl)
rutinoside
Jang et al.
(2018)
novel compounds. In this area of science, TLC and MS are highly precise and sophisticated
techniques that work with atmospheric pressure ionization MS (TLC-API-MS) and matrixassisted laser desorption ionization MS (TLC-MALDI-MS) (Cretu and Morlock, 2014;
Sajewicz et al., 2011).
3.2.7 Levels founds of glycosidic derivatives of flavonoids in foods/plants
Huge quantitative studies been done on glycosides and the aglycones, but the daily use of
flavones varies among person to person and countries even, e.g., in Europe, America, and
China, daily intake estimates are 0.7–9.0, 1.1–1.6, and 1.9–4.2 mg/d, respectively (Cassidy
et al., 2014; Jennings et al., 2013; Pounis et al., 2016; Sun et al., 2015; Wang et al., 2014;
Zamora-Ros et al., 2011). The content of polyphenolics in chamomile and parsley has been
examined, and reported to possess extremely higher flavone amounts. The apigenin
O-glycosides concentrations in dry plant parts are noted to be as high as 5320 mg/100 g in
chamomile flowers, while in parsley leaves, they were 1350 mg/100 g (Lechtenberg et al.,
II. Phenolics
70
3. Analysis of polyphenolics
2007; Svehlikova and Repcak, 2006). Mint family (Lamiaceae) is enriched with the flavone
O-glycosides, but the concentration of the polyphenolics remained variable among the species. In dried peppermint leaves, luteolin glycosides concentrations were found variable
(42–3070 mg/100 g) just because of variable sources of plant materials and analytical techniques used (Areias et al., 2001; Fecka and Turek, 2007). In kumquats (Fortunella crassifolia),
apigenin was noted to be about 21 mg/100 g of fresh weight (Sakakibara et al., 2003), but the
juice of kumquat (Fortunella japonica) has even less than 1 mg/100 g of apigenin (Barreca et al.,
2011). On the other hand, naringin in the peels of grapefruit was 100 times higher than in the
juice of the fruits (Wu et al., 2007). Numerous flavones in cereal crops and legumes have been
cited in Table 3.2.4. Diversity in the concentrations of flavones was found among different
fresh and green foods. For instance, vegetables in the asteraceae and apiaceae families have
presented highest concentrations of flavonoids (Hostetler et al., 2017).
TABLE 3.2.4
Sr.
no.
The concentrations of various flavones in cereal and leguminous plants.
Concentration
(mg/100 g)
Reference
Apigenin O-glycosides
Apigenin
Luteolin O-glycosides
Luteolin
2531
Carnat et al. (2004)
Trigonellafoenumgraecum (Fabaceae)
Apigenin
Luteolin
731
512
Wojdyło et al. (2007)
3
Mentha xpiperita
(Lamiaceae)
Apigenin O-glycosides
Luteolin O-glycosides
5.6–27
42–3070
Areias et al. (2001) and
Fecka and Turek (2007)
4
Origanum vulgare
(Lamiaceae)
Apigenin
Luteolin
15.6–19.4
901–1137
Bhagwat et al. (2014)
5
Camellia sinensis
(Theaceae)
Green, black, and
oolong tea
Apigenin C-glycosides
Luteolin C-glycosides
41.4–246.8
2.5–21.9
Engelhardt et al. (1993)
6
Alium cepa L.
(Liliaceae)
Quercetin/quercetin glucosides (7,4diglucoside, 3-glucoside, 4-glucoside)
6633 (red
onion)
1671 (yellow
onion)
Kwak et al. (2017)
7
Vigna mungo L.
(Fabaceae)
Vitexin
Isovitexin
42.31
84.61
Girish et al. (2016)
8
Chenopodium album
L. (Chenopodiaceae)
Quercetin
733.5
Arora and Itankar
(2018)
9
Murraya koenigii L.
(Rutaceae)
Quercetin-3-glycoside
Quercetin
Rutin
100–540
10–140
10–90
Sepahpour et al. (2018)
10
Cymbopogon citrates
L. (Poaceae)
luteolin-7-o-glycoside
10–60
Sepahpour et al. (2018)
Plant/family
Major compounds
1
Chamaemelum nobile
(Asteraceae)
2
II. Phenolics
3.2.8 Effects of food processing in glycosidic derivatives of flavonoids
71
3.2.8 Effects of food processing in glycosidic derivatives of flavonoids
Food may be processed using different strategies. A number of various energy media may
be applied for the processing of food. These may include use of heat, air, water, oil, and electromagnetic radiations. These processes usually have an impact on the bioactive compounds
present in the food. Some of the polyphenolic compounds such as proanthocyanidins and
anthocyanins are destabilized during the process of heating (Talcott et al., 2003). The antioxidant activity of these phenolic compounds may be lost during the instability of these compounds (Kader et al., 2002; Rossi et al., 2003). The presence of brownish or yellowish pigments
during the food processing confers different biological changes in the phenolic compounds of
food (Clifford, 2000).
Blanching is the first step in food-processing procedure, which is commonly used to deactivate the enzymes and soften the food products. This process leads to the degradation
of many phenolic compounds because these compounds depend upon the presence of different enzymes such as glycosidases, peroxidase, and polyphenoloxidases. A number of
byproducts such as anthocyanidins, sugars, o-dihydrophenols, o-quinones are produced
by the action of these enzymes. Contrary to these observations, some investigators reported
the increase in the total flavonoids and phenolics contents during the process of blanching in
beauty apple peels when these were boiled in water for 10–20 s (Wolfe and Liu, 2003). Some
researchers suggest that antioxidant activity could be retained if the green leaves of sweet
potato are blanched for one minute in boiling water (Chu et al., 2000).
Cooking is another common food-processing step, which has variable effects on the antioxidant capacity of phenolic compounds of food. In an experimental study, the total phenolic
content of raspberries and blueberries was increased by 50% and 53% when these were heated
at 100°C for 28 and 22 min, respectively (Sablani et al., 2010). On the contrary, the total phenolic contents were reduced in dehydrated plums; however, the antioxidant capacity increased in dried plums compared with fresh ones (Piga et al., 2003).
Food may be processed by the process of baking in which the food items are heated up to
120°C. During this process, a number of chemical reactions may occur. These include
caramelization (an oxidation process between ascorbic acid and reducing sugars), Strecker
degradation (interaction of amino acids with dicarbonylic compounds), Maillard reaction
(between amino acids and reducing sugars), oxidation of phenolic antioxidants to their polymers, hydrolysis of esters and glucosides. It is further reported that the total phenolic content
of purple wheat bran remained unchanged when it was baked at very high temperature of
177°C for 20 min (Li et al., 2007).
There is a wide variation in the whole phenolics content of the vegetable foods processed
by microwave cooking. It was observed in an experimental study that total phenolics content
was slightly decreased in some vegetables such as peas, cauliflower, Swiss chard, and spinach
when these were cooked under microwave at 800 W, but there was a remarkable decrease in
the total phenolics content of these vegetables after boiling in a hot water for 6–13 min (Natella
et al., 2010).
There was an increased total phenolics and flavonoids contents of apple mash under microwave heating set at 1500 W ( 40–70°C) for an average 10 min (Gerard and Roberts, 2004).
Similarly, black raspberries showed great variation in anthocyanin concentration during the
food processing. There was 42% and 51% reduction in the total anthocyanin content when
II. Phenolics
72
3. Analysis of polyphenolics
black raspberries were processed in canned-in-water or syrup at 87.3–93.3°C for 4 min,
respectively (Hager et al., 2008). But the blackberry products reduce 17.8% and 10.5% in
anthocyanin content in the same scenario (Hager et al., 2010). A significant reduction in anthocyanin content was seen in strawberry (28%) puree and blackberry (3%) when these were
processed for 2 min at 70°C (Patras et al., 2009).
The anthocyanin content was decreased in some of the other food-processing procedures.
For example, the anthocyanin content was decreased by 41% in red cabbage under boiling at
94–96°C for 3 min and 29% decreased under steaming for 10 min (Volden et al., 2008). Similar
results were seen in blueberry products when these were pasteurized at 90°C for 1.5 min (Lee
et al., 2002); on the contrary, the anthocyanins were found stabilized in black carrots during
boiling at 70–90°C (Kırca et al., 2007). The possible reason for stability of anthocyanins at high
temperature might be due to diacylation of anthocyanin structure.
Some other food-processing techniques like steaming and flaking were supposed to decrease the caffeic acid, tocotrienols, and some avenanthramides in oat groats, but vanillin
and ferulic acid were increased during these procedures. Some phytochemicals such as tocopherols, p-coumaric and ferulic acids, tocotrienols, acids of vanillin were increased in
oat during the process of autoclaving at 100–120°C for 16 min (Bryngelsson et al., 2002).
The severity of heat application in food-processing procedures has a vital role in determining
the stability of anthocyanins. Depending upon the intensity and duration of heat, the anthocyanins content behaves differently in various food-processing procedures (Wolfe and
Liu, 2003).
3.2.9 Pharmaceutical applications of glycosidic derivatives of flavonoids
Flavonoid analogues along with metal complexes also play a potential role in agriculture,
industrial, and pharmaceutical chemistry (Kumar et al., 2009). Following are the various
applications of flavonoids.
3.2.9.1 Medicinal uses
Many of the current-day medicines rely on using flavonoids to treat various human and
animal diseases. Flavonoids are being applied in both traditional and modern medication systems across the globe. The biological potential of flavonoids is hidden mainly in its structure
and concentration. The flavonoids, especially the C-glycosyl flavonoids, show diverse biological activities such as hepatoprotective, antioxidant, antiinflammatory, antiallergic, antiviral,
antithrombotic, anticarcinogenic potential (Xiao et al., 2016), antipyretic, antibacterial, and diuretic activities (Fan et al., 2015a). It is worth mentioning that di-C-glycosyl flavonoids are of
special interest because of their quick absorbance in the intestinal tract and are finally delivered to different organs without any structural alterations (Xiao et al., 2016). Of them, most of
the activities are mainly due to the strong antioxidant nature, inhibition of the production of
reactive oxygen species (ROS), ability to transfer electrons, chelation of the microelements
particularly iron and copper that help amplify the production of ROS in the cells, activation
II. Phenolics
3.2.9 Pharmaceutical applications of glycosidic derivatives of flavonoids
73
of the antioxidant enzymes, reduction of α-tocopherol residues, and inhibition of the expression of oxidases (Heim et al., 2002; Middleton et al., 2000; Pietta, 2000). Flavonoids are a huge
group of polyphenolic compounds with diversity in structures having strong antioxidant effects (Perez-Gregorio et al., 2014). The efflux of ROS helps the cancerous or tumor cells to apoptosis. The majority of plant secondary metabolites are limitless source of anticancer drugs,
such as vincaalakaloids, camptothecin, taxol, and podophyllotoxin analogues (Hosseini and
Ghorbani, 2015). The flavonoid phytochemicals comprise a huge collection of phenylalanine
derivatives belonging to polyphenol class, including the subclass of flavonols, which are well
represented by kaempferol, quercetin, and rutin (Iriti and Varoni, 2013). The C-glycosyl flavones (vitexin, isovitexin, and quercetin) isolated from black gram husk have protected the
erythrocytes and DNA from ROS-mediated damage and shown strong anticancer activity by
upregulating Bax and caspase-3 expressions in HeLa cells (Girish et al., 2016). The flavonoid
rutin (quercetin 3-O-β-D-rutinoside) sensitizes triple-negative breast cancer cells during chemotherapies and reverse the multidrug resistance by inhibiting efflux pumps (adenosine
triphosphate-binding cassette transporters), activating the apoptosis and cell-cycle arrests
(Iriti et al., 2017).
The Mexican people widely use Opuntia ficus-indica as a food, as well as treatment of multiple health conditions particularly skin aging and inflammations, and these properties are
mainly due to frequent occurrence of flavonoid isorhamnetin glycosides (Antunes-Ricardo
et al., 2015). The fruits of Juniperus communis showed the presence of mono and biflavonoids
have been traditionally used to cure acute and chronic skin conditions ( Jegal et al., 2016).
Quercetin glycosides (isoquercitrin, rutin) are polyphenol flavonoid compounds occurring
extensively within fruits and vegetables, and they are well known to have antioxidant and
neuroprotective effects (Magalingam et al., 2016). In another recent study, it is reported that
kaempferol glycosides fraction was purified from unripe Jindai-soybean (Edamame) leaves
and successfully reduces the diabetic and obesity effects in C57BL/6J mice. However, the
background phenomenon of antidiabetic potential was mainly the rise in lipid metabolism
along with downregulation of SREBP-1c and PPAR-γ (Zang et al., 2015).
Osteoporosis results from an imbalance between bone resorption and bone formation, and
this is being cured in traditional medication system by applying Herba epimedii in China
(Khosla et al., 2008; Sambrook and Cooper, 2006; Zhao et al., 2016). Moreover, icariin is the
major constituent in the plant Herba epimedii (Kapoor, 2013), which is proved to strengthen
the bones by stimulating the differentiation of osteoblasts while suppressing the differentiation of osteoclasts and bone resorption activity in vitro (Huang et al., 2007; Ma et al., 2011;
Song et al., 2013; Zhang et al., 2012).
3.2.9.2 Uses as food
Many of the flavonoids are naturally included in our diets. A huge number of polyphenolic
compounds (approximately 8000) have been reported to occur in plants (fruits, vegetables),
beverages (tea, coffee, and red wine), and chocolates (Geleijnse and Hollman, 2008). Opuntia
ficus-indica plant, which is enriched with isorhamnetin glycosides flavonoids, is being used as
a good source of food in Mexico (Antunes-Ricardo et al., 2015).
II. Phenolics
74
3. Analysis of polyphenolics
Plant flavones are normally complexed as 7-O-glycosides with either acetyl or malonyl
moieties (Martens and Mith€
ofer, 2005). The rooibos tea plants are enriched with flavones
(luteolin 8-C-glucoside or orientin and luteolin 6-C-glucoside or isoorientin), and extensively
have been used in China and all over the world (Bramati et al., 2003). Many other teas (oolong,
black and green) are said to possess isoorientin and orientin along with range of apigenin
mono- and di-C-glycosides (Engelhardt et al., 1993). It is noteworthy that Bergamot juices
have shown the maximum concentration of total flavones glycosides, including both flavone
O- and C-glycosides (Caristi et al., 2006; Gattuso et al., 2006). Many mint (Lamiaceae) species
have been found to possess kaempferol O-glycosides, but the concentrations vary among different species particularly the luteolin glycosides and used as a spice or condiment to prepare
different sort of foods (Areias et al., 2001; Fecka and Turek, 2007).
3.2.9.3 Tanning of leather
The association of plant-derived polyphenolic compounds with proteins has been focused
on last few decades, particularly in the fields of beverage industry, human and animal health,
medicine, and especially in leather production (Schropfer and Meyer, 2016). The artificial
transformation of animal skins or hides to finished leather, traditionally involves the use
of plant-based natural polyphenolic compounds (Brodowska, 2017). In addition to the tanning or coloring of the animal hides, the choice of polyphenolic compounds is based on their
ability to protect the molecular forms of hide-forming collagen fibers (Bohm, 1999).
The history of using vegetable tannins is thought to be the oldest known agents for the
leather formation (Brodowska, 2017) . They are divided into three main classes based on their
chemical structures, including hydrolysable tannins made up of esters of gallic acid and/or
glucose moieties, condensed tannins which are composed of catechin monomers, while the
tannins with iridoid or secoiridoid structure have a cyclopentanpyrane molecule with two
oxygen atoms (Haslam, 1966; Roux et al., 1975). Moreover, chromium (III) and aluminum
(III) salts, and dual function glutaraldehyde are frequently used in dying of the animal skins
since decades (Brodowska, 2017). Characterizing the nature of intermediary and final products during tanning is highly desirable in attempting to improve tanning efficiency and get
superior quality of the finished leather. With the onset of standardization, focus has been paid
to the physical characteristics of the finished leather products particularly the shrinkage,
and its associated shrinkage temperature has not depicted any obvious analysis to underlying chemical and physicochemical transformations (Musa and Gasmelseed, 2012). In the production of various leathers, vegetable tannins hold a crucial position and are considered as
vital tanning agents while dealing with nonchrome tanning. Commercial vegetable tannins in
use did not affect the quality of the various leather products; hence, the invention and exploration of new tannins from vegetables is of utmost desire. A unique spectroscopic signature
left by the tannins in the finished leather products, which led to identify the origin and type
of the tannin used herein. The standard procedures, which are using complexes of tanning
reagents, will impart distinct markers on the finished leather products. These markers
will reflect the procedure of the chemical processing as well as molecular mechanisms by
which tanning translates unprocessed leather into a finished marketable product (Romer
et al., 2011).
II. Phenolics
3.2.10 Main conclusions
75
3.2.9.4 Natural plant pigments
A huge list of natural pigments has been reported, but the carotenoids are regarded as the
crucial and important pigments comprising tetrapyrrole derivatives of natural phenolic compounds in plants (Tapas et al., 2008). Flavonoids are the major group of plant-origin pigments
and occur mainly in plants kingdom. The manipulation in genetic flavonoid materials in recent years had modified the flavonoid production pathways in a way leading to promotion in
production of novel compounds. Moreover, it is possible to keep a normal range of plant
colors in the best practicable genetic background, which possess certain features, including
stature, chilling hardiness, and resistance to multiple diseases. In addition, engineering of
novel flower colors that needs the association of anthocyanins with flavones or flavonol glycoside copigments is quite possible nowadays. It is further said that alteration in flower colors
may be achieved by manipulating the genetic control of vacuolar pH (Bohm, 1999).
The anthocyanins produce purple, red, or blue colors mainly in diverse class of fruits and
vegetables (Markakis, 1982; Salerno et al., 2016). These colored anthocyanins can easily be
found in eggplant, red cabbage, lettuce, and onions (Bridle and Timberlake, 1997; Clifford,
2000). The anthocyanins along with flavonoids have specialized structures that are capable
to scavenge ROS by acting as strong antioxidants, reduce inflammations, show antitumor
properties, and help fight against Parkinson’s and Alzheimer’s diseases, and diabetic retinopathy (Galvano, 2005; Hou, 2003; Kong et al., 2003; Nabavi et al., 2015; Wang and Mazza,
2002; Yin et al., 2015). Onions are rich in anthocyanins especially 3-(300 -glucosyl6-malonylglucoside), 3-(600 -malonylglucoside), 3-(300 -glucosy-glucoside), and 3-glucoside.
Cyanidin are the frequent polyphenolics in different varieties of onions, while many minor
anthocyanins make their place along the major pigments (Donner et al., 1997; Fossen et al.,
1996). Furthermore, quercetin-3-O-rutinoside, quercetin 4-O-β-glucoside, and quercetin
3,4-O-β-diglucoside constitute about 80% of the total flavonoids reported in onion
(Bonaccorsi et al., 2005; Hollman and Arts, 2000; Murota and Terao, 2003).
3.2.10 Main conclusions
Fruits and vegetables are rich in a huge quantity of bioactive phytochemicals, which have
been well documented for their preventive roles against diverse human ailments and diseases. The beneficial effects of these phytochemicals against key diseases in humans are well
elucidated especially in last two decades due to the easy access to high-tech methodology.
Moreover, the glycosidic flavonoids can be used as complementary medicine to protect
and maintain human health with increasing age. These food-based medicines, being chemopreventive agents, are considered to be safe and reliable for aging-related issues in human
body. In view of this, flavonoids are playing a crucial role, and one needs to explore more
novel flavonoids from unknown flora, especially in the third world, to combat the nutritional
deficiencies. In summary, glycosides of flavonoids are potential candidates as complementary medicines for the prevention and treatment of different types of human diseases, foods,
and some industrial usages. Current data led us to focus on the exploration of cheaper and
exploitable sources of flavonoid glycosides to fulfill the future demands in food and pharmaceutical industry.
II. Phenolics
76
3. Analysis of polyphenolics
Acknowledgment
This chapter is the outcome of an in-house financially nonsupported study.
Author contributions
All authors have directly participated in the planning and drafting of the manuscript and approved the final version
for submission.
Conflict of interest
The authors declare no conflict of interest.
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3.3.1 Phytochemistry of isoflavonoids
85
S U B C H A P T E R
3.3
Isoflavonoids
Ovais Sideeqa,b, Fazlullah Khanb,c, Muhammad Ajmal Shahd, Kamal Niaze
a
School of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran bInternational
Campus, Tehran University of Medical Sciences (IC-TUMS), Tehran, Iran cDepartment of
Toxicology and Pharmacology, Faculty of Pharmacy, The Institute of Pharmaceutical Sciences
(TIPS), Tehran University of Medical Sciences, Tehran, Iran dDepartment of Pharmacognosy,
Faculty of Pharmaceutical Sciences, Government College University, Faisalabad, Pakistan
e
Department of Pharmacology and Toxicology, Faculty of Bio-Sciences, Cholistan University of
Veterinary and Animal Sciences (CUVAS), Bahawalpur, Pakistan
3.3.1 Phytochemistry of isoflavonoids
Among the subclasses of flavonoids, isoflavonoids consist of a 15-carbon (C6-C3-C6) backbone, positioned as 1,2-diphenylpropane skeleton (Reynaud et al., 2005). The main characteristic of isoflavonoids is the 3-phenylchromen-4-one core structure, in which a phenyl ring, a
6-membered heterocyclic ring, is attached to 15-carbon atoms with another phenyl ring at the
C3 position, therefore named as A-ring, C-ring and B-ring, respectively, as shown in Fig. 3.3.1.
Based on the arrangement of substitution on the A and B rings, there are different classes of
isoflavonoids (Zhang et al., 2010).
The chemical structure of isoflavonoids or isoflavones is very similar to human sex hormone estrogen (found in females as estradiol and in males as testosterone) (Sharma and
Ramawat, 2013). The diversity of subgroups of isoflavonoids is based on the number and
8
FIG. 3.3.1 Basic structure of isoflavonoids (2-phenyl-2,3-dihydro-4Hchromen-4-one).
1
O
2
7
2′
3
1′
6
3′
4
5
O
6′
3-Phenylchromen-4-one
4′
5'
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3. Analysis of polyphenolics
HO
O
O
O
H3C
O
O
OH
OH
Daidzein
Formononetin
HO
HO
O
O
OH
H3C
O
O
O
OH
OH
Glycitein
Genistein
H3C
O
OH
-
O
O
O
OH
HO
FIG. 3.3.2
O
O
Biochanin-A (olmelin)
CH3
O
Biochanin-A
(5-Hydroxy-3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-olate)
Structure of main representatives (daidzein, formononetin, glycitein, genistein, and biochanin A).
complexity of possible substituents on the basic structural skeleton (methoxyl, aromatic or
aliphatic acids, prenyl, methylenedioxy, or isoprenyl (Sharma and Ramawat, 2013).
Isoflavonoids can be found in plants as water-soluble compounds, predominantly as
b-glucosides (genistin, daidzin, glycitin), or as acetyl-b-glucosides and malonyl-b-glucosides
(Fig. 3.3.2). There are four categories of dietary isoflavonoids: (1) aglycones (without attached
glucose), (2) glucosides or glucones, (3) acetylglucosides or acetylglucones, and (4)
malonylglucosides or malonylglucones (Sharma and Ramawat, 2013).
Some of the main isoflavonoids are genistein, daidzein, glycetein, biochanin A, and
formononetin (Tsao, 2010). The common name of 5,7-dihydroxy-3-(4-hydroxyphenyl)4H-1-benzopyran-4-one is genistein (Fig. 3.3.2) (Węgrzyn et al., 2010).
II. Phenolics
3.3.2 Biological activities of isoflavonoids
87
3.3.1.1 Properties of isoflavonoids
Isoflavonoids show antioxidant potential, often being a reason of positive effects on human
health (Dixon and Pasinetti, 2010). Isoflavonoids are considered useful chemosynthetic
markers (Reynaud et al., 2005). Their estrogen-like properties have been a growing interest
among pathologists (Reynaud et al., 2005). Relating their biological activities, isoflavonoids
share structural similarities with estrogen-17β-estradiol (E2) (Miadoková, 2009). Although
isoflavoids exhibit many chemical properties, their participation in redox reactions is one
of the significant properties. The free radical scavenging (chain-breaking antioxidants) mechanism is also attributed to isoflavonoids (Sharma and Ramawat, 2013). Soybean foods, after
fermentation, release aglycone isoflovonoids by removing the glucosidic group (Barnes,
2010). Genistein (40 ,5,7-trihydroxyisoflavone) and daidzein (40 ,7-dihydroxyisoflavone) are
abundantly available and are most studied members among isoflavonoids (Alves et al.,
2010). Methylated derivatives of genistein and daidzein are biochanin A (5,7-dihydroxy-40 methoxyisoflavone) and formononetin (7-hydroxy-40 -methoxyisoflavone) (Alves et al.,
2010). Synthesis of isoflavonoids occurs by phenylpropanoid route, which is specific of
legumes (Dastmalchi and Dhaubhadel, 2014). Successive hydrogen abstraction at C3 of
benzopyran ring, migration of C2 to C3 in aromatic B-ring, and hydroxylation of C2 in flavanones develop isoflavonoids (Simons et al., 2012). 13 Subclasses of isoflavonoids have been
investigated as a result of C-ring oxidation and attachment and presence of other and extra
heterocyclic rings (Simons et al., 2012). Substituting C5-isoprenoid (or prenyl) group is called
prenylation. Prenylation of isoflavonoids leads to increase in lipophilicity, which in turn optimizes affinity toward biological membranes, thus improving communication with target
proteins (Simons et al., 2012). Glycitein and genistein-derived metabolites are found in human urine (Uehara, 2013).
3.3.2 Biological activities of isoflavonoids
Isoflavonoids, apart from showing the phytoestrogen activity, promote several biological
processes like getting involved with cell-cycle regulators. They also inhibit the apoptosis that
is carried out by mitochondria-dependent pathway. Isoflavonoids also inhibited tyrosinekinase. They are involved in free-radical scavenging mechanism (Ming et al., 2013; Rietjens
et al., 2013; Sumien et al., 2013; Wang et al., 2002). Genistein has been shown to play a role in
preventing the prostate and breast cancer (Kładna et al., 2016). Genistein possesses tremendous health favorable responses by inhibiting the transcription factors NFκB, AP-1 and Akt
signaling pathways (Qian et al., 2012; Siow and Mann, 2010; Vina et al., 2011). Genistein has
been suggested as a dietary supplement for menopausal women to relieve the estrogen side
effects (Rusin et al., 2010). The derivatives of genistein lead to many antifungal and
antibacterial phenomena (Rusin et al., 2010). Genistein has shown to participate in the treatment of mucopolysaccharidoses and cystic fibrosis (Malinowska et al., 2009). Isoflavonoids
prevent atherosclerosis by inhibiting the oxidation of LDL, which is essential in atherogenesis
(Patel and Barnes, 2010). The antioxidant properties of isoflavonoids, mainly genistein and
daidzein, may explain their anticancer functions (Sharma and Ramawat, 2013). Soy
derived-isoflavonoids have been shown to improve glucose tolerance in diabetic animal
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3. Analysis of polyphenolics
models, thus decreasing the levels of blood glucose. This role has been thought to alter the
type-2 diabetes metabolism (Cho et al., 2010). Formononetin has displayed the induction
of angiogenesis (Li et al., 2015). Genistein has been reported to stop the maturation of melanoma cells in humans synthetically. Genistein has also been shown to inhibit DNA damage as
a result of UV-light introduced oxidation (Koirala et al., 2014). Genistein and genistin display
proapoptotic activity in disorganizing the cell cycle by being involved in stopping of G1 or
G2/M phase of cell cycle (Koirala et al., 2014). Degradation of microbes in GI tract serves
a source for isoflavonoid metabolites. These metabolites are included in different subclasses
of isoflavonoids such as dihydrodaidzein and dihydrogenistein, respectively (Simons et al.,
2012). Biochanin A inhibits production of TNF-α and IL-6 in macrophages stimulated by LPS
(Ming et al., 2015). It has been demonstrated in LPS-activated human vascular endothelial
cells that biochanin A inhibits expression of inflammatory advocator (Ming et al., 2015).
Formononetin has shown vasorelaxing effects as it causes relaxation via endothelium/nitric
oxide (NO)-dependent and endothelium-independent reactions (Wu et al., 2010). In addition
to many useful biological activities, isoflavonoids have also been related to cause unhealthy
responses. Some of these responses have been related to hormone levels, development of female reproductive tract, and complications in sex differentiation of brain (Chrzanowska et al.,
2015). Isoflavonoids derived from soy downregulate androgen receptor (AR) and prostate
specific-antigen (PSA). They have also displayed the action of inhibiting mammalian target
of rapamycin (mTOR) and cause growth reduction in several prostrate cancer cell lineages
(Mahmoud et al., 2014). Genistein prompts cell degradation or autophagy in carcinomasuffering cells of ovary (like A2780, CaOV3, and ES2) and in cancer cells of human colon
(HT-29) (Mahmoud et al., 2014). Two isoflavonoids, i.e., neovestitol and vestitol, extracted
from Brazilian Red propolis show antiinflammatory and antimicrobial activities (Bueno-Silva
et al., 2013). During pregnancy and lactation, genistein exposure leads to disruption in development of reproductive system depending on the dose. Genistein enhances fertility by aiding
acrosomal reaction when used at low dose, but suppresses male fertility by inhibiting acrosomal reaction on high dosage (Zhang et al., 2013). Table 3.3.1 sums up effects of major
isoflavonoids.
3.3.3 Current and potential industrial applications of isoflavonoids
The protective role of soy-derived isoflavonoids against postmenopausal breast neoplasm
has been established in recent studies (Wang et al., 2013). Dietary isoflavonoids, which are
mainly derived from soy, may influence the learning and memory (Wang et al., 2013). The
extract of soy isoflavonoids caused inhibition of UVB-induced keratinocyte death and suppression of UVB-induced intracellular H2O2 release, causing a reduction in oxidative stress
(Wang et al., 2013). The use of isoflavonoids makes them important food additives to prevent
menopausal-related symptoms (Brodowska, 2017). The use of antimicrobial properties of soy
isoflavonoids shows their attractive nature than artificial polymer or chemical-based coatings, thus playing a credible role against pathogenic biofilms (Dhayakaran et al., 2015). Many
isoflavonoids confirm the antiplatelet activity. Tectorigenin, an isoflavonoid, has been well
studied and cleared for having potent antiplatelet effects (Applová et al., 2017). Genistein
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3.3.4 Possible interactions properties of isoflavonoids
TABLE 3.3.1
89
Major isoflavonoids show different biological activities.
Isoflavonoid
Effects
References
Genistein
Antioxidant
Anticancer
Cardiovascular
Antidiabetic activity
Kładna et al. (2016) and Sharma and Ramawat (2013)
Diadzein
Antioxidant
Antiosteoporotic
Antic
Immunostimulator
Sharma and Ramawat (2013) and (Miadoková (2009)
Formononetin
Cardiovascular
Antioxidant
Estrogenic
Wu et al. (2010) and Li et al. (2015)
Biochanin A
Antiinflammatory
Antiproliferative
Antioxidant
Antiviral
Anticancer
Kole et al. (2011) and Ming et al. (2015)
Glycitein
Antiinflammatory
Therapeutic
Lee et al. (2010) and Yoshida et al. (2001)
demonstrates radiation safety in normal tissues and radiation sensitization tissues with
tumor. Thus, it is being investigated for use in radiation therapy ( Johnke et al., 2014). Improving serum lipid values and decreasing the LDL have been associated to some soy
isoflavonoids (Ogbuewu et al., 2010). The bioactive isoflavonoids are phytochemicals with
nutraceutical activities (Prakash et al., 2012). As isoflavonoids are similar to 17-β-estradiol,
they are strong candidates for hormone replacement therapy (HRP). They attach to the estrogen receptors and show beneficial outcomes on skin (Nemitz et al., 2015). Genistein shows
dynamic control against photodamage caused by UVB on human skin (Nemitz et al.,
2015). Daidzein and its metabolite, equol, have shown the property of tumor growth inhibition. Daidzein has shown effective responses by inducing apoptosis in human MCF-2 breast
cancer cells that were cultured. Thus, it has been shown to be effective against breast cancer
when designing new drugs. Daidzein and equol could play a pivotal role (Liu et al., 2012).
Pure isoflavonoids, genistein and daidzen also, inhibit hemoglobin glycosylation. The inhibition of this process can be used to help in diabetes (Hosseini et al., 2015).
3.3.4 Possible interactions properties of isoflavonoids
Isoflavonoids interact with plants, bacteria, fungi, herbivorous insects, mollusks, and vertebrates (Prokudina et al., 2011). Isoflavonoids play a vital dual role while interacting with
plants and environments. In first line of defense, they acts as phytoalexins against pathogens
and stress or injury. They act as indicators or responsers in symbiotic relation in nitrogenfixing bacteria (Dastmalchi and Dhaubhadel, 2014). Metabolons are ordered protein
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90
3. Analysis of polyphenolics
complexes, which are formed by metabolic enzymes involved in flavonoids and
isoflavonoids synthesis. A study showed important relation of protein-protein interactions
in isoflavonoid biosynthesis with formation of dynamic metabolons (Waki et al., 2016).
Genistein, one of the isoflavonoids, affects the pharmacokinetics of Imatinib (cancer growth
blocker), which is also called selective tyrosine kinase inhibitor. It has been demonstrated that
multiple doses of genistein administrated in rat, induced the activity of CYP3A4 and lowered
the levels of imatinib in plasma (Wang et al., 2015). Treatment of Rhizobium tibeticum with
isoflavonoids hesperetin and apigenin prior to incubation affected the symbiotic relation
between Rhizobium tibticum and fenugreek (Trigonella foenum graecum). Their treatment
increased the nodulation and nitrogen fixation of fenugreek (Abd-Alla et al., 2014). When root
tips of pea are stimulated with infusion of plant pathogen, it leads to exudation of Pisatin, a
phytoalexin, derivative of isoflavonoid. Pisatin is known for significant antimicrobial effects
in legumes (Baetz and Martinoia, 2014). In the human colon, the isoflavonoid phytoestrogen
derivatives biochanin A, formononetin, and glycetin go under O-demethylation with involvement of Eubacterium limosum (Hur and Rafii, 2000). P450 are michrosomal cytochromes found
in human liver. Many isoflavonoids, especially genistein and daidzein, are competent to inhibit P450 (Kopecná-Zapletalová et al., 2017). Daidzein and genistein form complexes with
pepsin, a digestive enzyme, with high affinity to induce conformational modification in pepsin (Nan et al., 2016). A study conducted on nonstimulated guinea-pig trachea with histamine
induction shows synergy between isoflavonoids daidzein and hesperetin, which are
expectorants present in fruit peel of Citrus aurantium L. (Rutaceae) (Shih et al., 2016). Daidzein
and genistein have Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor. Both of these
isoflavonoids bind to catalytic spot of trypsin, an enzyme, and avoid the effect of trypsin. This
result was indicated to relieve the symptoms of pancreatitis (Zeng et al., 2016). Isoflavonoids
that contain a glycosidic bond are hydrolyzed by microflora (Lactobacillus, Bacteroides, and
Bifidobacteria) in intestines with the help of glycosidase production (Uehara, 2013). Synergy
between genistein and famous anticancer drugs like adriamycin, docetaxel, and tamoxifen
provides a powerful effect in usage of combination therapy (Spagnuolo et al., 2015).
Moreover, isoflavonoids and vitamin C supplement reduce LDL oxidation significantly
(Xu et al., 2015).
3.3.5 Techniques of extraction, purification, and fractionation of isoflavonoids
The extraction method of isoflavonoids depends on their different structures and the
source from which they are obtained, like heartwood, seeds, leaves, rhizomes, roots, etc.
(Raju et al., 2015). Extraction yield of isoflavonoids depends on the technique used in extraction, solvent of extraction, solvent pH, time of the extraction process, temperature, concentration of the sample and solvent, and components of the sample (Rostagno et al., 2010).
Traditional methods used in extraction like maceration, percolation, soxhilation have been
used for decades. Extension of newer techniques has prime role in the development of agriculture via traditional way. Some of these new and recent methods include Microwaveassisted extraction, solvent extraction, ultrasonication, superficial fluid extraction, ionic
liquid-based ultrasound-assisted extraction (ILUAE), solid-phase extraction (SPE), and
II. Phenolics
3.3.6 Techniques of identification and quantification of isoflavonoids
91
pressurized liquid extraction (PLE) (Raju et al., 2015). Extraction using ultrasound shows accelerated extraction decreasing the time and increasing the extraction productivity (Raju
et al., 2015). Separation of soy isoflavonoids from soybean seeds is usually done by applying
solvent extraction or column chromatography (Liu et al., 2013). Foam fractionation is adopted
as a separation technique to recover aglycone derivatives of isoflavonoids (Liu et al., 2013).
The products of foam fractionation, which is called foamate, are utilized as the raw material
to get isoflavonoid aglycones. The efficiencies are measured by response surface methodology (RSM) (Liu et al., 2013). To separate and enrich the needed compounds after extraction,
conventional and well-proportioned liquid-liquid extraction is applied. Liquid-liquid extraction employs aqueous two-phase extraction (ATPE). Extraction and enrichment are combined
by microwave-assisted aqueous two-phase extraction (MA-ATPE), getting aid from aqueous
two-phase system (ATPS) as extracting agent. MA-ATPE, compared to traditional techniques
of extraction, is dynamic and potent. MA-ATPE has been put into work in simultaneous extraction and enrichment of genistein and biochanin A from leaves of D. odorifera (Ma et al.,
2013). Expediting substrate-solvent mixture and simplifying the mass transfer, negative pressure cavitation extraction (NPCE) can be used (Yan et al., 2010). Four mains bioactive
isoflavonoids such as prunetin, tectorigenin, genistein, and biochanin A have been extracted
from D. odorifera leaves using deep eutectic solvent-based negative pressure cavitationassisted extraction (Costa et al., 2016). For the extraction of isoflavonoids from solid samples,
the method of solid-liquid extraction with a mixture of methanol and water, ethanol and
acetonitrile, which acts as a solvent, is considered (López-Gutierrez et al., 2014). Fragmentation of eight isoflavonoids, i.e., dadzin, puerarin, genistein7-O-β-D-glucoside, sophoricoside,
glycitin, calycosin 7-O-β-D-glucoside, ononin, and 6-methoxy genistein7-O-β-D-glucoside,
which can be found extensively in Chinese medicinal herbs, was explored with the use of
ESI-MS negative ion mode (Ablajan, 2011).
3.3.6 Techniques of identification and quantification of isoflavonoids
For identification and characterization of isoflavonoids, different systematic methods like
thin-layer chromatography (TLC), ultraviolet (UV), high-performance liquid chromatography (HPLC), and capillary electrophoresis have been used over the past. Combining the potency of HPLC with NMR, which gives details of structure and mass spectrometry (MS), has
provided an excellent hyphenated technique to identify phytochemicals from plants. Nowadays, reverse phase-HPLC (RP-HPLC) is found to be popular for analysis of herbal extracts.
Apart from identification and characterization of isoflavonoids, mass spectrometry allows
the identification and quantification of metabolites of isoflavonoids in plasma, urine, and
feces, providing a profile of constituents for pharmacokinetics. Fast-atom bombardment
(FAD), liquid secondary ion mass spectrometry (LSIMS), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption ionization
(MALDI) are accessible to ionize the molecules. Electrospray ionization-mass spectrometry
(ESI-MS) is designed to look into the metabolites of isoflavonoids in organic fluids (Raju
et al., 2015). In identifying the isoflavonoids and their conjugates in red clover in Lithuania,
ultra-high-performance liquid chromatography (UHPLC) combined with a diode array
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92
3. Analysis of polyphenolics
detector (DAD) and quadrupole time-of-flight mass spectrometry (q-TOF-MS) is a convincing technique (Taujenis et al., 2015). An offline combination of immune-affinity chromatography (IAC) and HPLC-ESI-MS provides a developed tool for spotting the simple
isoflavonoids (Prokudina et al., 2011). Orbitrap analyzer has been found efficient to detect,
identify, and quantify isoflavonoids acting as nutraceuticals in soy (López-Gutierrez
et al., 2014).
3.3.7 Levels found of isoflavonoids in plants/food-based plants
Isoflavonoids are mostly found in soybeans but are also present in legumes, like soy, red
clover, kidney beans, sprouts of mung bean, Japanese arrowroot (Kudzu). Soybeans are the
major dietary source of isoflavonoids (Zaheer and Humayoun Akhtar, 2017).
Red clover presents an amount of isoflavonoids 2–10 times higher as compared to soybean
seeds (Taujenis et al., 2015). There are reports of variable and diverse isoflavonoids content in
soy-derived foods (soy milk, tempeh, and tofu) and some leguminous plants like fenugreek,
red clover, black gram, lentils, and chickpeas (Sharma and Ramawat, 2013). They contain 4 mg
of isoflavonoids (daidzin, genistein, glycitin, sissotrin, and ononin) per gram of dry matter
(DM) in soybean and its products or extracts (Leuner et al., 2013). Biochanin A and
formononetin are present in high concentrations in red clover (5 mg/g). Considering
legumes, genistein has been reported in high concentration from Andira macrothyrsa
(598 μg/g), Pachyrhizus tuberosus 250 μg/g), Calopogonium muconoides (184 μg/g). Similarly,
biochanin A content has been measured from Cratylia argentea (76 μg/g), C. mucunoides
(53 μg/g), and from flowers of A. macrothyrsa (40 μg/g) (Leuner et al., 2013). Concentrations
are generally in the following order: genistein > daidzin > glycitein but are dependent on the
location of growth, climate, and diversity (Zaheer and Humayoun Akhtar, 2017). Mature soybeans contain genistein in a scale of 5.6–276 mg/100 g (Spagnuolo et al., 2015). Table 3.3.2
sums up some sources of isoflavonoids and their respective levels, while Table 3.3.3 shows
various plant families and the isoflavonoids found in them.
TABLE 3.3.2 Sources of isoflavonoids and their
respective levels (Alves et al., 2010).
Source
Level (μg/g)
Alfalfa sprouts
43
Peanuts
1.5
Barley
0.2
Apple
0.1
Broccoli
0.1
Cauliflower
0.1
Instant coffee
9
II. Phenolics
3.3.8 Effects of food processing in phytochemicals
93
TABLE 3.3.3 Various plant families containing
isoflavonoids (Reynaud et al., 2005).
Isoflavonoid
Family
Daidzein
Rutaceae
Malvaceae
Menispermaceae
Formononetin
Myristicaceae
Rutaceae
Biochanin A
Myristicaceae
Rosaceae
Rutaceae
Malvaceae
Asteraceae
Genistein
Iridaceae
Moraceae
Rosaceae
Myristicaceae
Rutaceae
Glycitein
Rutaceae
3.3.8 Effects of food processing in phytochemicals
To decrease the phytochemical loss in fruits and vegetables, food-processing methods are
highly delicate ways for food processors in order to improve phytonutrients chemistry. Stability of phytochemicals is greatly impacted, positively or negatively, when it comes to food
processing and storage settings. Food processing may also indicatively reduce the content of
phytochemicals. It has been reported in some cases that blanching affects enzyme polyphenol
oxidase by inhibiting it, causing an improvement in the anthocyanin stableness in processed
foods. Processing operations such as thawing, cutting, and shredding reduce glucosinolates
in cruciferous vegetables because of the presence of enzyme myrosinase, which impairs
glucosinolates throughout tissue disruption. Thermally processed fruits have been showing
higher contents of phytochemicals. By increasing the time of blanching and boiling continuously, there is reduction of phytochemicals in vegetable and fruit processing (Tiwari and
Cummins, 2013). Thermal degradation and lixiviating of substances into cooking media
can lead to loss of antioxidant phytochemicals. Cooking at high temperatures can form
prooxidants, as a result of Maillard reaction. A useful effect of food processing is the improvement of antioxidant phytochemical bioavailability (Hoffman and Gerber, 2015). With the degeneration of plant cell wall, cooking might affect the extractability and bioavailability of
phytochemicals. After boiling shifts, carotenoids are enhanced in the sample, which varies
with plants extracts. It is high in vegetables, which have low concentration of carotenoids
in crude tissue and whose fibrous organization is firm and fixed (Palermo et al., 2014). In feasible conditions, irradiation affects the composition of plant phytochemicals and sometimes
enhances their concentration (Alothman et al., 2009). Fermentation process remarkably
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3. Analysis of polyphenolics
influences and increases the bioaccessibility of phytochemicals by changing the esterified
compounds into their free form (Yeo and Ewe, 2015).
3.3.9 Trends and concluding remarks
Isoflavonoids are being studied intensively till date and have become a hot topic of
research. Since their determination in legumes and soy, they have been investigated to have
many biological effects both in plants and animals. Many isoflavonoids and their derivatives
(isoflavones, isoflavanones, isoflavans, rotenoids, pterocarpans, and phytoestrogens) have
been reported from different plant species. Main focus has been put on genistein,
formononetin, biochanin A, and daidzein. Many biological processes and mechanisms have
been attributed to isoflavonoids, including immunological effects, cardiovascular effects, and
antioxidant properties. In recent years, isoflavonoids have been reported to be used as biomarkers in tumors. They have shown anticancer, antidiabetic, therapeutic, and neurological
effects. Combination of isoflavonoids with vitamins, proteins, and other estrogens has shown
a boost in controlling many diseases such as osteoporosis and drug abuse. Current data led us
to focus on the exploration of cheaper and exploitable sources of isoflavonoids to fulfill the
future demands in food and pharmaceutical industry. Future directions lead us to a very stable contact with experimental and quantified use of isoflavonoids. Radiological use of
isoflavonoids has been a breakthrough and should be more highlighted to reduce the injury
effects caused by radiation. Similarly, the effect on attenuation of menopausal symptoms
should be more precise to avoid conflict. Some studies have also shown undesirable effects
(inhibit normal cell proliferation, arrest cell cycle, and apoptosis) of isoflavonoids. Thus, in
conclusion, isoflavonoids have dietary, biological as well as potent industrial applications,
which need more clinical studies need to be done.
Acknowledgment
This article is the outcome of an in-house financially nonsupported study.
Author contributions
All authors have directly participated in the planning or drafting of the manuscript and read and approved the final
version.
Conflict of interest
The authors declare no conflict of interest.
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S U B C H A P T E R
3.4
Lignans and flavonolignans
Muhammad Nadeema,b, Imran Taj Khana,b, Fazlullah Khanc,f, Muhammad Ajmal Shahd,
Kamal Niaze
a
Department of Dairy Technology, University of Veterinary and Animal Sciences, Pattoki, Lahore,
Pakistan bFood Chemistry Lab, University of Veterinary and Animal Sciences, Lahore, Pakistan
c
International Campus, Tehran University of Medical Sciences (IC-TUMS), Tehran, Iran
d
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Government College
University, Faisalabad, Pakistan eDepartment of Pharmacology and Toxicology, Faculty of BioSciences, Cholistan University of Veterinary and Animal Sciences (CUVAS), Bahawalpur,
Pakistan fDepartment of Toxicology and Pharmacology, Faculty of Pharmacy, The Institute of
Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran
3.4.1 Introduction of lignans and flavonolignans
The term “Lignan” is used to designate the group of dimeric phenylpropanoids in which
two C6-C3 are joined by C8 (central carbon atom). Lignans have been found to be present in
over 60 families of vascular foliage (Gordaliza et al., 2004). Lignans are a subcategory of
nonflavonoid polyphenols. Plants are a great sources of several foods; they are also a potent
reservoir of pharmaceuticals, pesticides, flavoring compounds and pigments, and these are
usually categorized as secondary metabolites, they can also perform many biological functions in living organisms, e.g., antioxidant, antimicrobial, anticarcinogenic, antiplatelet aggregation, modulation of hormone, and detoxification of enzyme systems (Kiyama, 2016; Ram
et al., 2015). These compounds may not be useful for the plants themselves; rather they play
an important role in building and maintenance of the interaction between plants and other
organisms of the ecosystem (Idso and Idso, 2001; Liu et al., 2015). Structurally, lignans are
characterized by a mainstay, which is altered in several ways leading to the formation of over
700 different types of naturally present lignan varieties (Xu et al., 2014). On the basis of architectural perspectives, lignans can be categorized into arylnaphthalene, aryltetralin,
dibenzylbutane, dibenzylbutyrolactone, tetrahydrofuran, and furofuran. Lignans have been
found to be present in over 60 families of vascular foliage. Flaxseed (linseed) is regarded as the
richest source of lignans: secoisolariciresinol, lariciresinol, pinoresinol, and matairesinol have
has been found in appreciable amounts; however, secoisolariciresinol is present in highest
II. Phenolics
3.4.2 Main representative of lignans and flavonolignans
99
concentration. On an average, flaxseed contains about 3.7 mg/100 g lignans. Sesame, whole
grains, legumes, black tea, soymilk, coffee, apricots, strawberries, peaches, garlic, carrots
and asparagus, vegetables of Brassicaceae contain lignans in reasonable amounts.
Flavonolignans may be defined as naturally occurring phenols, which are made up of a
part of flavonoid and a part of lignan. Flavonolignans are the most comprehensively investigated group of lignans, due to their several medicinal applications and pharmacological activities. Flavonolignans were first isolated from Silybum marianum (milk thistle), a well-known
therapy for the treatment of several chronic and several liver-related disorders (Abenavoli
et al., 2010; Kidd and Head, 2005). Flavonolignans isolated from the fruit of milk thistle were
pharmacologically tested, “silymarin” a commercial pharmaceutical product is also prepared
from milk thistle (Pandey and Rizvi, 2009). Milk thistle originated from Mediterranean region; this plant has been used in the treatment of liver-related disorders since thousands
of years. The word milk thistle is given due to the existence of milk veins. On average, milk
thistle contains 1.5%–3.7% silymarin on dry weight basis. Silymarin is a blend of flavonoid
multiplexes (Chen et al., 2012). Flavonolignan has distinctive chemical structure; it is made
up of two units. First one is taxifolin (flavonol belongs to flavonoids), while the second is
phenylpropanoid unit (Kurkin, 2003).
3.4.2 Main representative of lignans and flavonolignans
Flaxseed (linseed) is regarded as the richest source of lignans: secoisolariciresinol,
lariciresinol, pinoresinol, and matairesinol have been found in appreciable amounts; however, secoisolariciresinol is present in highest concentration (Table 3.4.1).
Flavolignans are widely distributed in plants, they are found in seven plant families. However, Silybum and Asteraceae are regarded as the most abundant sources of flavolignans; about
17 different types of flavolignans have been isolated from these families of plants (Table 3.4.2).
Flavolignans are widely distributed in the following families of plant kingdom:
a. Asteraceae
b. Berberidaceae
c. Chenopodiaceae
d. Chenopodiaceae
e. Fabaceae
f. Poaceae
g. Scrophulariaceae
Flavonolignans have high medicinal and functional value; the biologically active substances must be extracted from the fruit in such a way that it causes minimum or no loss
of the flavonolignans. For the concentration of flavonolignans, a simple and straightforward
method has been developed (MacKinnon et al., 2007). According to this method, the
harvested fruits are carefully dried, followed by the removal of outer coat or bran layer,
the inner fruit/endosperm contains flavonolignans, the concentration may vary and be as
high as 3.7%.
II. Phenolics
100
TABLE 3.4.1
3. Analysis of polyphenolics
Chemical structures of nutritionally significant lignans.
Name of lignan
Molecular formula
Molecular weight
Structural formula
OH
Secoisolariciresinol
C20H26O6
362.422 g/mol
OH
Food
References
Flaxseed, wheat bran, silver
fir wood
Tsao (2010)
Sesame seeds, Brassica
vegetables, and white fir
Yahia et al.
(2010)
Flaxseed
Milder
et al. (2005)
Oil seeds, whole grains,
vegetables, and fruits
Manach
et al. (2004)
Sesame oil
Davies
(2010)
O
O
HO
HO
Lariciresinol
C20H24O6
360.4 g/mol
HO
O
OH
O
O
OH
Pinoresinol
C20H22O6
358.38 g/mol
OH
O
H
O
O
H
O
HO
Matairesinol
C20H22O6
358.39 g/mol
H3C
CH3
O
O
O
HO
OH
O
O
Sesamin
C20H18O6
354.35 g/mol
O
O
H
H
O
O
O
II. Phenolics
3.4.2 Main representative of lignans and flavonolignans
TABLE 3.4.1
101
Chemical structures of nutritionally significant lignans—cont’d
Name of lignan
Molecular formula
Molecular weight
Structural formula
O
Hydroxymatairesinol
C20H22O7
374.384 g/mol
H
HO
O
H
Food
References
Flaxseed, sesame seed,
barley bran, and oat bran
Fischer
et al. (2011)
Flaxseed, sesame seed,
barley bran, oat bran, wheat
bran, and rye bran
Smeds
et al. (2007)
Lignan precursor converted
by intestinal bacteria
Prasad
(2000)
Lignan precursor converted
by intestinal bacteria
Heinonen
et al. (2001)
H
OH
H3C
O
O
CH3
OH
CH3
Syringaresinol
C22H26O8
418.442 g/mol
OH
O
O
CH3
H
O
O
H
H3C
O
CH3
O
HO
Enterodiol
C18H22O4
302.36 g/mol
OH
HO
OH
HO
O
Enterolactone
C18H18O4
298.33 g/mol
O
HO
OH
II. Phenolics
102
TABLE 3.4.2
3. Analysis of polyphenolics
Chemistry of flavonolignans found in milk thistle.
Name of flavolignan
Molecular formula
Molecular weight
Silybin A
C25H22O10
482.44 g/mol
Structural formula
Abs
O
OH
HO
O
O
O
OH
OH
Silybin B
C25H22O10
482.44 g/mol
OH
O
Abs
OH
O
OH
O
O
O
OH
HO
O
OH
Abs
Isosilybin A
C25H22O10
482.44 g/mol
O
HO
O
OH
O
O
OH
HO
O
Isosilybin B
C25H22O10
482.44 g/mol
OH
Abs
O
HO
O
O
OH
O
OH
HO
O
OH
OH
Silychristin
C25H22O10
482.44 g/mol
OH
O
O
O
HO
OH
OH
OH
II. Phenolics
O
Abs
103
3.4.3 Biological activities of lignans and flavonolignans
TABLE 3.4.2
Chemistry of flavonolignans found in milk thistle—cont’d
Name of flavolignan
Molecular formula
Molecular weight
Structural formula
Isosilychristin
C25H22O10
482.44 g/mol
Abs
OH
O
OH
O
OH
HO
O
OH
O
OH
Silydianin
C25H22O10
482.44 g/mol
HO
OH
O
O
HO
H
H
O
H
OH
H
O
O
OH
3.4.3 Biological activities of lignans and flavonolignans
Intestinal bacteria convert lignan precursors to enterolignans, enterodiol, and
enterolactone (Milder et al., 2007). A pharmacokinetic study was performed to measure
the bioavailability of secoisolariciresinol administered at 0.9 mg/kg body weight. About
40% of secoisolariciresinol was available to the body in the form of enterodiol and
enterolactone. Peak concentration of enterodiol and enterolactone reached after 73 and 56 h
of ingestion. Considerable amount of lignans was available to the human body as enterodiol
and enterolactone, conversion of lignan precursors to enterodiol and enterolactone varies
from individual to individual (Heinonen et al., 2001). Lignans have a high functional value;
consumption of diet containing lignans can help to reduce the risk of cardiovascular diseases.
The existence of enterodiol and enterolactone has the capability of correcting dyslipidemia;
these also have hypotensive, antioxidant, and antiinflammatory properties (Prasad, 2000).
Flaxseed, barley, wheat bran, nuts, sesame seeds, legumes, fruits, and vegetables are rich
in lignans. In 1889, finding of 12-year study revealed those with high level of enterolactone
II. Phenolics
104
3. Analysis of polyphenolics
had significantly lower risk of cardiac failure than those having low levels (Park et al., 2005;
Prasad, 2000). The highest concentrations of lignans have been found in flaxseed, which is a
great source of omega-3 fatty acids and fiber. It provides 800 times more dietary lignans as
compared to other food items. Clinical studies have shown that supplementing foods with
30–50 g flaxseed per day for a period of 4–12 weeks led to the reduction of 8%–14% LDL cholesterol (Patade et al., 2008). In another potential study, the effect of lignan intake on breast
cancer was analyzed; women taking dietary lignan showed 17% less risk of breast cancer as
compared to the low quartile (Touillaud et al., 2007). Findings of a case study conducted in
United States revealed that women consuming high amount of dietary lignans had very low
risk of endometrial cancer (Lof and Weiderpass, 2006). Enterodiol and enterolactone can help
to prevent hormone subtle cancer (Ganorkar and Jain, 2013). No association has been found
between serum and urine concentration of enterolactone and prostate cancer (Ward et al.,
2008). Concentration of enterolactone in urine was positively correlated with bone mineral
density (Xu et al., 2015). Lignans are anticarcinogenic, hypotensive, cardiac-protective, decrease cholesterol, and increase stay time of the food in stomach (Imran et al., 2015) shown
in Table 3.4.3. Lignans have antioxidant capacity; therefore, they can minimize the risk of type
I diabetes by reducing the oxidative stress (Toure and Xueming, 2010). It can also inhibit phosphoenolpyruvate carboxykinase enzyme responsible for glucogenesis in liver in type II diabetes (Wall et al., 2015).
For the treatment of liver, spleen, and gall-bladder-associated disorders, silymarin has
been used since thousands of years (Surai, 2015). Silymarin has antioxidant, hepatoprotective,
anticarcinogenic, antiinflammatory, and antidiabetic properties (Vargas-Mendoza et al.,
2014). For the treatment of liver disorders, silymarin is used in toxin-induced liver damage,
chronic inflammatory diseases of liver ( Jahan et al., 2015). Silymarin was tested in plateletactivating-induced hypersensitivity; the study was conducted on guinea pigs (Sindhu et al.,
2017). Silymarin considerably reduced the bronchospasm in sensitized animals. The results of
this investigation showed that silymarin can be used for the treatment and prevention of
asthma. Silymarin has been known to have immunostimulatory properties (Alhidary et al.,
2017). The impact of silymarin was tested using glutathione level as a biological marker along
TABLE 3.4.3
Main biological activities of lignans.
Biological activity
References
Inhibition of viruses
Fang et al. (2015)
Anticarcinogenic
Alphonse and Aluko (2015)
Deterrence of cancer
Soonwera and Phasomkusolsil (2017)
Antiinflammatory
Spilioti et al. (2014)
Inhibition of microorganisms
Raghavendra et al. (2015)
Inhibition of oxidation
Song et al. (2016)
Immunity booster
Putri et al. (2018)
Liver protector
Chen et al. (2017)
II. Phenolics
3.4.4 Current and potential industrial applications of lignans and flavonolignans
105
with cellular proliferation; results showed that silymarin reestablished the level of glutathione with improved cellular proliferation (Fakurazi et al., 2008). Silymarin has been used since
centuries to increase the production of milk in nursing mothers (Zuppa et al., 2010). Results of
an investigation revealed that milk thistle considerably increased the production of milk in
lactation (Foong et al., 2015). Kazazis et al. (2016) showed that a dose of a silymarin
420 mg/day was found to be nontoxic.
3.4.3.1 Therapeutic perspectives of flavonolignans
1. Hepatoprotective
2. Hyperprolactinemia
3. Anticancer
4. For the treatment of obsessive-compulsive order
5. Antiinflammatory
6. Antiasthma
7. Immunostimulant
3.4.4 Current and potential industrial applications of lignans and flavonolignans
The protective role of soy-derived lignans and flavonolignans against postmenopausal
breast neoplasm has been established in recent studies (Flower et al., 2014). Dietary lignans
and flavonolignans, which are mainly derived from soy, may influence the learning and
memory (Borah et al., 2013). The extract of lignans and flavonolignans caused inhibition of
UVB-induced keratinocyte death and suppression of UVB-induced intracellular H2O2 release, causing a reduction in oxidative stress (Pan et al., 2009; Roubalová et al., 2017). The
use of lignans and flavonolignans makes them important food additives to prevent
menopausal-related symptoms (Đilas et al., 2009). The use of antimicrobial properties of
lignans and flavonolignans shows their attractive nature regarding artificial polymers or
chemical based coatings, thus playing a credible role against pathogenic biofilms (Bai
et al., 2015; Puupponen-Pimi€
a et al., 2005; Rukayadi et al., 2008). Many lignans and
flavonolignans confirm the antiplatelet activity. Silymarin, one of the flavonolignans, has
been well studied well and cleared for having potent antiplatelet effects (Ferreira et al.,
2010). Lignans and flavonolignans demonstrated radiation safety in normal tissues and radiation sensitization tissues with tumor. Thus, it is being investigated to be used in radiation
therapy (Bai et al., 2015; Davatgaran-Taghipour et al., 2017). Improving serum lipid values
and decreasing the LDL have been have been possible due to some lignans and
flavonolignans (Wallace, 2007). The bioactive flavonolignans are phytochemicals with nutraceutical activities (Achilonu and Umesiobi 2015). As lignans and flavonolignans are similar in
structure, they are strong candidates for hormone replacement therapy (HRP). They attach to
the estrogen receptors and show beneficial outcomes on skin (Korkina et al., 2008; Sharma,
2006). Lignans and flavonolignans show dynamic control against photodamage caused by
UVB on human skin (Korkina et al., 2008). Lignans and their metabolite, equol, have shown
property of tumor growth inhibition. Lignans have shown effective responses by inducing
II. Phenolics
106
3. Analysis of polyphenolics
TABLE 3.4.4
Lignans industrial and domestic applications.
Source of lignans
Potential application
Flaxseed
Flaxseed flour can be used for the fortification of bread, buns, cookies, pasta, noodles,
muffins, macaroni, spaghetti, whole wheat muffin, salad dressing
Sesame
Bakery products, cookies, crackers, cancer preventive, cardioprotective
Antioxidant for the stabilization of sunflower oil, soybean oil, and olein-based butter
Wheat bran
Fortification of flat unleavened bread “chapati,” bread, cookies, muffins, buns, etc.
Ground barley
Preparation of flat unleavened bread “chapati,” cookies, whole wheat biscuits
apoptosis in human MCF-2 breast cancer cells that were cultured. Thus, designing new drugs
shows to be effective against breast cancer; lignans, daidzein, and equol could play an important role in the suppression of tumor cell proliferation (Liu et al., 2012; Steiner et al., 2008).
Pure lignans, flavonolignans, isoflavonoids, genistein, and daidzein inhibit hemoglobin glycosylation. The inhibition of this process can be used to help in diabetes (Hosseini et al., 2015;
Steiner et al., 2008).
Flavonolignans are composed of a flavonoid and a lignan through oxidative process
(Pyszková et al., 2016; Svobodová et al., 2007). Oxidative link establishes between radicals
of flavanol taxifolin and coniferyly alcohol, which results in the adduct formation. Attachment of a phenol nucleophile can lead to the cyclization on the quinone methide produced
from coniferyl alcohol; the generated molecule in this state is known as silybin.
Milk thistle in original form cannot be used for industrial applications; it is mainly used in
the pharmaceutical industry for the preparation of pharmaceutical products. The technology
for the development of flavonolignans (3.7%) with lower surface layers has been established
(Li et al., 2013). Silymarin, a concentrated flavonolignan fraction, can be used for the following
industrial applications: Bread, cookies, pasta, cakes, noodles, fortification of wheat flour for
“chapati,” poultry feed, livestock feed, and chocolates (Table 3.4.4).
3.4.5 Techniques of extraction, purification, and fractionation of lignan
and flavonolignans
3.4.5.1 Classical methods of extraction of lignans
Still most of the phenolic compounds are extracted from plants by simple solvent extraction technique, which make use of organic solvents, which may be mixed with water in different ratios; further they may have different polarities (Ghisalberti, 1993). Classical methods
of extraction involve percolation, ultrasound-assisted extraction, turbo extraction, etc. In maceration, material is soaked in solvent, stirred, and then mixed; it is a simple technique, but it
requires more solvent. Percolation method is superior to maceration in terms of higher yield
of bioactive compounds. Soxhlet extraction is one of the simplest extraction techniques; it requires lower amount of solvents, but heat-sensitive bioactive compounds may be degraded in
the course of extraction.
II. Phenolics
3.4.5 Techniques of extraction, purification, and fractionation of lignan and flavonolignans
107
3.4.5.2 Microwave-assisted extraction
Zhang and Xu developed a microwave-assisted method for the complete extraction of
lignans, including the secoisolariciresinol diglucoside (SDG) in oil flaxseed meal (Zhang
and Xu, 2007). Microwave-assisted method involves the alkaline hydrolysis for 2 h with
0.25 M sodium hydroxide at ambient temperature followed by measurement on HPLC.
The method comprises two stages: in the first, sample is soaked in extraction solvent at
standard 80-W 5-min sonication with no sonication at 25 and 60°C for 1 h and 15 min. In
the second stage, microwave-assisted method was polished to increase the yield of SDG
by the concentration of ethanol (%), ratio between sample and solvent (g/mL) and
irradiation time.
3.4.5.3 Use of HPLC for the characterization of SDG
Analytical characterization of most of the lignans and SDG is usually performed by HPLC
(Popova et al., 2009). For analysis of lignans, reverse-phase HPLC method is generally preferred over the GC-based methods, as extensive sample preparations, purification, and derivatization are required for GC analysis (Mazur et al., 1996). GC-MS and HPLC methods were
used for the characterization of lignans ( Johnsson et al., 2000; Sicilia et al., 2003). This method
involves the use of acetonitrile 5%, phosphate buffer 0.01 M with pH 2.8. For the separation,
RP column is used and detection is carried out with UV detector at 280 nm.
3.4.5.4 Techniques for characterization of lignans
• Structural analysis
• Thin-layer chromatography
• HPLC
• Gas chromatography and mass spectroscopy
• Nuclear magnetic resonance
• Near infrared
• FTIR
3.4.5.5 Flow diagram for the extraction of lignans from flaxseed
Flaxseed and sesame are the richest sources of lignans; general method for the extraction of
lignin from these two sources is given in Figs. 3.4.1 and 3.4.2.
3.4.5.6 Fractionation of lignans
On the basis of solubility in different solvents, lignans may be fractionated into various
fractions. For this, alcohols and organic solvents (their mixture) are usually used (Markom
et al., 2007; Naczk and Shahidi, 2004). Fractionation of lignans is based on the philosophy that
low-molecular-weight lignans are solubilized in organic solvents, while the high-molecularweight lignans will be associated with residue (Moya et al., 2011). Low-molecular-weight
II. Phenolics
108
3. Analysis of polyphenolics
Flaxseed
Grinding (100 mesh size)
Oil extraction by
mechanical expression/solvent extraction
Cake for animal feeding and other uses
Extraction with alcohol
HPLC analysis
FIG. 3.4.1
Lignans
Evaporation for drying
Extraction with hydrolyzing agent
Extraction of lignans from flaxseed.
Methanol
Sesame seed oil
Residual oil
Temperature 70°C
Methanol extracted material
Crystals (about 95% pure)
Crystallization with methanol at 4°C (petroleum ether)
Methanol extract
Saponification using alcoholic KOH
Unsaponifiable matter
Unsaponifiable matter washed with ether
FIG. 3.4.2
Layer of ether
Extraction of lignans from sesame oil.
lignans have more fractions with weaker H-bonding as compared to high-molecular-weight
lignans. Lignans can be converted into different fractions using ether, ethyl acetate, methanol,
acetone, and dioxane water. They recorded a negative association between antioxidant capacities of lignan with the dissolving capacity of the solvent. Ultrafiltration is a technique in
which lignans can be fractionated into lower- and higher-molecular-weight fractions without
II. Phenolics
3.4.6 Techniques of identification and quantification of lignan and flavonolignans
109
using solvents/reagents (Westcott and Paton, 2001). Different ceramics membranes were
used to fractionate the liquor of Miscanthus sinensis. Lower-molecular-weight fractions had
5-kDa value; chemical composition of both fractions was not different. It is established that
lignan produced by ultrafiltration technique had higher antioxidant and antimicrobial activity (Garcı́a et al., 2010). It is reported that low-molecular-weight lignans can be produced by
ultrafiltration technique with large number of applications in functional and conventional
foods (Kajla et al., 2015).
The methods employed to extract, purify, and fractionate these natural products such as
flavonolignans are quite common, and samples are basically analyzed by high-performance
liquid chromatography and mass spectrometry (MS). It has been reported that sequential
extractions are often adopted while using solvents of increasing polarity, then purified by column or thin-layer chromatography or preparative-layer chromatography. Classical techniques involve organic solvents, such as chloroform, ethyl acetate methanol, chloroform,
and hexane, and include maceration, ultrasound-assisted extraction, and Soxhlet extraction,
which could interfere quantitatively as well as qualitatively in the extract composition
depending on the type of solvent, the organic solvent volume, the temperature, and the extractive process duration. Regarding the identification of the extracted compounds, GC-MS
and NMR analyses are reliable. There is no real, particularly recommended procedure as long
as flavonolignans widely differ in polarity and solubility and the use of an appropriate
method in each case is highly required (Graf et al., 2007; Ionescu et al., 2017).
3.4.6 Techniques of identification and quantification of lignan
and flavonolignans
For identification and characterization of lignan and flavonolignans, different systematic
methods like thin-layer chromatography (TLC), ultraviolet (UV), high-performance liquid
chromatography (HPLC), and capillary electrophoresis have been used over the past. Combining the potency of HPLC with NMR, which gives details of structure and mass spectrometry (MS), has provided hyphenated technique to identify phytochemicals from plants.
Nowadays reverse phase-HPLC (RP-HPLC) is found to be popular for analysis of herbal extracts. Apart from Identification and characterization of lignans and flavonolignans, mass
spectrophotometry mandates identification and quantification of metabolites of lignans
and flavonolignans in plasma, urine and feces, providing a profile of constituents for pharmacokinetics. Ionizing the molecules, fast-atom bombardment (FAD), liquid secondary ion
mass spectrometry (LSIMS), electrospray ionization (ESI), atmospheric pressure chemical
ionization (APCI), and matrix-assisted laser desorption ionization (MALDI) are accessible.
Electrospray ionization-mass spectrometry (ESI-MS) is designed to look into the metabolites
of lignans and flavonolignans in organic fluids (Del Rı́o et al., 2012; Eklund et al., 2008; Opletal
et al., 2004). In identifying the lignans and flavonolignans and their conjugates in red clover in
Lithuania, ultra-high-performance liquid chromatography (UHPLC) combined with a diode
array detector (DAD) is a convincing technique (Chen et al., 2011; Pang et al., 2016). An offline
combination of immune-affinity chromatography (IAC) and HPLC-ESI-MS provides developed tool for spotting the simple lignans and flavonolignans. Moreover, Orbitrap analyzer
II. Phenolics
110
3. Analysis of polyphenolics
has been found efficient to detect, identify, and quantify lignans and flavonolignans acting as
nutraceuticals in soy (Makarov et al., 2009).
3.4.7 Effect of processing and storage on lignans and flavonolignans
content in foods
Various studies have been performed to assess the effect of processing and storage on
lignans and flavonolignans content of foods. About 30% decline in lignans content was
recorded when vegetables were fried and boiled (Dhingra et al., 2012; Herranz et al.,
1981), and it also affected flavonolignans concentration. It has been found that lignans and
flavonolignans content of asparagus and mung bean sprouts decreased after boiling. A study
reported a small decrease in lignans and flavonolignans content of cooked vegetables. Lignan
and flavonolignans contents of white and brown rice slightly decreased after boiling
(Lewandowska et al., 2013). Effect of cooking on lignan and flavonolignans content of legumes was evaluated, lignan contents of legumes were not influenced by the cooking
(Wanasundara and Shahidi, 1998). Lignan contents of roasted and unroasted peanuts were
compared; roasted peanuts had higher concentration of lignans as compared to nonroasted
peanuts (Wanasundara and Shahidi, 1998). Effect of baking temperature and subsequent storage on secoisolariciresinol in bread was determined; it was recorded that secoisolariciresinol
was not changed by the baking temperature and storage (Edel et al., 2016). When the lignan
and flavonolignans content of rye was checked before and after baking, it was found that concentration of lignans was not changed by the baking temperature (Brodowska et al., 2014).
Olive oil was exposed to accelerated oxidation at 160°C for 25 h to find the effect of oxidation
on lignans and flavonolignans, and it was found that concentration of lignan and
flavonolignans was only mildly decreased, while other bioactive compounds were seriously
deteriorated; lignans can stand microwave heating up to 6–8 min (El-Gazayerly et al., 2014;
Wallace, 2007). Some natural occurring products such as oilseed, nuts, and bread have higher
amount of lignans (Table 3.4.5).
TABLE 3.4.5
Comparison of lignans content of oilseeds, nuts, and breads.
Oilseeds and nuts
Lignan content (μg/100 g)
Breads
Lignan content (μg/100 g)
Flaxseed
301,129
Flaxseed bread
12,474
Sunflower
39,348
Multigrain bread
6,744
Cashew
891
Wheat bread, whole grain
121
Peanut
629
Wheat bread, white
18
Poppy seed
94
Wheat, white flour
27
–
–
Rice, white, boiled
7
Reference
Smeds et al. (2007)
Source: Smeds, A.I., Eklund, P.C., Sj€
oholm, R.E., et al., 2007. Quantification of a broad spectrum of lignans in cereals, oilseeds, and nuts. J. Agric.
Food Chem. 55(4), 1337–1346.
II. Phenolics
111
3.4.8 Level of lignans and flavonolignans in food/plants-based products
3.4.8 Level of lignans and flavonolignans in food/plants-based products
On average, flaxseed contains about 3.7 mg/100 g lignans (Tsao, 2010). Sesame, whole
grains, legumes, black tea, soymilk, coffee, apricots, strawberries, peaches, garlic, carrots
and asparagus, vegetables of Brassicaceae contain lignans in reasonable amounts (Landete,
2012). Intestinal microflora converts the lignans into enterolignans, enterodiol, and
enterolactone. Their level in serum and urine is used as a marker of intake of dietary lignans.
Different levels of lignans and flavonolignans are shown in Table 3.4.6.
TABLE 3.4.6
Lignans content in some foods.
Food type
Lignans (μg/100 g)
Food type
Lignans (μg/100 g)
Flaxseeds
379,012.3
Dried apricots
400.5
Soybeans
269.2
Alfalfa sprouts
44.8
Soy nuts
122.2
Pistachios
198.9
Tofu
30.9
Dried dates
323.6
Tempeh
29.6
Sunflower seeds
210.3
Miso paste
63.9
Chestnuts
186.6
Soy yogurt
46.6
Dried prunes
177.5
Soy protein powder
16.5
Olive oil
142.6
Sesame seeds
7997.2
Soy sauce
14.3
Flax bread
7239.3
Rye bread
142.9
Soy bacon bits
35.1
Walnuts
85.7
Black bean sauce
10.5
Almonds
111.7
Multigrain bread
4785.6
Cashews
99.4
Soy milk
12.3
Winter squash
113.3
Doughnuts
31.4
Hazelnuts
77.1
Protein bar
19.6
Green beans
66.8
Soy veggie burger
15.2
Collard greens
97.8
Miso soup
2.8
Broccoli
93.9
Mung bean sprouts
128.7
Strawberries
48.9
Soy bean sprouts
2.2
White beans
33.5
Garlic
583.2
Peaches
61.8
Reference
Thompson et al. (2006)
II. Phenolics
112
3. Analysis of polyphenolics
3.4.9 Trends and concluding remarks
Lignans are a subgroup of nonflavonoid polyphenols. Recently, novel technologies have
been used for robust and efficient separation of lignans and flavonolignans, including a
fused-core technology in HPLC column from turmeric extracts. For isolation and purification
of these phytochemicals, HPLC, LC-MS, TLC, PLC, and MS are used. Advanced instruments
like preparative HPLC are used to speed up the purification process. Different techniques
such as GC and HPLC coupled with different detectors, namely, can be used to identify
and pure lignans and flavonolignans molecules. Plants are the vital source of several food
nutrients, as they are also a potent reservoir of pharmaceuticals, pesticides, flavoring compounds, and pigments. These are usually classified as secondary metabolites, which can also
be used for many biological functions in living organisms. Flavonolignans are the most prominently investigated group of lignans, due to their medicinal applications and pharmacological activities. The use of lignans and flavonolignans is reduced to the pharmaceutical
applications in changing lifestyles; functional foods supplemented with lignans and
flavonolignans should be prepared. Technology for the extraction, preservation, optimization
of dose for the supplementation of foods should be done. Further, clinical trials with foods
fortified with lignans and flavonolignans should be performed for setting guidelines for
the development of functional foods containing these bioactives.
Acknowledgment
This article is the outcome of an in-house financially nonsupported study.
Author contributions
All authors have directly participated in the planning or drafting of the manuscript and read and approved the final
version.
Conflict of interest
The authors declare no conflict of interest.
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3.5.1 Phytochemistry of stilbenoids
117
S U B C H A P T E R
3.5
Stilbenoids
Aadil Javeda, Hafiz Muhammad Imran Umarb, Fazlullah Khanc,d, Kamal Niaze
a
Department of Biotechnology, Graduate School of Natural and Applied Sciences, Ege University,
Izmir, Turkey bDepartment of Plant Breeding and Genetics, Faculty of Agriculture, Ege
University, Izmir, Turkey cInternational Campus, Tehran University of Medical Sciences
(IC-TUMS), Tehran, Iran dDepartment of Toxicology and Pharmacology, Faculty of Pharmacy,
The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran,
Iran eDepartment of Pharmacology and Toxicology, Faculty of Bio-Sciences, Cholistan University
of Veterinary and Animal Sciences (CUVAS), Bahawalpur, Pakistan
3.5.1 Phytochemistry of stilbenoids
The hydroxylated derivatives of stilbenes are called stilbenoids and there are two general
isomers of stilbenes (E/Z), and chemically these are 1,2-diphenylethylenes. Naturally,
stilbenoids can occur in monomeric and oligomeric forms along with their glycosylated derivatives as well. The major carbon skeletal backbone of these compounds occurs in the form
of C6-C2-C6 unit (1,2-diphenylethylene), but other oligomeric and hydroxylated structures
that arise from this chemical moiety are popular such as the one of resveratrol, α-viniferin,
and astringin,. Resveratrol, which is considered a model stilbenoid, has been studied for
its properties in a thorough fashion and a lot of biological and chemical information about
stilbenoids come from investigations performed on this compound. It was originally isolated
from the roots of a berry Veratrum grandiflorum in 1940. Other polymeric stilbenes have been
found in other food sources, e.g., derivatives of resveratrol in sprouted peanuts Arachis
hypogea (Shen et al., 2009). A lot of debate has been carried out in the past regarding the classification of stilbenes and their derivatives. However, according to latest reports, stilbenoids
can be characterized by the number of monomeric units contained and another group that
contains glycoside moieties based on their novel chemistry. Aglycones (piceatannol,
pinosylvin, pterostilbene, and resveratrol) and glycosides (astringin and piceid) are the
main representatives of stibenoids (Fig. 3.5.1). Oligomeric stilbenes are produced normally
by coupling that occurs between homogeneous or heterogeneous monomeric stilbenes, which
gives rise to a variety of skeletons, diverse oligomerization, and increase in complexity of
overall configuration. These oligostilbenes are further classified into two groups in
which the first group contains at least a heterocycle of five-membered oxygen moiety
II. Phenolics
118
3. Analysis of polyphenolics
OH
OH
OH
HO
OH
OH
Pterostilbene
Resveratrol
OH
OH
HO
O
OH
O
OH
Abs
Astringin
Piceid
Abs
OH
OH
O
O
HO
OH
HO
OH
OH
HO
H
O
HO
H
H
O
HO
OH
H
OH
H
FIG. 3.5.1 Various representatives of stilbenoids aglycones (piceatannol, pinosylvin, pterostilbene, and resveratrol) and glycosides (astringin and piceid).
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3.5.2 Biological activities of stilbenoids
119
(trans-2-aryl-2,3-dihydrobenzofuran), while the second group lacks this moiety. Later, this
classification was upgraded to a five-group system in which each group was further divided
into two subgroups of the previous classification. These groups are based on resveratrol,
isorhapontigenin, piceatanol, oxyresveratrol, resveratrol and oxyresveratrol (coupled heterogeneous), and miscellaneous oligomers (Niesen et al., 2013). The central structure of a stilbene
compound gives variable opportunities for polymerization; however, the chemical nature of
diphenylethylene does not give rise to certain modifications that can be turned into monomers that are novel in chemistry. A newly discovered monomer is a methoxy-prenylated
derivative along with cudrastilbene, which was isolated from the roots of Cudrania tricuspidata
from different locations in Asia. The most common monomeric form of stilbenoids is resveratrol as we will discuss the properties of this compound later in this chapter. There are several
bonding patterns of CdC and CdO combinations that can come from the polymerization of
these monomeric units. Among them, the most common moiety is benzofuran as it is observed in almost all of the isolated dimers that have been discovered recently. Other
prenylated dimers of resveratrol include arahypin 6 and arahypin 7, which were produced
by inducing peanut seed (Arachis hypogaea) to a fungal infection from Aspergillus caelatus
(Niesen et al., 2013). Other recently isolated dimers include longusol A and B, having a
benzofuran ring that connects resveratrol monomers with their carbon skeleton structures
reported as opposite stereoisomers. Plants belonging to genus Gnetum have been known
as a rich source of oligomeric stilbene derivatives, as macrostachyol C and D; both were isolated from these plants. Furthermore, Gnetum macrostachyum was also used for isolation of
macrostachyol B, the structure of which is unique as it contains a C-bridge resulting in a bicyclic internal ring system. For stilbene tetramers, Upuna borneensis was reported recently to
be exploited to isolate upunaphenols O and P, which consist of resveratrol dimers;
ampelopsin A, and cis viniferin. One of the most recently isolated stilbene hexamers was
taken from the acetone extract of Vatica albiramis stems and its structure contains a tetramer
of vacticanol A and a dimer unit. Another important category of stilbenoid is norstilbenes and
a structure called longusone A, which is a stilbene oligomer, contains an additional hydroxyl
group in the ortho position on the southern disubstituted ring and was isolated from Cyperus
longus. This compound has a unique tropilene moiety, which is absent in other natural stilbenes, which is why it was described in the category norstilbene (Niesen et al., 2013).
3.5.2 Biological activities of stilbenoids
Through substantial research that has been carried out on stilbenoids, they have been characterized as potential antibacterial, antioxidant, and anticancer agents; owing to their functions as moderators of NFKB and hemeoxygenase moderators shown in Fig. 3.5.2.
Stilbenoids, such as tertastilbenes (kobophenol A and B) extracted from Carex kobomugi
and Carex pumila, showed inhibitory potential against Staphylcoccus aureus (Kawabata
et al., 1989). Production of reactive oxygen species (ROS) as a result of biological redox processes has been known for a long period of time. These species cause oxidative stress, which
further mediates physiological imbalances in the body, further leading to several human
chronic diseases such as cancer, diabetes, and cardiovascular diseases. The exact mechanism
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3. Analysis of polyphenolics
Neuroprotective (¯ Amyloid plagues in
the brain, ¯ cerebral infarct volume, ¯
neuronal ROS generation, inhibit
cholinesterases)
Depigmentation (¯ Melanin
production, inhibit tyrosinases
activity
Cardioprotective
(Activate AMPK
upregulate eNOS)
FIG. 3.5.2
Ischemia-reperfusion injury (
Antioxidant enzymes, ¯ oxidative
stress, and inflammation; TNF-a
IL-1b)
Anticarcinogenic (Inhibit
angiogenesis and proliferation
of cancerous cell lines, apoptosis)
Stilbenoids
Regulate blood
pressure (Lower
systolic blood
pressure at high
dose)
Atherosclerosis (¯ Oxidative
stress, and inflammation; TNF-a,
IL-1b, inhibit oxidation of LDL
in endothelial cells)
Obesity (Inhibit lipogenesis, lipolysis, activate AMPK, SIRT
and PGC-1a)
Platelets aggregation
(Inhibit cyclooxygenase
enzyme)
Various mechanisms of stilbenoids biological activities.
of action of stilbenes in scavenging these free radicals (ROS) or to stimulate NADPH oxidase
and xanthine oxidase suppression is still under investigation (Frombaum et al., 2012). However, the phenolic structure of stilbenes has been implicated to have antioxidant properties.
Another important biological activity of stilbenoids was recently discovered according to
which these compounds can inhibit topoisomerase II, which functions in unwinding of coiled
DNA during the process of cellular transcription. A study, which investigated 40 stilbenes for
exploring the mechanism behind the aforementioned inhibition of topoisomerase II, found
that α-viniferin (oligomeric silbene) was highly dynamic in nature (Baikar and Malpathak,
2010). This compound also halted the cell-cycle progression in human colon cancer cells during S phase, NFKB, a protein complex, which has been characterized to be involved in cell
survival and proliferation (Fig. 3.5.2). It has attracted the interest of researchers as several diseases, including autoimmune, inflammatory, viral infections, and cancer due to its improper
regulation. Resveratrol and its derivatives have been investigated to study their interaction
with NFKB and their role in modulating this potent protein complex is now well understood
(Bagul et al., 2015). Neurodegenerative diseases like Parkinson’s and Alzheimer’s have been
characterized by improper function within hemeoxygenases and resveratrol being a wellstudied stilbenoid has been known to act as a hemeoxygenase moderator, which normally
processes the breakdown of heme to change iron, carbon monoxide, and bilburin (Richard
et al., 2011). Resveratrol has also been implicated to have positive effects in combating problems like type II diabetes and myocardial ischemia. A lot of research regarding stilbenoids
and their role in aging has been performed, which resulted in data that showed potential
antiaging properties of resveratrol and its analogues (Reinisalo et al., 2015). The application
of these compounds extended the lifespan of budding yeast (Saccharomyces cerevisiae) by the
mechanism that imitates caloric restriction. Similar results have also been obtained in honey
bee (Apis mellifera), fruit fly (Drosophilla melanogaster), and nematodes (Caenorhabditis elegans).
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121
The experiments that were conducted on mice later affirmed that the resveratrol mimicked
the consequential effects of calorie restriction, e.g., reduced inflammation, lowered albuminuria, decreased cataract formation, increased motor coordination, stable mineral density in
bone, and increased aortic elasticity. Despite aging being a complex issue, the antiaging effects of stilbenoids show great promise as evidenced in multiple studies, showing positive
effects on multiple physiological processes (Quideau, 2004). As already mentioned,
stilbeoinds belong to a class of compounds that have multiple pharmaceutically relevant capabilities; however, antiinflammatory and antioxidant activities of stilbenoids stand behind
almost all the other pharmacological effects of these potent compounds.
3.5.3 Current and potential industrial applications of stilbenoids
Natural stilbenoids are nowadays commercially marketed as nutraceuticals. Stilbenoids
(including pterostilbene, gnetol, piceatannol, and resveratrol) at molecular level target
cyclooxygenases, nuclear factor kappa B, nitric oxide synthase, interleukins, and tumor necrosis factor α among many others. Along with their inherent antioxidant activity and this
antiinflammatory action, these compounds have positive health effects against not only cardiovascular disease, but also cancer, diabetes, and neurodegenerative diseases (Dvorakova
and Landa, 2017). In food industry, stilbenoids have been associated with a phenomenon
called “French paradox” as the consumption of these types of compounds in the form of
wines has been proposed to prevent development of cardiovascular diseases (Stervbo
et al., 2007). The protective role of trans-resveratrol and its different derivatives was elucidated by various studies around the world. These compounds regulate not only the nitric oxide production but also the synthesis of eicosanoids, hence preventing the aggregation of
thrombocytes (Giovannini et al., 2001; Olas et al., 2001). Stilbenoids from other sources like
peanuts, pistachios, peanut butter, and chocolates are also considered to have health benefits,
so their consumption in optimal quantity is recommended (Guerrero et al., 2010). Considering the potential and influence of stilbenoids, especially on the physiology of plants, agriculture sector is impacted most in terms of their application. For example, antifungal effects of
resveratrol in different leaves and berries are of utmost importance while considering the industrial applications of stilbenoids ( Jeandet et al., 2002; Adrian and Jeandet, 2006). The phytoalexin effect of these compounds has been known for a long time, as they act in response to
stresses, injury, or after an attack by pathogens like nematodes, bacteria, fungi, or herbivores,
therefore contributing to active defense mechanisms (Adrian and Jeandet, 2012). Genetically
modified plants produced by transferring stilbene synthase (STS) gene for overproduction of
stilbenoids like trans-resveratrol are used for controlling disease and promotion of overall
health in some economically important plants (poplar, tomato, and tobacco) (Delaunois
et al., 2009). As the influence of stilbenoids is increasing in pharmaceutical industry due to
their excellent biological properties and interactions, a large number of patented products
can be seen in the market as well. For example, recently a safe, novel, and readily bioavailable
stilbenoid (3,5-dimethoxy-3,40 -dihydroxystilbene) has been marketed due to its ability to stop
adipogenesis and inhibit the accumulation of lipids in the body. There are other compositions
of similar fashion, including 2,30 ,50 ,6-tetrahydroxy-trans-stilbene, which have been marketed
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3. Analysis of polyphenolics
for their nutraceutical and cosmeceutical effects ( Jayaraman et al., 2015). As the phytophysics
and phytochemistry of stilbenoids and their related compounds is being recorded continuously, the feasibility of their synthesis is projected as being easy and stable, both chemically
and thermally. Apart from the biological activities discussed in previous section, these compounds are suggested to have a prominent role in material sciences as well as more data about
their optical, electric, and optoelectric properties is being gathered (Satheeshkumar et al.,
2015). Recently, novel moieties obtained by stilbenoids are being investigated to increase
and broaden the spectrum of their industrial applications. For example, dendrimers synthesized from stilbenoids have been investigated for their potential application as additives for
dye-sensitized solar cells (Ravivarma et al., 2017).
3.5.4 Possible interactions of stilbenoids
Stilbenoids, as discussed earlier, are compounds that occur naturally and are simple; however, they result by complex biosynthesis processes and their functions are also ecologically
complex as well. Their interactions with other compounds and biological moieties are of great
importance in elucidating their applicability (Kumar et al., 2012). A structure activity study,
for example, was carried out in 1995 that characterized the interaction of stilbenes (based on
combretastatin) with tubulin, which showed that trans-stilbenes have an affinity and can bind
to tubulin (Woods et al., 1995). Resveratrol, which is a polyphenol, is suggested to have an
irreversible inactivating role for cytochrome P450. It has been depicted that naturally occurring stilbenes like resveratrol, resveratroloside, 40 -dihydroxy-3-O-methoxystilbene, 5,3dihydroxy-40 -O-methoxystilbene, and piceid inhibit the hydroxylation of testosterone (androgen) by inhibiting cytochrome P450 (Regev-Shoshani et al., 2004). An analogue of the stilbene
pawhuskin A, when investigated for directed ortho metalation reactions, was found to be a
have high selectivity for delta opioid receptor, as an antagonist. Further docking studies also
confirmed that this stilbene can adopt a conformation that is quite similar to naltrindole,
which is a popular antagonist for this important receptor (Hartung et al., 2015). Tetramer stilbenes of resveratrol (hopeaphenol and vitisin A) have shown inhibitory effects on the enzyme
activity of angiotensin-I-converting enzyme (ACE). Their affinity toward the enzyme as calculated by docking calculations implied that antihypertension effect of these compounds can
be a further potential application for stilbenoids in future (Su et al., 2015). More recently, in the
quest for finding potential anticancer agents, stilbene analogues have been investigated for
their potential in inhibiting the expression of P-glycoprotein (ATP binding cassette) transporter, which functions by mediating the efflux process by hydrolysis of ATP and is
expressed highly in cancer stem cell population. Stilbenes are now considered as a thirdgeneration inhibitors of P-glycoprotein due to their phenylpropanoid nature (Tripathi and
Misra, 2016). Cyclooxygenase-2 (COX-2) is an enzyme that is responsible for inflammation
and pain. COX-2 inhibition by targeting reduces the risk of peptic ulceration and is employed
as the function of celecoxib, rofecoxib, and other drugs to work in an antiinflammatory manner. 4-nitro-30 ,40 ,50 -trihydroxy-trans-stilbene has been characterized as COX-2 inhibitor in a
fairly recent study and computational tools of compound docking, commercial ELISA test,
and QSAR assay have given essential evidence to suggest its ability to inhibit this enzyme.
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The cytotoxicity of this stilbene, when compared with the celecoxib, was found to be elevated,
suggesting its potency as well (Regulski et al., 2018). Piceatannol and resveratrol have been
reported to inhibit monoamine oxidase (MAO) that is found in human adipose tissue and are
shown to have antilipolytic action, thus act to limit lipotoxicity that is associated with obesity
(Les et al., 2016). Trans-resveratrol has also been investigated for their role in the reduction of
telomerase activity and it is proposed that this compound has an ability to inhibit the expression levels of telomerase reverse transcriptase in human melanoma cells. Moreover, it also
binds to all DNA model systems and can discriminate between duplex and G-quadruplex
DNA in biophysical investigations (Platella et al., 2017).
3.5.5 Techniques of extraction, purification, and fractionation of stilbenoids
Polyphenols like stilbenes have a variety of health benefits and that is the reason a plethora
of research and investigation have been carried out to develop certain extraction, purification,
and fractionation methodologies for these compounds. Conventionally, the approach for extraction that is employed, is heating with a solvent like ethanol, which is followed by filtration, which further leads to the concentration and purification parts of the processes. It is now
considered as an expensive and a time-consuming procedure that requires large quantities of
solvent, and a lot of scientific efforts have been done to improve in this area of phytochemistry. Other traditional methods include maceration at laboratory temperature, extraction at
elevated temperature, fluidized-bed extraction, Soxhlet extraction, microwave-assisted extraction, and accelerated solvent extraction (Soural et al., 2015). There have been some experiments, which focused on the selective extraction of stilbenoids like resveratrol and piceid by
employing molecularly imprinted polymers (Zhuang et al., 2008). The extraction recovery of
trans-resveratrol was reported to be 83%; however, for another compound named emodin, the
selectivity was found to be elevated to 99% in the same investigation. Another strategy for
extraction called superficial fluid extraction (SFE), utilizing carbon dioxide for the medium
of extraction, has been employed for extracting substances of nonpolar origin from different
plants (Beňová et al., 2010). The yield of emodin from this extraction procedure when compared with conventional Soxhlet extraction was found to be 2.5 times higher. Furthermore,
the extraction time period from SFE was reported to be five times shorter as well. However,
in case of resveratrol, the efficiency of this method was less effective than Soxhlet method. In
order to increase the extraction efficiency of resveratrol, biotransformation procedures were
attempted. Enzyme hydrolysis technology, high-speed countercurrent chromatography, and
eco-friendly extraction procedures were then reported to be effective in recovering resveratrol and piceid. Still, there are some drawbacks to each of these methods when it comes to
being expensive and time consuming; however, these are still proposed as simplistic and
spontaneous methodologies for extraction (Wang et al., 2013). High-pressure preparative
chromatography has been considered one of the standard methods for purification of compounds like stilbenoids. However, methods that employ such chromatographic purification
are not only costly but also require pretreatment with traditional silica gel columns as well,
which makes the procedure time consuming and less efficient. For relevant excellent sample
recovery of such compounds, high-speed counter current chromatography (HSCCC), which
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3. Analysis of polyphenolics
is one of the continuous and efficient provisions of separation technologies, utilizes liquid
chromatography, requires no support from a matrix, and reduces the loss of sample on
the support, which is irreversible in nature (Liang et al., 2011). Yang et al. (2012) used HSCCC
for the purification of 12,13-dihydroxyeuparin from Radix Eupatorii Chinensis. The mentioned
techniques have also been employed for fractionation of stilbenoids; however, there are certain techniques that are used specifically for fractionation of the extracts prior to the purification or either identification of these compounds. Recently, Chiou et al. (2016) utilized
medium-pressure liquid chromatography (MPLC) for fractionation of the extracts and
semipreparative HPLC for recovery of the isolated stilbenes from bioelicited peanut sprout
powder for the purpose of investigating longevity extension in mice.
3.5.6 Techniques of identification and quantification of stilbenoids
As the concentration of stilbenoids and other related compounds in plants is relatively low,
modern and sensitive techniques are required for the determination and quantification of
these compounds. When purification and concentration steps are required for a given sample,
solid-phase extraction is performed on those samples. For this purpose, common C-18 cartridges are employed in laboratories for obtaining satisfactory concentrations. For monomers
like resveratrol, a cartridge of polymeric polystyrene-divinylbenzene has been extensively
used for their determination. For polyphenols, the approach that has been utilized mostly
is liquid-liquid extraction. Both liquid and gas chromatographic approaches have been
employed in methods that are coupled with various detection systems. The usage of ultraviolet (UV) detection with electrochemical detection, for analyzing resveratrol in different plant
extracts, has also been proposed. For trans-resveratrol detection in red wine, chemiluminescent detection systems have also been used for their direct analysis. For determination of phenolic compounds of stilbenoid origin, e.g., cis/trans-piceid and cis/trans-resveratrol with high
selectivity, several UV and fluorescence detector systems have also been utilized. In order to
develop a simple and simultaneous separation, identification and quantification of phenolic
compounds like stilbenoids, reverse-phase high-performance liquid chromatography (RPHPLC) on semimicro scale has also been engaged as a straightforward method for whole
berries of Vitis vinifera. For confirmation of peak identification, photodiode array detector
(PDA) of 2.5-μL cell coupled with a mass spectrometry detector equipped with an
electrospray ionization source (ESI) was used in this study (Nicoletti et al., 2008). For accurate
measurements, liquid chromatography coupled with mass spectrometry (MS) of triple, quadruple, and quadruple time-of-flight (QTOF) systems have also been reported in the literature. As a rule of thumb, reverse-phase columns (C-18) and a mobile phase of an organic
solvent like methanol or acetonitrile are the usually observed chromatographic techniques
with UV and diode array detector (DAD) systems, for analyzing these compounds. Moreover,
for high selectivity and sensitivity, MS is employed for determination of such compounds.
More recently, variable wavelength detector (VWD) and fluorescence detector (FLD) have
also been reported as rapid identification systems without previous treatments as compared
to previously employed gas chromatography-mass spectrometry (GC-MS) (LópezHernández and Rodrı́guez-Bernaldo de Quirós, 2016). The specificity of these techniques if
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125
compared to modern techniques, which incorporate triple quadrupole mass spectrometry
(HPLC-QqQ), is limited due to the fact that different compounds with similar mass or even
spectroscopic properties may elute in a single run of chromatography. HPLC-QqQ system
also allowed multiple compounds to be simultaneously quantitated in complex matrices in
a shorter period of time. For improving the chromatographic separation, ultra-highperformance liquid chromatography (UHPLC) has been employed, which resulted in
enhanced resolution and faster separation as it utilizes low particle size and high column
pressure as compared to conventional chromatographic methods. Recently, UHPLC coupled
with triple quadrupole MS that was operated under multiple reactions monitoring (MRM)
mode has been employed for faster and accurate identification and quantification of
stilbenoids (Hurtado-Gaitán et al., 2017). Arraki et al. (2017) employed UHPLC-MS for identification and quantification of stilbenoids from seven Tunisian red wines and identified
piceatannol, piceid, a-viniferin, e-viniferin, isohopeaphenol, and hopeaphenol from Mornag
appellations of these wines.
3.5.7 Levels of stilbenoids found in plants or food-based plants
Naturally occurring stilbenoids have a limited but heterogeneous distribution in the plant
kingdom and are known for their complexity in structure and also in their biological activities. Vitaceae family has been identified to prominently contain stilbenes and their derivatives, particularly a species named Vitis vinifera, which is popular for the production of
grape vines, has been known as the richest source of stilbenoids. Other families in the plant
kingdom that have been recognized as natural sources of this class of compounds are
Fabaceae, Gnetaceae, and Dipterocarpaceae among others (Rivière et al., 2012). The biosynthesis of resveratrol and related compounds is limited to a range of plant species that are included in human consumption, namely, grapes, bilberry, pine, mulberry, and peanuts. The
range of quantity of these compounds in plants and food sources has also been considered
heterogeneous as the methods that are used for extraction, identification, and quantification
are not entirely invariable in nature. An investigation of grape berry skins was carried out to
find its stilbene content and it was observed that cis-piceid was found 92.33 μg/g, trans-piceid
was 42.19 μg/g, and trans-resveratrol was 24.06 μg/g (Romero-Perez et al., 2001). Peanuts
have been considered as a major source for these compounds, especially resveratrol and
piceatannol. The amount of these compounds that was obtained from the calluses of Arachis
hypogaea was reported to be in the range of 2.17–5.31 μg and from 0.25 to 11.97 μg, respectively
(Ku et al., 2005). In another study performed on grapes, levels of stilbenoids like resveratrol
and e-viniferin were reported to be around 4.8 g/kg dry weight (dw) of the Pinot Noir canes
(Gorena et al., 2014). In human diet, the quantity of stilbenes is proposed to be quite low as
compared to other compounds from plant sources. One of the popular compounds from
stilbenoids, resveratrol which is thought to have anticarcinogenic effects, has been reported
in red wine to be in the range 0.3–0.7 mg aglycones/L and 15 mg glycosides/L (Vitrac et al.,
2002). In a comprehensive review by Donnez et al. (2009), the roots of Japanese knotwood
Poligonum cusidatum was recognized as the source, which contained the most abundant levels
of naturally occurring resveratrol. The roots of this species have been used for hundreds of
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3. Analysis of polyphenolics
years for treating inflammation in traditional Asian herb medicine. A lot of research has also
been carried out in exploring the transgenic plants for the production of these compounds;
however, levels of stilbenes and their isomeric forms not only depend on the plant species,
they also vary from the tissues and organs of these species as source materials. The ripening
stages can also affect the levels of stilbenoids, as reported in case of tomato and apple
(Nicoletti et al., 2007). The overall content of stilbenoids has been reported to be dependent
on the species of plant, the biological aspect (activity) of the pool of enzymes and precursors in
biosynthetic pathways, and also on the differences in these secondary metabolic pathways
leading to the production of these compounds (Giovinazzo et al., 2012).
3.5.8 Effects of food processing on stilbenoids
Polyphenols, including stilbenoids, are natural antioxidants and function in lowering the
inflammatory response and can assist in modulating the cellular signaling pathways. The
overall stability of the structure of these compounds and their antioxidant potential is
reported to be tempered when induced by cooking, postharvest processing, and storage
(Rusu et al., 2018). Major effects of heating on these moieties include loss of phytochemical
content, altered rheological and organoleptic abilities, and damage to nutritional quality.
Processing techniques usually involve microbial inactivation, yield improvement and enzyme inactivation. There are certain alternatives to heating that include high-intensity light
pulses, pulsed electric fields, flash vacuum pressure, and high hydrostatic pressure ( JimenezSánchez et al., 2017). The fermentation process alone, during the production process, can influence the quality of natural stilbenoids. Vrhovsek et al. (1997) studied the influence of two
strains of yeast on the cis/trans-resveratrol and glucoside isomers of resveratrol in wine. It was
reported that the yeast that contained higher levels of β-glucosidase activity increased the
concentrations of cis/trans-resveratrol and decreased the concentrations of resveratrol glucosides. Nonflavonoid polyphenols, including stilbenes present in cranberries, contribute to the
biological effects of this fruit. Although resveratrol has been studied a lot from wine and
grapes, cranberry is also considered a rich source of this compound with 0.2–0.4 mg/L.
The effect on lyophilized and powdered cranberries was investigated and the concentrations
of different stilbenoids were relatively closer; still a lot of research is required to properly document the effects of these processing techniques on these phytochemicals (Pappas and
Schaich, 2009). When the effects of grape processing on the antioxidant ability of antioxidant
phenolics were investigated, it was reported that the red wines that were produced by mash
heating and fermentation, contained the highest concentrations of stilbenoids and with
increased antioxidant ability (Netzel et al., 2003). The processes of oxidation and gammaradiation can break the chemical bonds inside polyphenols and result in discharging soluble
phenols of lower sizes, which can lead to an overall increase in antioxidant rich phenolics. UV
radiation effects, when investigated for resveratrol content in grapes that were irradiated,
caused the stilbene concentration to increase while it did not induce any change in the content
of other phenolics. UV-B, UV-C irradiation showed increase in the resveratrol content of the
irradiated grapes, while it did not induce any significant changes in the other phenolic
compounds. However, the p-hydroxybenzaldehyde content was observed to be decreased
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127
in tomatoes that were treated with gamma-radiation (Alothman et al., 2009). The processing
of strawberries to produce jam has been reported to result in a loss of up to 70% of anthocyanin content, while blanching and pasteurization of purees caused the reduction in anthocyanins up to 43%. However, blanching processes have also been known to inactivate certain
enzymes such as polyphenol oxidases, which consequently caused the improvement in the
stability of anthocyanins (Tiwari and Cummins, 2013). Pulse electric field (PEF), as mentioned
earlier, is an alternative to heating for processing different foods and its effect was investigated on three different types of grapes. The study reported that the highest PEF energy treatment yielded the best results for stilbenes, anthocyaninins, and total phenolics. The grape
variety that gave the best outcome was Tempranillo, the second one was Graciano, and then
followed Grenache. Furthermore, when these samples were tested for antioxidant potential, it
was observed that lowest energy treatments of PEF resulted in increased antioxidant capacity
for these compounds (Elez-Martı́nez et al., 2017).
3.5.9 Trends and concluding remarks
The natural production or extraction of stilbenoids is quite insufficient to meet the overall
demand in the market and for this particular reason there is a need to develop novel methods
for their production on a larger scale. Currently, there are three methods for their production,
namely, extraction from plant raw materials, chemical neosynthesis, and biotechnological
production. The first two methods have been reported to produce insufficient yield; therefore,
biotechnological production of these products is one of the trendy topics of research regarding stilbenoids (Chu et al., 2018). For this purpose, plant cells and in vitro cultures of hairy
roots combined with metabolic engineering have been utilized under sterile conditions for
large-scale collection of trans-resveratrol and its derivatives. Moreover, there are certain compounds or materials that are added to these processes for enhancement of the production of
stilbenoids, called elicitors. Most commonly used elicitors for producing trans-resveratrol are
components of polysaccharides, chitosan, heavy metals, cyclodextrins (CD), β-glucan, and a
combination of other signaling molecules like salicylic acid, jasmonic acid, and methyl
jasmonate. These molecules push the biosynthetic pathways for the production of stilbenes
and in turn act as inducers in the biosynthesis reactions (Lu et al., 2016). Bioactive secondary
metabolites such as stilbenes have also been produced by grape vine cell cultures and
agrobacterium-mediated transformation has been reported to be used for incorporation of
key genes belonging to the metabolic pathways into these cells for improvement of the production of target stilbenoids ( Jeandet et al., 2016). More recently, metabolic engineering
approaches that exploit the effect of overexpressing certain genes like sts genes, calciumdependent protein kinase cpk genes and rol genes under the control of double cauliflower
mosaic virus (CaMV) 35S promoter from different sources have been reported for stable
and enhanced production of trans-resveratrol (Chu et al., 2018). Stilbenoids, as mentioned
in previous sections, a well-known class of naturally found phytochemicals, are stress metabolites that are normally synthesized in response to an infection and are defense compounds
having diverse actions on biological and cellular mechanisms that are applicable to human
health and disease. Such pharmacological capabilities of stilbenoids have motivated the
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synthesis of analogues so that their activities can be improved and turned into products. A
major example of this phenomenon is tamoxifen, which has a chemical core structure of
1,1,2-triphenylethylene and has been proved to be a potent and efficient in treatment of breast
cancer ( Jordan, 2006). Furthermore, 30 -hydroxystilbene, which is synthetic stilbene, has been
reported to be more efficient than resveratrol in inhibition of resistant leukemia cells. Similarly, 3,4,5-trimethoxy-40 -bromo-cis-stilbene has been observed to inhibit the proliferation
of human colon cancer cell more effectively than resveratrol is proposed to be a chemotherapeutic agent for colon cancer (Rimando and Suh, 2008). In light of the unique properties and
interactions of stilbenoids with biological moieties, these compounds have great potential in
medicine, agriculture, and cosmetics as well. However, a lot of research is still required in
order to understand the exact mechanism of action of these compounds. One last concluding
remark would be that as the scientific literature was found to contain a lot of studies on resveratrol, there are other compounds belonging to this class of phytochemicals that need the
attention of scientific community to better understand their applications as well.
Acknowledgment
This work was the outcome of a financially nonsupported study and an in-house effort.
Author contributions
All authors of this manuscript have contributed in planning, literature review, organizing, writing, reading,
rereading, and proof reading of this manuscript.
Conflict of interests
All authors of this manuscript declare and reveal no conflict of interest regarding the publication and distribution of
this manuscript.
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S U B C H A P T E R
3.6
Tannins (hydrolysable tannins, condensed tannins,
phlorotannins, flavono-ellagitannins)
Mohammed Bulea, Fazlullah Khanb,c, Muhammad Farrukh Nisard, Kamal Niaze
a
Department of Pharmacy, College of Medicine and Health Sciences, Ambo University, Ambo,
Ethiopia bInternational Campus, Tehran University of Medical Sciences (IC-TUMS), Tehran,
Iran cDepartment of Toxicology and Pharmacology, Faculty of Pharmacy, The Institute of
Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran
d
Department of Physiology and Biochemistry, Faculty of Bio-Sciences, Cholistan University of
Veterinary and Animal Sciences (CUVAS), Bahawalpur, Pakistan eDepartment of Pharmacology
and Toxicology, Faculty of Bio-Sciences, Cholistan University of Veterinary and Animal Sciences
(CUVAS), Bahawalpur, Pakistan
3.6.1 Phytochemistry of the tannins
Tannins dominate quantitatively the vast plant secondary metabolites, being the fourth
most abundant compounds in the plant vascular tissue next to cellulose, hemicelluloses,
and lignin. A fifth of plants dry weight content can be tannins, while there might be variation
in amount in relation to environmental conditions (Adamczyk and Simon, 2017). Traditionally, plants rich in tannins are used in leather tanning; consequently, their name is coined
from their property to bind and precipitate proteins. Tannins are a group of structurally complex polyphenols comprising hydrolysable tannins, proanthocyanidins (syn. condensed tannins), and phlorotannins (Suvanto et al., 2017). The hydrolysable tannins (HTs) consist of
hydrolysable ester bonds between the main chemical scaffolds, whereas in the condensed tannins the monomers are connected via CdC or CdOdC bonds. Condensed tannins are the
ones that bear the major medicinal properties attributed to tannins. On the other hand, tannins formed by conjugations between proanthocyanidines (PACs) and hydrolysable tannins
via CdC bonds are sometimes classified “complex tannins” (Bedran-Russo et al., 2014).
HTs are widely distributed in nature and they are characterized by multiple esters of gallic
acid with glucose. Currently, more than 500 glucogalloyl derivatives have been identified
(Blenn et al., 2011). Tannins structures commonly possess 5–7 aromatic rings and consist
of 12–16 phenolic substituents per 1000 relative molecular mass unit. HTs as well consist
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3.6.1 Phytochemistry of the tannins
133
of esters of phenolic acids, such as gallic acid in gallotannins (GTs) or other phenolic acids
such as in ellagitannins (ETs), and a polyol, usually glucose. A more complex group of
HTs is formed when the galloyl groups are further esterified or crosslinked via oxidation
(Lamy et al., 2011). The simplest HTs are gallotannins that comprise polygalloyl esters of glucose, which is widely found in red oak, fringe cups, staghorn sumac, pedunculate oak, and
others. On the other hand, ellagitannins are formed through intramolecular coupling,
resulting in CdC and CdOdC linkages between galloyl residues of glucogalloyls. Among
the bioactive tannins, ETs are widely found in some fruits like Fructus chebulae, Phyllanthus
emblica, and Punica granatum L. (Cai et al., 2017).
The condensed tannins CTs (also known as proanthocyanidins, PAs) are prevalent in human nutrition, like grape seeds, apples, berries, red wine, chocolate, cocoa, and others. CTs
are more abundantly found in plants than the HTs and they have a rather complex structure
as compared to HTs (Cai et al., 2017). Proanthocyanidins are polymers that are formed by
monomeric flavan-3-ols units that are connected via C-4 ! C-8 bonds, and sometimes
through C-4 ! C-6 linkages (Smeriglio et al., 2017). The presence of asymmetric centers at
the C-2 and C-3 positions of the monomeric units has resulted in four isomers (+)gallocatechin, ()-epigallocatechin, (+)-catechin, and ()-epicatechin. In addition, the several
hydroxyl groups that exist in the CTs structure are responsible for the strong interaction of
CTS with proteins and metal ions. Proanthocyanidins, which are found widespread in vegetables, are mainly composed of catechin and epicatechin units (procyanidins). Whereas
propelargonidin or prodelphinidin containing (epi) afzelechin or (epi) gallocatechin basic
units are less common and found in grains such as barley, broad beans, red kidney beans,
red currants, pinto beans, black tea, and cinnamon (Cai et al., 2017; Smeriglio et al., 2017). Tannins like phlorotannins (Fig. 3.6.1) are mostly found in marine algae and they are
phloroglucinol oligomers produced in brown algae and have tannin activities. In the beginning, isolating phlorotannin was possible only after methylation or acetylation of free phenolic hydroxyl groups in the molecules. Afterwards, free phlorotannins of similar biological
activity were isolated from the edible brown algae Ecklonia kurome, such as eckol, an
antiplasmin inhibitor, and phlorofucofuroeckol A. Additionally, the other derivative
phlorofucofuroeckol-B possessing antiallergic activity was isolated from Eisenia arborea,
and it has long been used by Japanese traditional healers for gynecopathy. This revealed
the masking effect of acetylation and methylation on the pharmacological activities of the
phlorotannin derivatives (Okuda and Ito, 2011).
Structurally, tannins are diverse phenolic compounds exhibiting complex structure. Both
HTs and CTs have reactive OH groups, which form complexes with proteins, including enzymes and polymers such as cellulose and hemicelluloses (Ncube and Van Staden, 2015). Various researchers have studied the nature of interaction between tannins and proteins,
including the size, the stereochemistry, the nature of the protein, and the suitable medium
for interaction (ionic strength and pH) to happen. In general, these interactions could occur
through covalent or ionic bonds, hydrophobic interaction, or hydrogen bonding. The stability
of tannin-protein complex depends on the number of bound tannins and the number of repeated amino acid sequences (Lamy et al., 2011). These tannin-protein interactions are dynamic and time dependent. Furthermore, the conformational flexibility of both the tannin
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3. Analysis of polyphenolics
6¢
5¢
4¢
3¢
1¢
2¢
(B)
(A)
(D)
(F)
n
(C)
(E)
(G)
FIG. 3.6.1
Structure of representative tannins. (A) Tannin general structure, (B) simple gallotannin, (C) simple
ellagitannins, (D) condensed tannin, (E) tannic acid, (F) eckol, and (G) phlorofucofuroeckol A.
and the protein is a vital complementary factor for a stronger interaction. In addition to the
phenolic groups and the aromatic rings, through which the tannins act as multidentate ligands on the protein surface, increase in the number of polyphenols galloyl groups affects
positively the efficacy of protein binding (Ncube and Van Staden, 2015).
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3.6.2 Biological activities of tannins
135
3.6.2 Biological activities of tannins
Tannin-rich foods have potential antioxidant, cancer-fighting activity, and reduce the risk
of developing cardiovascular disease as well, although their consumption inhibit nonheme
iron bioavailability (Kitunen et al., 2017). The oxidation reduction reaction that occurs during
the interaction of phenolic hydroxyls with metal ions reduces the absorption of metal ions
such as calcium and iron. Yet an outside of tissues interaction between calcium and tannins
has a blood-pressure-lowering effect (He et al., 2015). Moreover, tannin-collagen interaction
forms complexes that are expected to be stabilized mainly through hydrogen bonding between the protein amide carbonyl and the phenolic hydroxyl as well as the covalent and hydrophobic bonds. Such complexes are substantially important in dental application,
particularly the polyhydroxylated PACs. This is mainly because the polyhydroxylated PACs
can form an insoluble matrix with carbohydrates and proteins (Bedran-Russo et al., 2014). On
the other hand, the interaction of tannins with saliva proteins decreases saliva lubricity results
in feeling of dryness by causing contraction of tongue epithelial tissue. This phenomenon is
referred to as convergence or astringency. The study of fruit astringency has obtained considerable attention since astringent substances have appreciable biological activity such as
antibacterial, antiviral, antiinflammatory, antioxidant, anticarcinogenic, antiallergenic,
hepatoprotective, vasodilating, and antithrombotic activities (He et al., 2015).
Studies have demonstrated that tannins are implicated in the antiulcer properties of several herbal products. Particularly, in peptic ulcer disease, tannins act by precipitating the
microproteins and thus form a protective pellicle that prevents absorption of toxic substances, and boosts resistance to the proteolytic enzymes, a related activity against H. pylori.
Moreover, in experimental models, such compounds that are isolated from secondary tannic metabolites showed both in vivo and in vitro activity against peptic ulcer disease (De
Jesus et al., 2012). In addition, the anticancer activities of tannins against a range of cancer
cells have been investigated. In most of the cell lines such as hepatocellular carcinoma
(HCC), breast cancer, lymphocytic leukemia, lung cancer, and epidermoid carcinoma, tannins demonstrated cytotoxic activity in a dose-dependent fashion. The structural activity
relationship studies pointed out the number of galloyl moieties in tannins have direct relation to higher anticancer efficacy (Cai et al., 2017). Besides, as the degree of polymerization
of tannins increases, which results in increase in number of hydroxyl groups, the free radical
inhibitory activity of the resulting compound will be stronger. Furthermore, tannins can
readily react with oxygen radicals and release a large number of free hydrogen due to
the phenolic hydroxyl groups present in their structure. Being strong free radical scavengers, tannins can reduce the risk of aging and related diseases (cardiovascular disease, aging, cataracts) (He et al., 2015). Tannins have strongly displayed cardioprotective activity
through inhibiting elastin-degrading enzymes, inducing stabilization of pericardial tissue,
and decreasing calcification of the glutaraldehyde-fixed aortic wall (Sieniawska and Baj,
2017b). Furthermore, in vitro studies depicted that procyanidin-containing cocoa inhibits
platelet aggregation, which is a common pathology to cardiovascular diseases. It was also
reported that tannins from Arbutus unedo (or “Strawberry Tree”) leaves inhibited thrombininduced platelet aggregation. Leaves of this tree are traditionally utilized as a remedy for
high blood pressure (Macáková et al., 2015).
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3. Analysis of polyphenolics
The antibacterial activities of polyphenols such as tannins, some flavonoids, and xanthones
revealed that they are effective against Methicillin-resistant Staphylococcus aureus (MRSA)
(Hatano et al., 2005). Several plants-based phenolic antimicrobial agents are available in certain food known for their tannin, essential oils, resins, and glycosides content. For instance,
the bioactive component in cacao and tea is catechin, gallic acid in berries, allicin in garlic,
eugenol in cloves, thymol and carvacrol in oregano, eugenol and thymol in sage, and allyl
isothiocyanate in mustard (Anon, 2001). Moreover, antimicrobial hydrolysable tannins and
proanthocyanidins exist in different medicinal plants, vegetables, and fruits. These secondary
plant metabolites act as antibacterial agent through different mechanisms, including disintegration of bacterial cell wall, interfering bacterial metabolism, and by inhibiting extracellular
enzymes. Their antiviral activity is also reported to be related to the basic tannin structure.
Mechanistically, the tannins have been demonstrated to interfere with the viral adsorption
of HSV and HIV, which is an action attributed to the tannins’ ability to bind to viral surface
proteins. In addition, most active ellagitannins and several proanthocyanidins have been
reported to be potent reverse transcriptase inhibitors (Smeriglio et al., 2017).
3.6.3 Current and potential industrial applications of tannins
Pycnogenol (PYC) is mainly extracted from the bark of the French maritime pine. It consists
of catechin, taxifolin, procyanidins of different chain lengths composed of catechin,
epicatechin, phenolic acids, and their glucose esters or glucosides. The nutritional supplement PYC contains pharmacologically active components that have potential protective actions against age-related chronic diseases. (Fitzpatrick et al., 1998). Moreover, PYC has
been used in treating circulation problems, allergies, asthma, attention deficit hyperactivity
disorder (ADHD), ringing in the ears, high blood pressure, diabetes, muscle soreness, endometriosis, menopausal symptoms, osteoarthritis, painful menstrual periods, erectile dysfunction, and retinopathy (Sieniawska and Baj, 2017a). Today, numerous plant products are
consumed daily for their potential health benefits as nutraceuticals and dietary supplements
containing hydrolyzable and condensed tannins. Popular products such as Healing America
Ellagitannin capsules; EllagicActive tablets; and Buckeye Nutritionals Black Raspberry
Ellagitannins 60 capsules are standardized blends comprising ellagitannins, punicalagins,
and polyphenols. Other products containing pomegranate extract such as Pomegranate Pills
with Ellagic Acid from Juice and Seeds capsules, PomeGuard capsules, Pomegranate Standardized capsules, Living Pomegranate capsules, and endothelial defense with full-spectrum
pomegranate capsules have been used to reduce the risk and prevent atherosclerosis. Furthermore, other supplements containing tannins are commonly available on market, including
decaffeinated green coffee extract as antiobesity supplement, green tea extract as antioxidant
supplement, Swedish Birch bark extracts as antioxidant and antifungal, cranberry extract as
antibacterial agent (Sieniawska and Baj, 2017b).
Tannins are commonly used as food additives and food preservatives. In accordance with
the Commission Implementing Regulation (EU) No. 872/2012 (2012) tannic acid (TA) is a
food-flavoring agent. Although berries are the richest dietary source of tannins, the total tannin content of HTs in food ranges from 1 mg/kg to 2 g/kg in chestnuts and in blackberries,
respectively. Presently, the interest in HTs for different scientific and commercial purposes
II. Phenolics
3.6.4 Possible interactions of tannins
137
has increased. The astringency of HTs is their most studied property and known to have important effects in food quality in different ways. Various processed food items have been
found to have HTs, including aged wines and spirits or other food products, to which
HTs have been added so as to improve their antioxidant activity; this also includes meat, fish,
or wine (Arapitsas, 2012). The astringent characteristic of tannins has increased their importance in the search for potential drug candidates for the treatment of noninsulin-dependent
mellitus. In particular, the tannins of fruits, vegetables, and beverages origin have gained significant ground. HTs, as well as procyanidins, reduce starch digestion through forming complex with human α-amylase. Thus, this inhibiting enzyme is also studied as effective
antidiabetic mechanism of functional foods. In this regard, tannins hinder carbohydrate digestion, glucose release, and absorption while keeping postprandial hyperglycemic secretion
attenuated. Additionally, they interact with adipose tissue, inhibiting adipogenesis and directly enhancing insulin activity (Sieniawska and Baj, 2017a). However, the tannin-protein
interaction is not limited to digestive enzymes, and tannins could be bound by other proteins
in the food. Therefore, microencapsulation technique to prevent such unintended interactions
and to sustain the functionality of tannins has been studied in a tannic acid-bovine serum albumin (BSA)-porcine pancreatic a-amylase system. It was demonstrated that the inhibition of
the unprotected α-amylase by tannic acid was considerably reduced by increasing the amount
of BSA; this action was diminished by microencapsulation of the tannic acid (Barrett et al.,
2018). On the other hand, a chewing gum made of tea polyphenols is demonstrated to be active against viral influenza infections and to inhibit spreading of this virus. Correspondingly,
procyanidin monomers and oligomers containing cocoa procyanidins and cocoa extracts are
claimed to modulate cytokine gene production, protein levels, and proved to be useful to individuals with asthmatic condition or viral infections or at risk of viral infections (Sieniawska
and Baj, 2017a).
3.6.4 Possible interactions of tannins
Structurally, tannins comprise hydroxyl groups at various positions and other functional
groups like carboxyl (OdC]O), which are responsible for complex formations with macromolecules and proteins. The interaction between protein and tannin is thought to be mainly
due to the formation of hydrogen bonds between the hydroxyl functions of the tannin and the
carboxyl groups of the protein. Furthermore, the contribution of other interactions such as
hydrophobic interactions between the aromatic ring of tannin and hydrophobic regions in
the protein structure stabilizes the complex formed between the tannin and the protein
(Wang et al., 2016). These interactions explain the binding properties of tannins to proline residue; the tannin receptor site, which has been reported to be mostly due to hydrophobic interactions and hydrogen bonding. Therefore, the complex formation of tannic acid (TA) with
proteins rich in proline (e.g., salivary protein, gliadin from gluten, and kafirin from sorghum)
is due to similar interactions between TA and these proteins (Barrett et al., 2017). In addition
to proteins, tannins are capable of forming precipitates with alkaloids. Tannins can also react
with metals to form complexes. Thus, in spite of the various biological activities that tannins
have, they can’t be administered directly as herbal injections because of their strong binding
properties with proteins and metals ions. It has been reported that in addition to interacting
II. Phenolics
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3. Analysis of polyphenolics
with prescription drugs upon oral administration, tannins caused allergies and pain when
injected. Consequently, some requirements in the Chinese Pharmacopoeia recommend that
tannins be avoided in herbal injections. Likewise, the US FDA also recommended that tannic
acid is unsafe to be used on the skin for the treatment of diaper rash, prickly heat, and minor
burn or sunburn (Cai et al., 2017).
TA is a hydrolysable tannin that can interact with choline, an amine precursor of acetylcholine, to form complexes and it readily adsorbs to chitosan. Furthermore, TA can react with
a number of organic N-containing compounds, such as arginine (from all amino acids), nitrogen bases, polyamines, chitin, and chitosan (Adamczyk and Simon, 2017). In rat model study,
tannin-rich diet reduced vitamin A content in liver and utilization of vitamin B12. Besides,
iron absorption decreases due to the formation of insoluble matrix, in people who consume
tannin-rich sorghum (Macáková et al., 2014). Moreover, tannins in ruminant feed are linked to
decrease in milk yield and sulfur availability; toxic changes in the intestine, liver, spleen, and
kidney; mucus in urine; and fatal constipation (Kumar and Singh, 1984). On the other hand,
tannin-enzyme interactions follow a similar pattern as the interaction of tannins with
nonenzymatic proteins. Consequently, tannins were considered potential inhibitors of enzymatic actions, while some studies stated that minor decline in enzymatic activity could occur.
However, recent evidences have demonstrated that enzymatic activity is improved following
interaction with tannins applied in low concentrations. Though increased concentrations of
tannins caused reduction in the catalytic activity (Adamczyk and Simon, 2017). For example,
tannic acid, which is a hydrolysable tannin mostly available in plant-derived foods, inhibits 6β-hydroxylation of testosterone by CYP3A4 in rat and human liver microsome at 16.8 and
20.2 μM IC50 values, respectively (Basheer and Kerem, 2015). However, the FDA has placed
tannic acid under the generally recognized as safe (GRAS) list for use as direct food additive
(Wang et al., 2016). Yet, herbal drugs such as crofelemer containing tannin derivatives have
been reported to have interaction with liver microsomal enzymes. Crofelemer (Fig. 3.6.2) is a
herbal drug approved for treatment of diarrhea associated with various etiologies, especially
in relation to HIV antiretroviral drugs. It is a polyphenolic compound classified as proanthocyanidin (condensed tannin), which are abundantly available in natural products from plants
sources (Tradtrantip et al., 2010). This compound is extracted from the bark latex of Croton
lechleri, a plant known in the western Amazon regions of South America. In its structure,
crofelemer is a polymer, which comprises a linear chain of catechin, epicatechin,
gallocatechin, or epigallocatechin monomer units. Results of in vitro study on human liver
microsomes (CYP 1A2, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4 isozymes) demonstrated that
crofelemer inhibits a number of CYP enzymes (FDA, 2011).
Chemical structure of crofelemer (1. R ¼ H—procyanidin;
2. R ¼ OH—prodelhinidin).
FIG. 3.6.2
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3.6.5 Tannins techniques of extraction, purification, and fractionation
139
3.6.5 Tannins techniques of extraction, purification, and fractionation
Traditionally herbal medications are prepared by decoction of the plant material, i.e., by
extraction of dried plant in boiling water. The tannins in the plants, being able to retain their
original structure throughout the drying process, usually undergo hydrolysis during decoction, as in geraniin yielding corilagin, ellagic acid, and brevifolincarboxylic acid (Okuda and
Ito, 2011). Generally, extraction of tannins from plants is performed using solvents such as
ethanol, methanol, acetone, or aqueous mixtures of these solvents. Before starting the extraction, the plant material is washed with petroleum ether or dichloromethane to remove lipophilic impurities. Proanthocyanidins with low degree of polymerization or simple gallic acid
esters can easily be extracted with ethyl acetate since they are low-molecular-weight compounds. To proceed with the separation, the crude extract is further treated with Sephadex
LH-20 and RP materials with alcohol-aqueous-mixtures or acetone-aqueous-mixtures. Light
protection is important during extraction of proanthocyanidins since they are sensitive to
light, while safety is demanded to reduce the risk of hydrolysis during the extraction of
hydrolysable tannins (Serrano et al., 2009).
The conventional plant secondary metabolites extraction method has a long history; however, the use of microwave-assisted extraction (MAE) is a recent addition in the area. Quite a
tremendous research interest has been shown toward utilization of MAE for the extraction of
polyphenols and tannins from vegetables. In comparison to the conventional methods, MAE
proved to be advantageous since it requires shorter extraction period, uses less solvent, and
provides a better yield. The efficiency of MAE is due to the fact that microwave treatment has
mechanical effect, hence heating the solvent mixture directly. In addition, the high energy of
the microwave will have direct interaction with the free water molecules in the glands and
vascular systems, because of which rupture of the plant tissue followed by leakage of the active constituents into the solvent occurs (Al-Harahsheh and Kingman, 2004). The ruptured
cell wall leaks the constituents and as a result facilitates the transfer of the secondary metabolites to the surrounding solvent system. Therefore, the yield of the phytoconstituents will be
higher and the time required for extraction is minimized. The method of extraction using
MAE exposes the plant to the solvent via cell rupture, while the conventional heat-reflux extraction needs a series of permeation and solubilization to bring the secondary metabolite out
of the matrix (Mandal et al., 2007). The method used in MAE can be varied, based on the conditions of the study, including the time span, solvent used, or power extraction (Rhazi
et al., 2015).
Further purification of extracts (fractionation) using chromatographic techniques needs
few grams of the crude hydroalcoholic extract. A glass column (90 cm high and 5 cm in diameter) containing silica gel 60 (300 g) is required to run the chromatography for crude
hydroalcoholic extract. Initially, the column is prepared via dry packing with silica or packing
by gel slurry in hexane:ethyl acetate (EtOAc) (70:30). On the other hand, the crude
hydroalcoholic extract will be placed on the top of the column after mixing with silica gel
and drying it. Then elution of the crude extract starts with hexane:ethyl acetate (70:30) and
continues by increasing polarity of solvent by adding more EtOAc up to a proportion of
10:90. The column is washed with methanol at the end. The collected subfractions are joined
after a TLC check and then evaporated, lyophilized, and stored at 20°C in darkness
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140
3. Analysis of polyphenolics
(Zhao et al., 2011). Further analysis is done using ultra-high-performance liquid chromatography/mass spectrometry (UHPLC/+ESI-QqTOF MSMS) and high-performance liquid
chromatography (HPLC) to analyze the chromatographic profiles. Subsequently, after
performing the identification of the isolated compounds by 1D and 2D-NMR, spectroscopic
measurements structures will be determined (Dikti Vildina et al., 2017).
3.6.6 Tannins techniques of identification and quantification
Tannins’ chemical investigation emerged lately because of the lack of methods suitable for
isolation and structure elucidation. Yet the existing methods are not fit for analyzing all kinds
of tannins, particularly the highly polymerized ones. The Folin-Ciocalteau reagent test is popular one, but it’s not specific for tannins and gives positive result to other polyphenols too.
However, Hartzfeld et al. (2002) have utilized standard color reactions to selectively determine esters of gallic acid or ellagic acid (Hartzfeld et al., 2002; Serrano et al., 2009). For instance, proanthocyanidins turn into red products whenever reacted with mineral acids or
with Vanillin-HCl. Moreover, proanthocyanidins treated with dimethylaminobenzaldehyde
forms blue products. Apparently, these color reactions are used in spectrophotometric determinations and quantification of tannins (Serrano et al., 2009).
Folin-Ciocalteu’s phenol reagent has been used to estimate the total phenolic content. The
spectrophotometric determination of total polyphenols in a sample is performed by measuring the absorbance of a mixture of the sample solution, Folin-Ciocalteu’s phenol reagent, and
NaOH-Na2CO3 solution. Once the measure of the total polyphenols content of the original
sample is done, casein will be applied to adsorb the tannins in the sample and the total phenolic content of the resulting solution will be determined. Hence, the difference in absorbance
of the original solution and casein-adsorbed solution is used to determine the total tannins
content of the sample. All the results are expressed as g GAE/100 g dry weight of the extract
(Gong et al., 2014; Zhao et al., 2011). The analysis of highly polymerized tannins is difficult
with the currently existing methods. In this regard, high-molecular-weight HTs are frequently analyzed after undergoing acid hydrolysis to their corresponding products such
as gallic acid or ellagic acid (Serrano et al., 2009).
Various techniques were developed for quantitative and qualitative analysis of low-molecular-weight tannins like proanthocyanidins, including TLC, countercurrent chromatography,
different gel separation techniques, 13C-NMR, and normal phase and RP HPLC (Serrano et al.,
2009). HPLC-based analysis of proanthocyanidins in the presence of a nucleophile and following acid-catalyzed depolymerization is one among the methods that provide qualitative
and quantitative result. These results include the nature and proportions of proanthocyanidin
constitutive units and their average degree of polymerization (Pinasseau et al., 2016). On the
other hand, NMR-based analysis is important for in situ characterization of natural products
such as tannins. The 13C-NMR spectra of tannins have a fingerprint peak that makes the spectroscopic identification quite clear. In addition, the degree of extractions can be quantified
using NMR, which gives validated data than conventional gravimetric techniques (Romer
et al., 2011).
II. Phenolics
3.6.8 Effects of food processing on tannins
141
3.6.7 Levels founds of tannins in plants/food-based plants
Cranberry contains complex phytochemicals, including flavan-3-ols, anthocyanins, A type
procyanidins (PACs), benzoic acid, and ursolic acid. The flavan-3-ols of cranberry exists as
monomers, oligomers, and polymers. On weight basis, 85% of cranberry flavan-3-ols are composed of the oligomers and polymers known as condensed tannins or PACs. Among the
PACs in cranberry (2)-epicatechin is found in larger quantity while (+)-catechin and (epi)
gallocatechins are the other PACs but smaller in amount (Blumberg et al., 2013). Grape tannins are mainly found in the seeds, skin, and stem. Tannins in grape skin are high in concentration at early stage of development and declines over a period of time. These tannins in
different tissues vary in structure and content. Proanthocynidins in the seeds have
epicatechin subunits resulting in procyanidins thus formed, whereas skin proanthocynidins
contains additional epigallocatechin subunits, and consequently forms prodelphinidins as
well (He et al., 2015). Skin tannins are more polymerized than the corresponding seed tannins,
which have more galloylated units (13%–29%) in comparison to the tannins in the skin (3%–
6%). The grape stem tannins and proanthocyanidins in seed and skin (25.00 and 1.387 mg/g
dry weight, respectively) can change the phenolic content of wine by contributing large
amount of tannins (McRae and Kennedy, 2011), while other plants such as sorghum, tea, cranberry fruits, plums, apples, peaches, apricot, beans, and peanuts contain proanthocyanidin in
different proportions as shown in Table 3.6.1. Similarly, peanuts contain phenolic compounds
in the skin and hull. Despite the fact that the peanut skin is light as compared to the weight of
the nut, it encompasses a larger number of polyphenols than the nut. The polyphenol content
of the dry skin is reported to vary from 115 to 149 mg/g in relation to the extraction solvent
used. Another study demonstrated that the polyphenol content in peanut to be roughly between 90 and 125 mg/g of dry skin (He et al., 2015) as shown in Table 3.6.1. Generally, although our diet is reach in tannins (daily intake is approximately 0.1–0.5 g), they are
perceived less important, may be because of their structural complexity and polymeric nature
(Smeriglio et al., 2017).
3.6.8 Effects of food processing on tannins
Tannins, being considered antinutritional, have been removed by food-processing techniques. Various studies have demonstrated the effect of food-processing techniques on the
stability of tannins. The effect of cooking and processing is clearly displayed in proanthocyanidins, which are found in fresh plums and grapes and do not appear in prunes and raisins. This indicates that the proanthocyanidins were either hydrolyzed or polymerized
during the processing, thus becoming barely possible to extract or quantify (Serrano et al.,
2009). A study that has investigated the effect of thermal processing on proanthocyanidins
pattern of peaches using LC-MS reported 5%–24% decrease of monomers up to pentamers
and reduction up to 30% of hexamers and heptamers as well. In comparison, the effect is less
pronounced in grape seeds, causing reduction of 11%–16% in proanthocyanidins content. On
the other hand, the impact of storage is observed in canned peaches, which also resulted in
considerable loss of higher oligomers due to long period of storage and complete absence of
II. Phenolics
142
TABLE 3.6.1
3. Analysis of polyphenolics
Proanthocyanidins content of various plant species.
Samples
Species
Proanthocyanidin (mg/g dry
weight)
References
Mangrove
K. candel
106.35 21.16
Zhang et al. (2010)
R. mangle
219.27 63.11
Zhang et al. (2010)
Bordeaux Merlot
25.00 1.637
Chira et al. (2011)
Cabernet Sauvignon
28.232 5.793
Chira et al. (2011)
Bordeaux Merlot
0.724 0.142
Chira et al. (2011)
Cabernet Sauvignon
1.387 0.304
Chira et al. (2011)
12.76 1.15
Queiroz et al.
(2018)
Dahongpao
210.09 12.55
Chen et al. (2018)
Tieluohan
201.42 5.35
Chen et al. (2018)
Baijiguan
155.29 6.42
Chen et al. (2018)
Shuijingui
199.58 4.77
Chen et al. (2018)
Cranberry fruit
Vaccinium oxycoccus L.
13.3–36.7
Blumberg et al.
(2013)
Plums
Prunus domestica
3.2–33.4
Smeriglio et al.
(2017)
Blueberries
Vaccinium myrtillus L.
8.7–27.4
Smeriglio et al.
(2017)
Vaccinium corymbosum L.
31.1–33.5
Smeriglio et al.
(2017)
Apple
Malus domestica Borkh.
4.6–27.8
Smeriglio et al.
(2017)
Peaches
Prunus persica L.
2.9–11.0
Smeriglio et al.
(2017)
Apricot
Prunus armeniaca L.
0.8–7.3
Smeriglio et al.
(2017)
Beans
Black turtle bean (crude
extract)
40.6
Singh et al. (2017)
Black soybean (crude
extract)
20.6
Singh et al. (2017)
Seed coat (6 peanut
varieties)
29.7–84.7
Singh et al. (2017)
Raw kernel (6 peanut
varieties)
2.88–4.73
Singh et al. (2017)
Cotyledons
1.74–3.75
Singh et al. (2017)
Grape (seeds)
Grape (skin)
Sorghum powdered drink
mix
Tea
Peanuts
II. Phenolics
3.6.9 Trends and concluding remarks
143
those with structure larger than tetramers (Hong et al., 2004; Serrano et al., 2009). Similarly, in
cranberry, which is mainly processed and consumed as juice, loss of polyphenols due to thermal processing is high. Particularly, anthocyanins loss is very high reaching up to 50%, while
the heat-resistant flavonols and PACs are the least lost (Blumberg et al., 2013). Furthermore,
investigations on the food processing in the making of cookies and bread from sorghum bran
indicated large amounts of procyanidins are lost, especially the higher-molecular-weight
polymers. It was demonstrated that processing of sorghum affects the overall content
resulting in 42%–84% procyanidin content retained in cookies and only 13%–69% in bread
(Serrano et al., 2009). Dehulling is another main method used in food processing to reduce
the fiber content of grains such as barley, wheat, peas, beans, and rice. Cowpea seeds have
been studied for the effect of dehulling on their total condensed tannin content. The total tannin content of whole cowpea seeds was 0.3–6.9 mg/g and that of the seed coat was 7.2–
116 mg/g. The study showed that dehulling eliminated 98% of the tannin in raw cowpeas
(Yasmin et al., 2008). On contrary to thermal processing, freeze-drying at very low temperature is an effective means to avoid the loss of tannins during the food processing (Serrano
et al., 2009).
3.6.9 Trends and concluding remarks
In this chapter the chemical, structural, biological, pharmacological properties as well as
the level in plants and the industrial applications of tannins were discussed. In general, tannins are very widespread and important secondary plant metabolites with multiple phenolic
hydroxyl groups, which are responsible for their biological activity. They have been utilized
as dietary supplements, nutraceuticals, functional food additives, and pharmaceuticals.
Tannin-containing products are mostly claimed to have an array of biological activities, including antioxidant, anticancer, antidiabetic, antiallergy, antimutation, antiaging, and antimicrobial. A recent study on the interaction of common tannins in food products,
procyanidin B3, and proline rich fraction of wheat extract, which is mostly associated with
onset of Celiac Disease (CD), showed that proanthocyanidins are important as nutraceuticals.
This study elucidated different soluble B3-peptide complexes comprising immunoreactive
peptides, with varying size and diversity, emphasizing the importance of dietary proanthocyanidins in modulating gut diseases. Besides, the complex forming property of proanthocyanidins plays a role in enzyme inhibition (pectinase, amylase, lipase, protease, and
β-galactosidase) that will result in reduced absorption of other biomolecules such as proteins
and carbohydrates (Smeriglio et al., 2017). Conventionally, tannin-protein binding has been
referred to as “antinutritional effect” of tannins. Traditionally, protein-tannin binding has
been cited as a major contributor to the antinutritional effect of tannins outside of mineral
metabolism. Tannins affinity toward protein mediates nonheme-iron chelation via salivary
proline-rich proteins (PRPs). The bioavailability of nonheme iron is increased by binding
of tannins to PRPs instead of the nonheme iron. It has been shown that in addition to increasing nonheme iron bioavailability, intensification of PRPs secretion in relation to tannins consumption improves protein availability in rats (Khan et al., 2015). Although products
containing tannins have long been used enormously due to their beneficial health effects
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144
3. Analysis of polyphenolics
mechanisms of their action, their interaction with biomolecules remains widely undiscovered
(Macáková et al., 2015). Tannins is extracted, purified, and fractioned via MAE, TLC,
UHPLC/+ESI-QqTOF MSMS, HPLC, 1D and 2D-NMR spectroscopy, while identification
and quantification of tannins is achieved via TLC, NMR, and RP HPLC. Thus far, the number
of studies done to assess the potential drug targets and mechanisms of actions of tannins
in vivo is also few in numbers. Hence, future studies need to focus on evaluating the properties of tannin complexes, including the bioavailability, metabolism, tissue distribution, adverse effects, and their interaction with other bioactive compounds and nutritional
supplements. In addition, the number of tannin derivatives that have been approved as drug
and dietary supplements is increasing in recent years. Therefore, further investigation is
needed to aid the traditional claims in order to facilitate the development of tannin-related
dietary supplements, nutraceuticals as well as to design and discover novel lead compounds,
drug candidates, and drug molecules.
Acknowledgment
All the authors of the manuscript thank and acknowledge their respective universities and institutes.
Conflict of interest
There is no conflict of interest.
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3.7.1 Phytochemistry of the curcuminoids
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S U B C H A P T E R
3.7
Curcuminoids
Aadil Javeda, Muhammad Asif Shahzadb, Fazlullah Khanc,d, Kamal Niaze
a
Department of Biotechnology, Graduate School of Natural and Applied Sciences, Ege University,
Izmir, Turkey bDepartment of Plant Biotechnology, Atta Ur Rahman School of Applied
Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad,
Pakistan cInternational Campus, Tehran University of Medical Sciences (IC-TUMS), Tehran,
Iran dDepartment of Toxicology and Pharmacology, Faculty of Pharmacy, The Institute of
Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran
e
Department of Pharmacology and Toxicology, Faculty of Bio-Sciences, Cholistan University of
Veterinary and Animal Sciences (CUVAS), Bahawalpur, Pakistan
3.7.1 Phytochemistry of the curcuminoids
The turmeric (Curcuma longa), an annually growing herb under the ginger family, is mostly
available in south and southeast tropical Asia. This plant has wide range of uses, but among
them the medicinal and culinary uses are at the top priority. Curcumin, a yellow color pigment, is the most effective ingredient of turmeric, and it was first extracted by Vogal in 1842
(Vogel and Pelletier, 1815). Lampe and Milobedeska were the pioneers to yield the structure
of curcumin (C21H2006) as diferuloylmethane (Aggarwal et al., 2003). In 1910, the feruloyl
methane skeleton of curcumin was also ascertained by Lampe (Lampe and Milobedzka,
1913). The molecular formula of curcumin is (C21H2006); the melting point is 183°C. The molecular weight is 368.37 g/mol. It is a yellow-orange powder, which is insoluble in water and
ether but soluble in ethanol, dimethyl sulfoxide, and acetone (Aggarwal et al., 2003).
Curcumin or curcumin-I is naturally available in the root of Curcuma longa, which is grown
commercially and is known as turmeric, a yellow-orange color dye. The main ingredient of
turmeric is curcumin and, in combination with other chemical ingredients, it is known as the
“curcuminoid” (Srinivasan, 1952). Three major types of curcuminoids present in turmeric are
demethoxycurcumin (curcumin-II), bisdemethoxycurcumin (curcumin-III), and another one
is newly discovered cyclocurcumin. The percentage of curcumin, in commercial curcumin, is
curcumin I (77%), curcumin-II (17%), and curcumin III (3%), respectively. The other
names of curcuminoid complex is Indian saffron, yellow ginger, yellow root, kacha haldi,
ukon, or natural yellow 3. Spectrophotometrically, in acetone, curcumin is able to absorb
430 nm (max). Optical density of 1% solution of curcumin is near about 1650 absorbance units.
Curcumin shows a fantastic yellow hue at pH 2.5–7 and red hue at pH >7. The fluorescence of
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curcumin has a broad band in acetonitrile (λmax ¼ 524 nm), ethanol (λmax ¼ 549 nm), or micellar
solution (λmax ¼ 557 nm) (Chignell et al., 1994). Upon irradiation, curcumin is able to produce
singlet oxygen (1O2) (λ > 400 nm) in toluene or acetonitrile (phi ¼ 0.11 for 50 μM curcumin);
moreover, curcumin can also quench 1O2 (kq ¼ 7 106 M1 s1) in acetonitrile. Lately, the activity of 1O2 quenching in curcumin was investigated by Das and Das more specifically (Das
and Das, 2002). Generically, curcumin is superoxide in toluene and ethanol, while it appeases
superoxide ions in acetonitrile (Toniolo et al., 2002). Iwunze and McEwan experimented together with fluorescence and absorptiometric techniques to study the interaction between
curcumin and biological radical stressors like curcumin and peroxynitrite (Iwunze and
McEwan, 2004). While investigating both techniques, workers noticed the asymptotical enhancement of signal up to the equivalency of concentration of peroxynitrite and curcumin.
Nonetheless, a variation in fluorescence wavelength was noticed due to the preliminary oxidation of the hydroxyl group as a consequence of the nitration of the phenoxyl group of
curcumin. Another reaction was conducted for the nitration of curcumin by peroxynitrite
with an association constant of 1.2 106/M s and 3.6 106/M s for the fluorescence and
absorptiometric techniques correspondingly (Iwunze and McEwan, 2004).
Curcumin has been observed to be phototoxic to mammalian cells in presence of oxygen
found in basophilic leukemia cell model of a rat (Dahll et al., 1994). Depending on the environment, curcumin shows a diversity of spectral and photochemical properties as a consequence of multiple or alternate pathways to demonstrate the photodynamic effects.
Photogeneration of singlet oxygen and reduction forms of molecular oxygen of curcumin under various conditions pertinently to cellular environment is a relevant example (Aggarwal
et al., 2003). Tonnesen used aqueous solution to evaluate the kinetics of pH-dependent
curcumin degradation (Tønnesen and Karlsen, 1985). Constant rate against pH eluded the
pKa norm of the acid protons. The graph also depicts the perplexity of curcumin degradation.
The investigators also examined curcumin stability under the exposure to UV/visible radiation (Tønnesen et al., 1986). These researchers recognized the main degradation products,
inquired the reaction mechanisms, and ascertained the half-lives of curcumin in different solvents and in the solid state. Moreover, they also evaluate the photobiological activity of
curcumin by bacterial indicator systems (Tønnesen et al., 1987). Although in less concentrations, curcumin shows phototoxicity for Salmonella typhimurium and Escherichia coli upon irradiation, which indicates effectiveness of curcumin as a photosensitizing drug likely to be
phototherapeutic agent of diseases like cancer and bacterial and viral infections (Aggarwal
et al., 2003).
3.7.2 Biological activities of curcuminoids
A wide range of biological activities of compounds that are classified as curcuminoids have
been reported, including antimicrobial, antiinflammatory, anticancer, neuroprotective, radioprotective, and cardioprotective activities (Amalraj et al., 2017). All three major forms of
curcuminoids have been reported to show significantly decreased urokinase plasminogen activator and anticancer activity by inhibiting collagenase (matrix metalloproteinases) in
in vitro human fibrosarcoma model (Yodkeeree et al., 2009). In a study involving 58 breast
cancer patients, curcumin (20 μM)-mediated apoptosis caused maspin protein upregulation
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and indicated anticancer activity (Prasad et al., 2010). In 2012, Ye et al. (2012) reported that
curcumin (5–20 μM) reversed cis-platin resistance by inducing the human lung adenocarcinoma A549/DDP cell apoptosis via mechanisms involving caspase-3 and hypoxia-inducible
factor 1-alpha (HIF-1α). Bisdemethoxycurcumin (BDMC) (1–80 μM) induced apoptosis in
nonsmall-cell lung cancer (NSCLC) cells and inhibited the viability of NSCLC cells (Xu
et al., 2015). Curcumin (20–200 μg/mL) showed stronger antifungal activity in comparison
to DMC in pathogens of Candida albicans by virtue to methoxy group (Zhang et al., 2012).
Curcumin also showed antinephrotoxic activity in albino rats during histopathological examination by significantly decreasing the inflammation and apoptosis at 200 mg/kg
(Hismiogullari et al., 2015). Curcumin has also been reported in a study exploring the
antiviral activity toward influenza virus in canine kidney cells, where curcumin (30 μM)
interrupted the virus-to-cell attachment and inhibited the propagation of the virus (Chen
et al., 2010). The antioxidant activity of curcumin (0.05 mM) was recorded by 2,2-diphenyl1-picrylhydrazyl (DPPH) method, and it was shown that the reaction required the sequential
proton loss electron transfer (SPLET) mechanism (Galano et al., 2009). Later, the antioxidant
capability of curcumin (200 mg/kg) was correlated with its hepatoprotective, cardioprotective, and antiinflammatory effects in Wistar strain rats (Naik et al., 2011). Furthermore,
the antioxidant activity of curcuminoids was validated in Sprague-Dawley rats as these compounds (150 mg/kg) significantly reduced urinary biomarkers of stress induced by oxidation,
e.g., allanoin, 8-hydroxy-20 -deoxyguanosine, 3-nitrotyrosine, and m-tyrosine (Dall’Acqua
et al., 2016). The neuroprotective ability of curcumin (0.1% of 10 mM) has also been recorded
in a neuroblastoma cell line (SH-SY5Y) in which curcumin inhibited 6-OHDA-induced neurotoxicity ( Jaisin et al., 2011). In a clinical investigation on depressive disorder involving human subjects aged 18–65 given DSM-IV, curcumin (500 mg 2 times/day) was observed to be
more efficient in comparison to placebo for improvement in mood related symptoms associated with the disorder (Lopresti et al., 2014). In two different animal models (rats) of
Alzheimer’s disease, curcuminoids (3–10 mg/kg) and their individual components (CUR,
DMC, and BDMC) showed increased expression of synaptophysin, decreased GFAP levels
in the hippocampus, and maximal rescuing effect (Ahmed et al., 2010; Ahmed and Gilani,
2011). In another Alzheimer’s disease model of murine neuroblastoma (N2A cells), curcumin
was observed to be more active as compared to demethoxy curcumin (DMC) for inhibition of
amyloid-β precursor protein (APP) and Tau IRES activity at 1–40 μM concentrations, furthering the case of curcumin as a therapeutic for neurodegenerative disorders (Villaflores et al.,
2012). Cooney et al. (2016) comprehensively reported the antiinflammatory effects of
curcumin (0.2%, w/w) through multiple molecular pathways, decreased neutrophil migration, higher xenobiotic metabolic activity and barrier remodeling in mice models of inflammation. A recent study involving 118 subjects of type 2 diabetes mellitus showed that
curcuminoids (1 g/day for 12 weeks) reduced total cholesterol in serum with positive effects
on lipids (non-HDL-C and Lp (a) levels) (Panahi et al., 2018).
3.7.3 Current and potential industrial applications of curcuminoids
Owing to nontoxic behavior, curcuminoids containing natural plant extracts are popular
preparations in diseases and their management since ancient times, along with their health
promoting effects as shown in practice in traditional medicine (Rahmani et al., 2018). In the
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field of medicine, considerable amount of research in recent decades has indicated that
curcumin play a role in curing or managing diseases via modulation of various factors, including genes and enzymes that are involved in the processes leading to pathogenesis
(Tiwari et al., 2018; Rahmani et al., 2018). Two curcuma species, namely, Curcuma aromatica
and Curcuma longa are readily available in markets as medicinal herbs as natural antioxidants
containing curcuminoids. In food industry, these are also exploited as natural colorants due to
their characteristic yellow color. Recently, different methods have been used to enhance the
coloring effects of these extracts, including irradiation, which is considered to improve the
properties of Curcuma aromatica for their potential use in cosmetic industrial products and
manufacturing food at industrial scale (Dosoky and Setzer, 2018). There is an increasing concern in public regarding the use of synthetic chemical additives as preserving agents in foods.
Therefore, food industries are actively looking for preservatives that are natural and with no
adverse effects when added to different types of foods. Curcuma longa, a natural food preservative that is consumed mainly in Asia with its rhizome containing amounts of curcuminoids
having antibacterial properties, is considered to increase the shelf life of foods prepared from
chicken and potato (De and De, 2019; Gul and Bakht, 2015). In 2011, Lee and Choung (2011)
had already reported that curcuminoids analysis in manufactured food could be potential
tool for quality control as they studied commercial foods in Korean markets by employing
HPLC for determining three principle curcuminoids. Furthermore, curcuminoids have been
reported to have potential benefits in cosmeceuticals owing to their antioxidant and skinlightening properties as they inhibit hyaluronidase, elastase, and collagenase in vitro
(Griffiths et al., 2016). Moreover, curcumin gel when applied to skin showed improved appearance in photodamaged condition, including solar elastoses, actinic keratosis, and pigmentary changes (Heng, 2010). Curcumin is also being explored as a hair-coloring agent,
being environment friendly in nature (Boga et al., 2013).
Other industries in which curcuminoids have potential for use are soap, perfume, and cosmetic industries (Sasikumar, 2005). Apart from studied properties of curcumins and other
curcuminoids, turmeric extracts through which curcuminoids are extracted mainly have
huge cultural relevance in dermatology and Indian skin care as well (Gopinath and
Karthikeyan, 2018). Since curcuminoids show excellent biological properties, in future, attention on clinical applications of these compounds in various diseases especially cardiovascular, neurodegenerative, cancer, and diabetes would gain focus as a lot of research has already
clarified the value of these compounds being diet-derived agents. Although curcuminoids
show relatively lower systemic bioavailability, henceforth, improvement in this area is also
gaining attention from scientific experts. Different modern approaches including adjuvant
drug-delivery systems involving structural modifications have been proposed for enhancement of potentials of these compounds (Zangui et al., 2019).
3.7.4 Possible interactions of curcuminoids
Curcuminoids, lipid-soluble plant chemicals, are mostly present in roots of different
plants. Turmeric, which is a complex of curcumin and demethoxycurcumin, is widely used
for various purposes. To get the potential desirable effectivity, these compounds sometimes
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151
are used in drug and routine supplements (Aggarwal and Sung, 2009). They are often used as
bioenhancers in order to increase the bioavailability of drug, thus increasing efficacy and bioavailability of that specific drug (Anand et al., 2007). Curcumin interactions with drugs, yogurt, and different cells provide the basis of its beneficial effects in treatment of different
chronic as well as infectious disorders. In ancient Chinese medicine, curcumin had an important role in treatment of respiratory and digestive disorders (Araujo and Leon, 2001). Elderly
people and those suffering from any disorder often go for intake of both medicinal and allopathic medicines. This kind of interaction often leads to the formation of complex interactions
that may be fruitful or sometime hazardous for the health of the patient (Kocher et al., 2015).
Interaction of curcuminoids with drugs was known to increase the efficacy of drugs inside the
body after intake. Different kinds of disorders require a specific drug intake in appropriate
dosage to help coping with it. Many scientific studies have provided evident proof of positive
impacts of curcumin in drug efficacy and bioavailability. Curcumin also plays significant role
in increasing the residence time of drugs like cicloprolol, thus making it more effective. This
new horizon of research and experimentation is opening frontiers in medicinal herbs utilization for the treatment of disorders (Kocher et al., 2015). In vitro and in vivo studies have
shown that curcumin leaves great impact on transporters and enzymes, which play their role
in metabolization of foreign compounds and alter the pharmacokinetics of drugs. Another
study directed to study the effects of curcumin on docetaxel have also clearly reflected the
effects on the drug intake mechanism and pharmacokinetics of drug (Sun et al., 2016).
Curcumin also plays significant role in tumor suppression and arrest of uncontrolled proliferative cell division. Interaction of curcumin with antitumor drugs like carboplatin and
etoposide plays synergic role in reduction of cell proliferation in retinoblastoma
(Sreenivasan and Krishnakumar, 2015). Curcumin also has been found to lower the blood glucose level in obese and hyperglycemic patients. Interaction of gliclazide with dosages of
curcumin had great impact on reduction of blood glucose level, thus increasing the efficacy
of drug in its action (Vatsavai and Kilari, 2016). Drugs are somehow positively influenced by
the interaction of curcuminoids.
Curcuminoids, in addition to drugs, also interact with various molecules and biological
components in complex manner. This interaction helps it in carrying out its specific biological
activities. They interact with membranes, transporters, and ion channels to enhance cellular
activity of an organism. Studies reflect a synergic relation between ascorbic acid and
curcumin in dealing with cancerous cells. This interaction along with ascorbic acid helps
in restricting the growth of leukemia and colon cancer (Ooko et al., 2017). The essential role
of curcumin in cancer treatment outlines the importance of medicinal plants in our medical
sector. Interaction of curcumin with human serum albumin is also reported where it bonded
with the sites present on albumin, thus causing alteration in conformation of the protein.
Spectroscopic analysis reveals the binding mechanism of curcumin to human serum albumin.
It binds through both hydrophilic and hydrophobic interactions. (Mandeville et al., 2009).
Curcumin also show strong interaction with phosphatidylcholine extracted from egg. It occupies hydrophobic region and can form strong bonds with metal ions. Curcumin is also capable of inhibiting membrane-bound enzymes such as kinase and phospholipase (Began
et al., 1999). Interaction with glutathione leads to the formation of curcumin glutathione conjugate. This conjugate is formed by electrophilic and neutrophilic interactions. These complex
interactions helped in assimilation of medicine in intestinal region, thus increasing the effect
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of intake (Awasthi et al., 2000). Omega 3 fatty acids complex with curcumin plays important
role in anticancerous activity in pancreatic cancer. This interaction leads to apoptosis of pancreatic cancerous cells, thus reducing the existence of tumor cells (Fiala, 2015). Curcuminoids
show a variety of interactions with biomolecules as well as enzymes. These interactions often
increase the anticancerous activity of curcumin inside the body. Research in study of its interaction is imperative to understand the positive impacts that curcumin does in coping with
different disorders.
3.7.5 Techniques of extraction, purification, and fractionation of curcuminoids
Extraction of curcuminoids is mostly done through conventional and inexpensive methods
that often lead to minimal extraction. Starting by extraction, followed by purification and fractionation are important steps in obtaining pure and quantified amount of curcuminoids
(Yulianto et al., 2018). Curcumin, present in Curcuma longa L., is extracted through traditional
methods and often results in impure and lower quantity. Extraction of curcumin is very important as this compound has been used in countering the medical complications and abnormalities (Osorio-Tobón et al., 2013).
Curcuminoids extractions techniques have evolved from traditional to modern over a period of time. Advancements in modern technology have improved the extracted
curcuminoids qualitatively as well as quantitatively. Most conventional method for the extraction involves the Soxhlet Apparatus. It is time consuming as well as laborious and also
results in very small amount of desired product as compared to raw material. This technique
requires a large quantity of dried roots extracted from turmeric plant and is subjected to
heating in a solution for over 12 h. After heating, the extract is further cooled down and then
concentrated using rotary evaporator (Prasad et al., 2014). A comparatively novel technique
for extraction of curcumin is ultrasonic assisted extraction (UAE) that involves the indirect
sonification of raw material in ultrasound bath. This technique yields three times more extract
as compared to traditional method. All techniques involve use of ethanol as solvent for initial
treatment of plant material (Şahin, 2018). Extraction of higher quantity of curcuminoids is a
priority of researchers in order to carry out sustainable experimentation. This need has always helped in modification of techniques to increase the extract and minimize the time required to perform the procedure. Supercritical fluid extraction (SFE) technique involves the
use of modified carbon dioxide, which yields in 90% recovery of extracts. It involves easy separation and extraction of curcuminoid by coupling with supercritical fluid chromatography.
Both these coupled techniques result in an improved and better extract as end product
(Sanagi et al., 1993). This method is mostly suitable for highly volatile compounds that involve difficult process for their extraction. Another novel approach for extraction of phytochemicals is microwave-assisted extraction, which involves the internal cellular heating of
material, thus rupturing the cell wall of plants, leading to easier extraction and improved
quantity (Dandekar and Gaikar, 2002). Enzyme-linked ionic liquid extraction utilizes a developed carbamate ionic liquid along with amylase and amyloglucosidase enzyme to extract
curcumin from turmeric roots up to the purity of 96%. It also reduces the time requirements,
thus leading to a rapid extraction process (Sahne et al., 2017).
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153
Followed by extraction, curcuminoids are purified using different methods in order to
obtain the refined phytochemicals. Extract is often contaminated by impure material that
is not needed and plays no significant role in assisting the phytochemical activity. These
impurities may affect the normal bioactivity, thus resulting in reduction of quality of extracts. Countercurrent chromatography (CCC) is mostly used for the purification of
curcuminoids. Turmeric is composed of three curcuminoids that differ in their properties
of size and chemistry. Curcumin, desmethoxycurcumin, and bisdemethoxycurcumin are
main constituents of turmeric powder. High-speed countercurrent chromatography
(HSCC) uses a simple two-phase solvent system making use of chloroform, methanol, or
water (Pozharitskaya et al., 2008). Curcuminoids are separated on the film on the basis
of different chemical properties. This technique is time efficient and more specific, requiring
minimal solvent consumption. Purification through HSCC is simpler, reliable, and automatic as compared to column chromatography (Inoue et al., 2008). Industrial-scale purification requires fast and inexpensive method in order to purify massive amount of curcumin
from extracts. Industries use cooling crystallization, which enables them to purify on larger
scale at the expense of reduced yield. Powder is mixed with solvent and then heated for
some time in order to facilitate formation of crystals. These crystals are further purified
(Ukrainczyk et al., 2016). Magnetic purification is a novel technique that makes use of
maghemite nanoparticles to purify the compounds showing inclination to iron chelation.
This technique utilizes specific nanoparticles known as surface-active maghemite
nanoparticles (SAMN). These particles purify curcuminoids by filtering other unwanted
compounds through magnetic property (Magro et al., 2015).
Curcuminoids extracts from plants consist of different constituents in varying percentage. These compounds have different properties like mass, size, charge, and chemistry
(Revathy and Elumalai, 2016). They can be fractionated by use of separation techniques like
chromatography with required modification in the procedure. Thin-layer chromatography
(TLC) fractionates the curcuminoids on the basis of molecular mass of the different constituents. Curcumin, dimethoxy curcumin, and bisdemethoxycurcumin curcumin have molecular mass of 368, 338, and 308, respectively. Use of methanol as solvent for mixing of
samples prepares for running on chromatographic apparatus (Gupta et al., 1999). Silicagel-coated plates are used for running mobile phase. In 20 min, the spots appear on plates,
which are dried and further quantified through another procedure. Column chromatography (CC) is also used for the fractionation of curcuminoids that involves mixing of sample
with silica gel and then loading onto the column composed of silica gel mesh. Chloroform,
ethanol, and glacial acetic acid are used as developing solvent system. Yellow spots appear
on the mesh, which is filtered to obtain crystals (Pushpakumari et al., 2014). Another horizon in separation of curcuminoids encompasses the use of supercritical fluid chromatography (SFC). This technique involves elution with supercritical fluid CO2 followed by
separation on column film. Three curcuminoids separated on film are further quantified
(Song et al., 2015). This method is advantageous in terms of solvent consumption and easy
solvent removal. Two-dimensional flash liquid chromatography involves a novel technique
for efficient fractionation of curcuminoids followed by nuclear magnetic resonance (NMR)
for quantitative analysis that ensures the fractionation of pure compounds ( Jayaprakasha
et al., 2013).
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3. Analysis of polyphenolics
3.7.6 Techniques for identification and quantification of curcuminoids
Identification of curcuminoids is done by various available methods depending upon the
scale and desired purpose. Plant extracts from roots and other parts are firstly extracted and
purified. Purification is followed by identification and characterization of the sample. This
sample is tested for the confirmation of the presence of desired compound. Other unwanted
compounds may give false-positive result; thus, an effective methodology is imperative for
precise and accurate characterization of phytochemicals like curcuminoids. Turmeric powder has three different compounds varying in content. Quantification determines the
amount of compound present in a given sample. This amount represents the percentage
of target compound in the sample. Various techniques have evolved in the last decades
in order to ensure the efficient and specific analysis of the sample. For laboratory analysis,
one of the common techniques for identification of curcuminoids is ultra-high-performance
liquid chromatography (UHPLC). This technique is fast, specific, and reliable. UHPLC is
mostly coupled with mass spectrometry (MS) to ensure the identification and characterization of curcuminoids present in sample (Avula et al., 2012). The coupled technique improves the preciseness and results in reduction of false positives. High-pressure liquid
chromatography (HPLC) coupled with pressurized liquid extraction (PLE) help in the precise quantification and identification of curcuminoids. They are identified on the basis of
their content in a given sample, which is determined through pressurized liquid extraction
(Chao et al., 2018). High-performance thin-layer chromatography (HPTLC) is an accurate
and precise technique for rapid identification of curcuminoids. Silica gel-based plates act
as stationary phase while acetate/methanol as mobile phase. Modern laboratory techniques
are multipurpose as they are performed for simultaneous identification, purification, and
quantification of curcuminoids. Opting a technique for a single purpose often proves to
be expensive though it results in more accurate outcomes. Quantification of curcuminoids
is important in understanding the content of phytochemicals in plants and other sources.
The most conventional technique used for quantification involves spectroscopic analysis
of the sample. Spectroscopy can be modified by incorporation of near infrared, infrared,
or visible light depending upon the target sample. Near-infrared (NIR) spectroscopy was
used for quantification of curcuminoids in one of the studies, which showed positive results
and sample was quantified based on NIR spectra (Tanaka et al., 2008). Another study
reflected the use of HPLC and UV visible spectroscopy for quantification of curcuminoids.
Both techniques were specific in curcuminoids determination and time saving. Calibrations
of experiments were carefully designed in order to ensure the accurate results with minimal
error. The mobile phase was prepared from ammonium acetate with orthophosphoric acid
solution. Comparison of both methods resulted in very specific results, which prove that
both HPLC and UV visible spectroscopy can be used for precise determination of
curcuminoids in a sample (Kadam et al., 2018). Quantification of curcuminoids from other
samples than extracts can also be done through liquid chromatography coupled with mass
spectrometry (LC-MS). A study quantified the curcuminoids in equine plasma using respective technique. Metabolites produced after intake of curcumin were also determined
using the same technique (Liu et al., 2018).
II. Phenolics
3.7.8 Effects of food processing on curcuminoids
155
3.7.7 Levels of curcuminoids founds in plants/food-based plants
Curcuminoids are phytochemicals most associated to Curcuma genus under the family of
Zingiberaceae also known as ginger family. This genus spans about 100 species present mostly
in Indian, Australian, and African regions. Chinese ancient medicinal treatment was also
based upon curcuminoids as treatment of numerous medical problems. Three most common
curcuminoids are present in rhizome of Curcuma longa. Desmethoxycurcumin, bisdemethoxycurcumin, and curcumin are prominently classified as curcuminoids as they are present in
most plants as compared to other minute compounds (Rodrigues et al., 2015). Turmeric or
Curcuma longa is the most widely used medicinal herb because of its numerous biological activities and its powder form contains 3.14% of curcomonoids by weight. Turmeric is a yellow
pigment powder extracted from the plant and is used in cooking, medicines, coloring, and
oral medicines. Among curcuminoids, curcumin is mostly focused for its impressive activities. Other two are also present in most plants but they are not that prevalent as compared to
curcumin. Curmumin and curcuminoids are abundant in other plants such as Curcuma aromatic and Curcuma phaeocalis contains 0.11% and 0.89%, respectively. In addition to Curcuma
longa, curcuminoids are also present in another species named Curcuma xantohorrhiza. A study
determined the presence of all three curcuminoids in C. xanthorrhiza (Lechtenberg et al., 2004).
Curcuminoids were also determined in Curcuma mangga, which showed presence of nine different compounds in different percentage. Curcuma mangga contain DMC and BDMC of
450.53 and 329.45 μg/mL levels, respectively. The study made use of different analytical techniques to determine the presence of different molecules in rhizome of C. mangga. In addition
to three main curcuminoids, presence of calcatrin, zerumin, and scopoletin was determined
using spectroscopic and chromatographic techniques. Though little is known about the biological activities of other compounds, still it is used as vegetable in some areas of Thailand and
Indonesia (Liu and Nair, 2011). Ginger family is rich in different curcuminoids, both discovered and undiscovered. Unexplored curcuminoids are yet to be revealed and future research
is imperative for understanding the medicinal properties of plants. Curcuma zedoaria has also
been reported to have different curcuminoids, and this species of ginger has been widely used
for the treatment of cervical cancer in China. All three curcuminoids are present in rhizome of
C. xedoaria and mostly used for treatment of various medical problems (Syu et al., 1998).
Curcuminoids have also been quantified in Ziniber zerumbut in a study involving quantification by HPLC. It has been widely used as medicine and health supplement. The rhizome of Z.
zerumbut, though having different types of phytochemicals, had small amount of
curcuminoids (Matthes et al., 1980).
3.7.8 Effects of food processing on curcuminoids
The scientific disciplines concerning functional foods and nutraceuticals allowing the
study of healthy living and eating have shown many characterization studies involving turmeric rhizome, which is considered as a raw material for curcuminoids (Shi et al., 2016).
The effects of extraction procedures on the quantity and quality of oleoresins have been
debated; henceforth, curcuminoids are reported to play important role in quality of
II. Phenolics
156
3. Analysis of polyphenolics
turmeric-containing foods being phenolic secondary metabolites and affect the organoleptic
characteristics of the food and influence the taste, flavor, and aroma. The postharvest processing of the extracts can influence different enzymes, including phenylalanine ammonia-lyase
for biosynthesis and stability of curcuminoids (Tomás-Barberán and Espı́n, 2001). The conventional postharvesting technologies include cultural farming practice, washing for slicing
of freshly harvested rhizomes, sun drying, dried milled rhizomes by steam drying and
milling, grinding, boiling the rhizome in alkaline media prior to dehydration (Bambirra
et al., 2002; Govindarajan and Stahl, 1980; Green et al., 2008). Lee et al. (2010) reported that
stability of curcuminoids decreases with increase in pH and temperature during storage.
Residual curcuminoids levels were also reported to decrease in autoclave conditions of
temperature by 80%–90% with only 10% loss in powdered form. Free-radical-scavenging
ability of curcuminoids also reduced by 33.4% and 10.3% as shown by 2,20 -azinobis(3ethylbenzothialozinesulfonate (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical,
respectively (Lee et al., 2010). HPLC analysis revealed that heat processing of turmeric causes
curcumin loss up to 27%–53%, by showing maximum loss in pressure cooking conditions for
10 min (Suresh et al., 2007). Furthermore, new techniques for processing these compounds
have been developed for improving the overall yield. Ternary solid dispersion technique
has been reported to enhance the nutraceutical and dyeing properties of these compounds
as the total curcuminoid and curcumin content along with their antioxidant property were
positively affected by spray-drying process (Martins et al., 2013). Total phenolics content
of turmeric extracts have reported to increase after drying as does the polyphenol oxidase
enzyme. However, sun-dried samples of rhizome showed reduced curcuminoids levels
(Prathapan et al., 2009). Moreover, it has been shown that combined vacuum treatment
and sonication produces highest infusions of curcuminoids along with highest total color
difference when compared with other treatments. Subsequently, dehydration also resulted
in products with highest retention of these compounds (Bellary and Rastogi, 2014).
3.7.9 Trends and concluding remarks
Advanced methodologies have been routinely utilized for isolation by HPLC and massspectrometry techniques followed by characterization techniques, including power X-ray diffraction, thermal gravimetric analysis (TGA), and differential scanning calorimetry
(Heffernan et al., 2017). Recently, novel technologies have been used for robust and efficient
separation of curcuminoids, including a fused-core technology in HPLC column from turmeric extracts (Osorio-Tobón et al., 2016). Since lower bioavailability of curcuminoids is a major concern for their usability and exploitation of their various biological properties, different
formulations have been proposed, including a food-grade formulation of curcumin with
fenugreek dietary fiber as curcumagalactomannosides (CGM), which supplied more than
70% of absorbed curcuminoids in unconjugated form (Kumar et al., 2016). For enhancing
the solubility in aqueous media, encapsulation methods have been described recently, which
include the usage of polyethylene glycol (PEG) for the formation of encapsulated particles of
curcuminoids (Perko et al., 2015). It has also been shown recently that isolated curcumin or
combined curcuminoids decrease the fast blood glucose levels in individuals with
II. Phenolics
References
157
dysglycemia differentially from nondiabetic individuals hinting their potential future role as
adjuvants in dysglycemia therapeutics (de Melo et al., 2018). In silico methods have been
employed to construct the analogs of curcuminoids to efficiently utilize the antimicrobial effects and to explore the targeted effects as antimalarial agents (Din et al., 2017; Viira et al.,
2016). There have been many food matrix design strategies developed for enhancing the bioavailability of curcuminoids, including colloidal delivery and excipient systems
(McClements, 2015). Various nanoparticle-based approaches have been attempted to develop
delivery systems for improvement of bioavailability of curcuminoids (Mehanny et al., 2016).
Other novel approaches employed with curcumins include solid dispersion, microemulsion,
nanogels, micelles, dendrimers, and liposomes. Liposomes have been observed to be effective
and extensively studied as liposomal curcumin indicated both proapoptotic and growth inhibitory effects on investigated cancer cells (Feng et al., 2017). More than 120 clinical trials
involving curcuminoids for different diseases have shown likely false activity of curcumin.
Despite constituting various beneficial properties, curcumin was recently described as being IMPS (invalid metabolic panaceas) and PAINS (pan-assay interference compounds) candidate. Until 2017, no double-blinded placebo-controlled clinical trial was successful
involving curcumin. Various reports have indicated curcumin as an improbable lead due
to being nonbioavailable, unstable, and reactive compound (Nelson et al., 2017). Therefore,
it is imperative to consider these aspects of these compounds prior to establishing them in
circumstances or research questions regarding animal models and human subjects.
Acknowledgment
This article is the outcome of an in-house financially nonsupported study.
Author contributions
All authors have directly participated in the planning or drafting of the manuscript and read and approved the final
version.
Conflict of interest
The authors declare no conflict of interest.
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S U B C H A P T E R
3.8
Coumarin
Muhammad Shafiqa, Fazlullah Khanb,e, Muhammad Ajmal Shahc, Kamal Niazd
a
Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Nanjing
Agricultural University, Nanjing, PR China bInternational Campus, Tehran University of Medical
Sciences (IC-TUMS), Tehran, Iran cDepartment of Pharmacognosy, Faculty of Pharmaceutical
Sciences, Government College University, Faisalabad, Pakistan dDepartment of Pharmacology and
Toxicology, Faculty of Bio-Sciences, Cholistan University of Veterinary and Animal Sciences
(CUVAS), Bahawalpur, Pakistan eDepartment of Toxicology and Pharmacology, Faculty of
Pharmacy, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical
Sciences, Tehran, Iran
3.8.1 Phytochemistry of the coumarin
Coumarin (1,2-benzopyrone; or chromen-2-one) belongs to a class of compounds present
abundantly in nature (Murray et al., 1982). The word coumarin has been derived from a
French word “coumarou” means “tonka bean.” Coumarin is a heterocyclic compound, which
was first isolated by Vogel from tonka beans, a vernacular French name in 1820 (Dipteryx
odoranta Wild; Fabaceae Family) (Keri et al., 2015). Since then, structural characterization, isolation, synthesis, and its biological activities of coumarin were reported from plants, fungi,
and bacteria (Asif, 2015; Borges et al., 2005).
Coumarin is naturally available phytochemicals, which are found in plants and is characterized by a benzopyrone family of compounds, in which a benzene ring and a pyrone ring
are attached with each other (Ojala, 2001). This family has been divided into three classes,
benzo-γ-pyrones, benzo-α-pyrones, and the principal class of coumarin that mostly consist
of flavonoids. Such combination of α-pyrone and benzene helps in conjugation of π-π stacking
interactions, which make it extensive effectively as medicinal drugs. It holds synthetic accessibility and variability making fragile interactions with excess of enzymes and receptors in the
body. Interactions such as van der Waals, hydrogen bonding, electrostatic interactions, π-π,
hydrophobic and metal coordination with dynamic sites in the body are responsible for its
huge versatility and efficacies in biological activity ( Jameel et al., 2016). Coumarin 1 and
its derivatives have become remedy, such as the anticoagulants warfarin, phenoprocoumon,
acenocourmarin, which act as vitamin K antagonists. Choleretics armillarisin A,
hymecromone (umbelliferone), and novobiocin, which is a potent inhibitor of bacterial
DNA gyrase (Stefanachi et al., 2018). More than 300 coumarin derivatives have been reported
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3.8.2 Biological activities of coumarin
163
from nature, particularly from plant origin. Coumarin is extensively known for its biological,
biochemical, and therapeutic applications (Borges et al., 2005; Liu et al., 2012). Physically, coumarin is a white crystalline and volatile compound. It smells like custard vanilla and its melting point is 341–344 K (Penta, 2015). It is well soluble in organic solvents (ethyl alcohol, diethyl
ether, and chloroform), and in fats and oils as well.
3.8.1.1 Classification of coumarin
There are different ways to classify coumarin according to their structure, existence, and
synthesis; therefore, there are several classes of coumarin. Fig. 3.8.1 shows the classification of
coumarin based on their chemical structure.
1. Simple coumarin
2. Furanocoumarin isomers (psoralen and angelicin)
3. Pyrone-substituted coumarin
4. Pyranocoumarin
3.8.2 Biological activities of coumarin
Coumarin has numerous pharmacological activities, which depend on their chemical
structures and on the replacement pattern. Some of these pharmacological activities coumarin
include, antiinflammatory, anticoagulant, antibacterial, antiviral, antifungal, antihypertensive, antitubercular, anticancer, anticonvulsant, neuroprotective and as phytoalexins
(Sandhu et al., 2014; Stefanachi et al., 2018; Venugopala et al., 2013). The present chapter will
highlight the recent development in coumarin scaffolds for the drugs discovery against various biological activities, which are discussed now.
1. Antiinflammatory activity of coumarin
Couramins shows antiinflammatory effect in the treatment of edema (Aoyama et al.,
1992). It induces phagocytosis, and through proteolysis couramin eliminates protein and
edema fluid from the wounded tissue (Garcia-Argaez et al., 2000; Kontogiorgis and
Hadjipavlou-Litina, 2003). Another derivative, imperatorin, also exhibits antiinflammatory activity in lipopolysaccharide (LPS)-stimulated mouse macrophage both in vitro and
in vivo model. It blocks the expression of certain proteins, which stimulate nitric oxide
synthase and cyclooxygenase-2 in LPS-stimulated mouse macrophages (Huang et al.,
2012; Iranshahi et al., 2015). Another compound, esculetin, which was derived from
Cichorium intybus and Bougainvillea spectabilis also induce antiinflammatory effect in rat’s
colitis, which is stimulated by trinitrobenzene sulfonic acid (Evans, 2009; Kwon et al.,
2011). Esculetin inhibits the cyclooxygenase and lipoxygenase enzymes (Fylaktakidou
et al., 2004).
2. Anticoagulant activity of coumarin
A coumarin derivative, dicoumarol, which was found in sweet clover, has showed anticoagulant property (Venugopala et al., 2013). Coumarin is vitamin K antagonists that
induce their anticoagulant activity by interfering with the cyclic interconversion of vitamin K and its epoxide (Hirsh et al., 2001). This conversion cycle of vitamin K trigger
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O
O
O
O
Psoralen
Coumarins
O
O
O
O
O
O
Angelicin
Pyrone
O
O
O
O
Pyranocoumarin
FIG. 3.8.1
Chemical structure of coumarin, furanocoumarin isomers (psoralen and angelicin), pyrone-substituted
coumarin, and pyranocoumarin.
hepatic carboxylated and decarboxylated proteins production, which have less
procoagulant property (Venugopala et al., 2013).
3. Antibacterial activity of coumarin
Coumarin itself has very low antibacterial activity, while coumarin products, which
have long hydrocarbon substitutions such as ammoresinol and ostruthin, are considered
to be the most effective drug against Gram-positive bacteria such as Bacillus megaterium,
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165
Staphylococcus aureus, and Micrococcus lysodeikticus (Venugopala et al., 2013). Some of the
natural origin coumarin is isolated from plants and some from microorganisms. The coumarin derivative novobiocin, which was isolated from fungal metabolites, has showed
broad-spectrum antibacterial activity against Gram-positive microbes such as Staphylococcus aureus, Streptomyces pneumoniae, Streptomyces pyogenes and against Gram-negative bacteria such as Haemophillus, Pasteurella, and Neisseria meningitides (Venugopala et al., 2013).
4. Antifungal activity of coumarin
Coumarin bioactive derivative osthole is isolated from various plants such as Angelica
pubescens, Peucedanum ostruthium, and Cnidium monnieri. It shows extensive spectrum of
antifungal effects against certain important plant pathogens, i.e., Rhizoctonia solani, Botrytis Cinerea, Phytophtora capsica, and Fusarium graminearum (Wang et al., 2009). Previous
studies have tested many coumarin derivatives for antifungal property; among them,
the most effective ones are imperatorin, psoralen, and ostruthin (Bourgaud et al., 2006;
Iranshahi et al., 2015; Rosselli et al., 2009).
5. Antiviral activity of coumarin
Coumarin (benzo-a-pyrone) is considered to have a privileged core structure for
scheming novel agents, which show high affinity and specificity to diverse molecular targets for antiviral agents (Penta, 2015). Numerous natural products have been defined as
anti-HIV agents, and coumarin is among one of them. Inophyllum-A, inophyllum-B,
inophyllum-C, inophyllum-E, inophyllum-P, inophyllum-G1, and inophyllum-G2—all
these HIV inhibitory coumarin products—were separated from giant African snail,
Achatina fulica. Both inophyllum-B and P inhibited HIV reverse transcriptase activity
and both were found active against HIV-1 in cell culture (Venugopala et al., 2013).
6. Anticancer activity of coumarin
There are several phytochemicals, coumarin among them, which have attracted considerable importance in the last few years. Coumarin has not only been used to treat cancer, but they also have the potential to reduce the side effects, which are concerned with
radiotherapy (Sandhu et al., 2014). Studies have showed that certain coumarin products
like imperatorin and osthole have exhibited anticancer effects. Esculetin also have
showed antitumor activities during N-methyl-D-aspartate toxicity in culture neurons
(Sandhu et al., 2014; Yang et al., 2010; Yun et al., 2011). Coumarin, which is present in
cassia leaf oil, also has cytotoxic activity (Choi et al., 2001).
7. Antihypertensive activity of coumarin
Dihydromammea is a coumarin found in West Africa, separated first time from the
seeds of the tree Mammea Africana (Basile et al., 2009). It induced hypertensive effect in
a study carried out with rats (Nguelefack-Mbuyo et al., 2008).
8. Antitubercular activity of coumarin
Many plants have umbelliferone, which is obtained by the distillation of resins. Certain
compounds, i.e., umbelliferone, psoralen, phelladenol, and scopoletin, were separated
from the entire plants of Fatous pilosa. Among them, umbelliferone and scopoletin are
found to be most the active compounds against Mycobacterium tuberculosis (Cardoso
et al., 2011; Kawate et al., 2013; Keri et al., 2015).
9. Anticonvulsant activity of coumarin
Imperatorin have showed anticonvulsant effect in mice. Osthole also exhibited anticonvulsant effect in mice, but the ED50 values for both imperatorin and osthole are different in range (Amin et al., 2008; Venugopala et al., 2013).
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10. Neuroprotective activity of coumarin
Previously coumarin has been considered as essential and medicinally beneficial compound; however, its importance is no less in several neurodegenerative diseases (NDs)
such as Alzheimer’s, Parkinson’s, and Huntington’s diseases (Epifano et al., 2008).
Esculetin exhibited neuroprotective effects on cerebral part of the brain in a mice model
(Wang et al., 2012).
11. Coumarin as phytoalexins
Phytoalexins are coumarin derivatives, which have oxygenated property and are produced by plants in consequence to damage, i.e., fungal infection, chemical or physical
damage. The most common activity of phytoallexins is to reduce or kill the invading
agents such as bacteria, viruses, and insects (Metraux and Boller, 1986). Ayapin is one
of them, which was first time isolated from Eupatorium ayapana (Asteraceae). Afterward
it was separated from several other plants such as Artimisia apiacea, Helianthus annus,
Pterocaulon virgatum, and Pterocaulon polystachyum (Venugopala et al., 2013).
3.8.3 Current and potential industrial applications of coumarin
Coumarin has a broad variety of applications in industry, mostly due to its strong fragrant
odor. It is notably found in high levels in plant origin foods, occurring in fruits, seeds, roots,
stem, bark, leaves, and branches of a wide variety of plants, including Tonka bean, Lavender,
yellow-sweet-clover, bison grass, and woodruff. In food industry, it is used as a flavoring and
additive agent. It is used as fixative and enhancer in perfumes, and essential oils. It is a potential lavender agent and used in soaps, toothpastes, and hair gel preparations; in tobacco
products as a flavor and odor stabilizer; also used in paints and rubbers. It is an odor masker
and is used in electroplating to decrease the porosity and increase the fluorescence of various
deposits, such as nickel (Egan et al., 1990).
Coumarin is a group of several natural and synthetic compounds, but only few are of economic importance. These derivatives include 3,4-dihydrocoumarin and 6-methylcoumarin,
which are used as flavor-enhancing agents, which are commonly used in the perfume industry, and 7-hydroxycoumarin, which is used in sunscreens and fluorescent brighteners (Asif,
2015). 7-Hydroxycoumarin derivatives are also used as a substrate for florigenic enzyme. 7aminio-4-methylumbelliferone and other derivatives are used as laser dyes. 4Hydroxycoumarin is an important derivative, which is found in ruined hay and it is a precursor of warfarin and dicoumarol, both of which are antagonists of vitamin K (Egan
et al., 1990; Ito et al., 2004).
3.8.4 Possible interactions of coumarin
Coumarin is a chemical compound available in different plants. Coumarin is well known
due to its numerous pharmacological activities, such as: antibacterial, antifungal, anticancer,
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3.8.5 Techniques of extraction, purification, and fractionation of coumarin
167
antiinflammatory, and neuroprotective activities (Venugopala et al., 2013). But if it is used in
high dosage (higher than 0.1 mg/kg), it may have toxicological effects (hepatotoxic). The use
of coumarin as food additive was banned in 1954 by the FDA on reports of liver toxicity in
rats. Due to toxic effect in liver of humans, the European Commission limited coumarin from
nature origin as a direct food additive to 2 mg/kg in food per day, although side effects of
coumarin in humans are rare, and only related with clinical doses (Lachenmeier et al.,
2011; Lungarini et al., 2008). In October 2004, The European Food Safety Authority (EFSA)
reviewed coumarin to make an acceptable daily intake in foods. EFSA cleared coumarin that
it is not a genotoxic, and they recommended a tolerable daily intake of 0–0.1 mg/kg body
weight/day (Lachenmeier et al., 2011).
Coumarin has showed antiplatelet activity and due to its antioxidant properties, it may be
a promising medicine in future for use in combination with the current therapeutic agents
(Zaragozá et al., 2016). A recent review showed the use of natural and synthetic coumarin
as COX inhibitors (Revankar et al., 2017). Coumarin has extensive iron-chelating activity,
and the use of iron-chelating coumarin in high acidic conditions may be harmful in contrast
to neutral conditions (Mladěnka et al., 2010).
The following drugs may show high response to coumarin derivatives:
Alcohol (acute intoxication), aminosalicylic acid, allopurinol, amiodarone, anabolic
steroids, chloramphenicol, chloral hydrate, cimetidine, co-trimoxazole, clofibrate,
dextrothyroxine, danazol, trimoxazole, erythromycin, ethacrynic acid, glucagon,
fenoprofen calcium, ibuprofen, indomethacin, Influenza virus vaccine, mefenamic acid,
isoniazid, meclofenamate, methylthiouracil, miconazole, metronidazole, malidixic acid,
neomycin, phenylbutazone, propoxyphene, quinidine, sulfonamides, tetracyclines,
thyroid drugs, antidepressants, and vitamin E.
The following drugs may reduce the response to coumarin derivatives:
Alcohol (chronic alcoholism), barbiturates, corticosteroids, corticotropin, carbamazepine,
glutethimide, methaqualone, mercaptopurine, oral contraceptives containing estrogen,
rifampin, vitamin K, among others.
3.8.5 Techniques of extraction, purification, and fractionation of coumarin
The coumarin is commonly extracted from plants by extraction with some solvents such
as benzene, chloroform, methanol, ethanol, and some petroleum ethers or their mixture
(Lozhkin and Sakanyan, 2006). The most common extraction of coumarin is attained with
ethanol and its aqueous solutions. The solid extract is achieved when the evaporation of
extractant is completed; this extract is further treated with chloroform and petroleum ether
or diethyl ether for purification. It gives good yield of furocoumarin in crystalline form, if it
is treated with petroleum ether. For further purification of the extracts, boiled water, activated charcoal, ethanol-chloroform (3:97 mixtures) or ethyl-acetate, and butanol are
required.
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3. Analysis of polyphenolics
A method of saponification is used for further purification of coumarin from the supplementary substances. This technique depends upon on the capability of the lactone pyronering, which can be broken due to alkalis and as a result coumarinates are produced. But this
method has some limitations due to isomerization and dehydration of some hydroxylcoumarin and the production of some secondary products (Lozhkin and Sakanyan, 2006).
Successive procedures are usually designed for the isolation of total coumarin and then the
separation of single derivatives. Previous studies worked on crystallization, fractional distillation, and sublimation in high vacuum. But several coumarin derivatives show good solubility in organic solvents, even if they are recrystallized several times, they did not yield
significant outcomes. Due to this intention, successive development in coumarin chemistry
directed to the progress of several chromatographic methods, which are free of limitations
from those which were found in the previous techniques (Lozhkin and Sakanyan, 2006). In
the column chromatography, different sorbents and solvent systems were used. In particular,
the separation of hydroxyl- and alkoxy-coumarin substitute at the benzene cycle can be
performed via silica gel column consecutively eluted with hexane and hexane chloroform
mixture and chloroform-methanol with the ratio of (9:1, 8:2, 7:3), respectively. Now the previous step of the procedure was replaced by using an ethyl acetate benzene with an
aluminum-oxide column system with the ratio of (1:2) or benzene-chloroform with silica
gel column (1:1), chloroform-ethanol (99:1 to 90:10), and butanol-benzene (1:3, 1:4)
(Lozhkin and Sakanyan, 2006).
Furo-coumarin can be separated on an aluminum-oxide column adsorbent with ether;
chloroform-petroleum ether (1:2), petroleum ether, mixtures, or serially removed with chloroform ethanol systems or hexane-chloroform on silica gel column. Coumarin availability is
indicated by usual fluorescence of similar sorbent bands under ultraviolet irradiation or by
the affirmative reactions of specific chemicals (Lozhkin and Sakanyan, 2006).
It was also recommended to isolate coumarin, refine, and evaluate the ingredients by
means of column chromatography with some absorbents like phenolic and phenolic ligands.
The possible phases comprise water and aqueous-ethanol solutions. The most commonly
aqueous solutions, which are used, are mineral acids, neutral salts, and their mixtures. From
all these results of chromatographic systems, it was concluded that such results are highly
significant than those which were provided by the classical dextran sorbents, silica gel,
and polyamide (Askari et al., 2009).
The potential of isolation of coumarin with column-chromatographic technique is patterned by two other types of chromatography, i.e., thin-layer chromatography (TLC), and
sometimes with paper-chromatographic technique. These methods display similarity of
the separated particles and identify even small quantity of coumarin. Thin-layer chromatography is commonly operated on sorbfi plated or silufol and occasionally on aluminum oxide,
silica gel layers (Lozhkin and Sakanyan, 2006).
Aluminum-oxide is usually considered the best absorbent for TLC separation and isolation
of coumarin. The commonly used eluents are ethyl-acetate and petroleum ether (1:2), ethylcyclohexane and chloroform petroleum ether (3:1), and benzene-ethyl acetate. Coumarin
chemical structure extracted from plants can be measured by different techniques, i.e.,
Pechmann reaction, Perkin Condensation, and Knoevenagel reaction.
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3.8.6 Techniques of identification and quantification of coumarin
169
3.8.6 Techniques of identification and quantification of coumarin
3.8.6.1 The titrimetric method
For quantification of coumarin derivatives this method was used before, but now is rarely
used. This method depends upon on the capability of a lactone-pyrone ring, which are open
using alkalis. Some additional alkalis are titrated with sulfuric acid (H2SO4) or hydrochloric
acid (HCL) and yield results. Advantage of this method is that it does not need a reference
samples for coumarin, while the hindrances are the toxicity of mercury compounds and
low specificity (Lozhkin and Sakanyan, 2006; Szewczyk and Bogucka-Kocka, 2012).
3.8.6.2 Calorimetric methods
These quantitative techniques for determination of coumarin depend on their capability to
make quantitative determination methods of coumarin, which are based on their capability to
make constant colored derivatives (light brown to cherry color) when reacted with diazonium
salts. All these techniques were used for the isolation of coumarin from different plants. One
study reported that photocolorimetry method was used for the identification of a sincoumar
(synthetic anticoagulant) (Lozhkin and Sakanyan, 2006; Szewczyk and Bogucka-Kocka, 2012).
3.8.6.3 Spectrophotometry
This method depends upon on the absorbance capability of coumarin in the ultraviolet
(UV) spectral range. For the quantitative determination of coumarin, a band of high absorption (220–350 nm) makes it possible to use UV spectrophotometry method. From previous
studies, it was identified that some bands, which are very important, come in the range of
coumarin and furo-coumarin, and these bands are linked to the electron transition from bonding to antibonding molecular orbitals.
The best method, which are using for the group analysis of coumarin, is UV spectrophotometry. This method is very easy and simple and the ingredients particles can be identified
by changing in their absorption spectral range. Though the disadvantage of this technique is
that it requires the coumarin reference samples to give more precise identification of the target ingredients (Lozhkin and Sakanyan, 2006; Szewczyk and Bogucka-Kocka, 2012).
3.8.6.4 Paper chromatography
This technique is not used alone for the determination of coumarin, this method is used in
combination with other physio-chemical techniques for the analysis of coumarin, e.g., isolation of total coumarin by paper chromatography (petroleum ether; DMF in acetone) followed
by polarographic identification. The big disadvantage of this method is that it is a timeconsuming technique and it needs concentrated extracts due to the weak absorption ability
of the chromatographic medium (Swain, 1953).
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3.8.6.5 Thin-layer chromatography (TLC)
This chromatographic technique is free of some disadvantages, which are present in paper
chromatography. Thin-layer chromatography is similar to paper chromatography and can
gives a quick isolation of ingredients in a mixture sample, and it can be used for the determination of coumarin in different samples (Stahl and Schorn, 1961).
3.8.6.6 Gas chromatographic method
Gas chromatography was mostly used for the determination and quantification of
furocoumarin in crude plant extracts. Several reports of the chromatography pattern of
replaced furocoumarin suggested some laws:
(i) Furocoumarin with O-alkyl substituents as C-5 are removed after 8-hydroxy isomers;
(ii) From hydroxy to methoxy-coumarin, the holding time decreases (due to less adsorption
over hydrogen bonds); and
(iii) The logarithm of the relative holding time is a linear function of the molecular weight
(Furuya and Kojima, 1967; Vilegas et al., 1997).
To know the structure and assessing the holding time of analogous coumarin, GC method
can be used. This GC method can also be used for the identification of coumarin and its derivatives in the liver of human beings as well as in the lab animals (Ren et al., 2016).
3.8.6.7 High-performance liquid chromatography (HPLC)
For the identification of coumarin and furocoumarin compounds, HPLC is extensively
used nowadays (Table 3.8.1). Several coumarin (umbelliferon, scopoletin, daphnoretin)
and carditonic glycosides in seeds were determined by HPLC. HPLC was used in the isocratic
mode, the isolation and simultaneous quantitative and qualitative coumarin determination.
Some studies have proved that HPLC and mass spectrometry (MS) gives reliable results when
used in combination (Ahn et al., 2008; Zheng et al., 2010).
TABLE 3.8.1
(aqueous).
Coumarin level in the extract of Pterocaulon balansae
Coumarin
mg/g dried plant express in 5-methoxy-6,7methylenedioxycoumarin
1
1.90
2
1.02
3
4.54
4
2.33
5
1.01
6
0.58
7
3.33
II. Phenolics
171
3.8.8 Effects of food processing on coumarin
3.8.7 Levels founds of coumarin in plants/food-based plants
Coumarin is an important natural flavoring, which is isolated from different plant materials. The level of coumarin in flavored food and various natural sources is shown in
Tables 3.8.2 and 3.8.3. It is extensively used in food and pastries. Food safety authorities have
declared a maximum limit of 2 mg/kg for different foods and 10 mg/kg for alcoholic beverages due to some concerns with their toxicity (Lachenmeier et al., 2011; Lungarini et al., 2008).
3.8.8 Effects of food processing on coumarin
Coumarin is extracted from natural origin with different techniques. A study found that
the level of isocoumarin in carrots increased with increasing ethylene concentrations (0.5–
50 ppm) and with increasing temperature (0–15°C) (Lafuente et al., 1996). When the carrots
are treated before storage with ultraviolet radiation, it also increases the accumulation of coumarin and makes it resistant against fungal growth (Mercier et al., 1994). Light is also a factor,
which favors the accumulation of coumarin in M. glomerata plant. Plants, which are exposed
TABLE 3.8.2 Coumarin content in different flavors and flavored foods
(Sproll et al., 2008).
Product group
Coumarin level (mg/kg)
Cassia cinnamon
2880–4820
Cinnamon star cookies
2419
Other bakery products and breakfast cereals
9
Vodka flavored with sweet grass
4
Yoghurt
0.7–2
Quark cheese
0.7–4
Rice pudding
0.7–4
Mulled wine
4–40
TABLE 3.8.3
(Lake, 1999).
The concentration of coumarin in some natural sources
Source
Concentration (ppm)
Green tea
0.2–1.7
Bilberry
0.0005
Cinnamon leaf oil
40,600
Peppermint oil
20
Cassia leaf oil
17,000–87,300
Cinnamon bark oil
7000
Other types of cinnamon
900
II. Phenolics
172
3. Analysis of polyphenolics
to sunlight for longer period, have significantly high content in coumarin compared to those
which are exposed for shorter light periods (De Castro et al., 2007). It has been demonstrated
that various processing systems such as thermal treatment (pasteurization, baking, cooling,
and freezing), nonthermal treatment (high pressure, pulsed electric fields, and filtration), mechanical process (peeling, cutting, and mixing), and domestic techniques alter the main phytochemicals in plants and other food products (Irina and Mohamed, 2012).
3.8.9 Trends and concluding remarks
Numerous natural and synthetic coumarin analogues have been discovered or synthesized. Recently, the coumarin has drawn a great attention due to their wide therapeutic behavior. The intention of the present chapter was to know coumarin and its extensive uses in
medical and pharmacological chemistry. In this chapter, an overview was presented to know
various analytical approaches of isolation, identification, and quantification of coumarin,
which should meet the following perquisites: less time, inexpensive, accurate, and precise
for a variety of applications. Coumarin is extracted, purified, and fractioned via TLC, UHPLC,
HPLC, 1D and 2D-NMR spectroscopy, while identification and quantification of coumarin is
done via TLC, NMR, and HPLC. Such approaches may help medicinal chemist for further
screening them for novel therapeutic purposes.
Acknowledgment
This chapter is the outcome of an in-house financially nonsupported study.
Author contributions
All authors have directly participated in the planning or drafting of the manuscript and read and approved the final
version.
Conflict of interest
The authors declare no conflict of interest.
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Further reading
Smyth, M.R., Lawellin, D.W., Osteryoung, J.G., 1979. Polarographic study of aflatoxins B 1, B 2, G 1 and G 2: application of differential-pulse polarography to the determination of aflatoxin B 1 in various foodstuffs. Analyst
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3.9.1 Phytochemistry and classification of the phloroglucinols, xanthones, and anthrones
175
S U B C H A P T E R
3.9
Phloroglucinols, xanthones and anthrones
Iyad Ibrahim Shaquraa, Mohammed Buleb, Fazlullah Khanc, Kamal Niazd
a
Department of Health Management and Economics, School of Public Health, Tehran University
of Medical Sciences, Tehran, Iran bDepartment of Pharmacy, College of Medicine and Health
Sciences, Ambo University, Ambo, Ethiopia cDepartment of Toxicology and Pharmacology,
Faculty of Pharmacy, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of
Medical Sciences, Tehran, Iran dDepartment of Pharmacology and Toxicology, Faculty
of Bio-Sciences, Cholistan University of Veterinary and Animal Sciences (CUVAS),
Bahawalpur, Pakistan
3.9.1 Phytochemistry and classification of the phloroglucinols, xanthones,
and anthrones
3.9.1.1 Phloroglucinols
Phloroglucinol derivatives are a major class of secondary metabolites. They exist widely in
several families such as the Myrtaceae family, Guttiferae, Euphorbiaceae, Aspidiaceae,
Compositae, Rutaceae, Rosaceae, Clusiaceae, Lauraceae, Crassulaceae, Cannabinaceae, and
Fagaceae. Moreover, these compounds also occur in marine and microbial sources.
Phloroglucinol compounds can be classified into monomeric, dimeric, trimeric and higher
phloroglucinols, and phlorotannins (Singh et al., 2010) as shown in Fig. 3.9.1.
Regarding monomeric phloroglucinols, this group encompasses acryl phloroglucinols,
phloroglucinol-terpene adducts, phloroglucinol glycosides, halogenated phloroglucinols,
prenylated phloroglucinols, and cyclicroup polyketides. For acyl phloroglucinols, it is considered the largest category of compounds among phloroglucinols of natural characteristics. It
comprises more than 100 simple acylated phloroglucinols in addition to various types of derivatives. Regarding their vast array of biological activities, numerous research efforts have
been endeavored to synthesize these compounds. Most congeners in phloroglucinol-terpene
adducts have been isolated from different species of Eucalyptus. They are classified, based on
the presence of the chroman ring, into two groups: euglobals (chroman-containing adducts)
and macrocarpals (without chroman-containing adducts). With regards to phloroglucinol
glycosides, more than 50 glycosides of phloroglucinol and its derivatives occurred in natural
sources. Phlorin, phloroglucinol β-D-glucoside (Fig. 3.9.2), is the simplest phloroglucinol
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3. Analysis of polyphenolics
Lysidiside C
HO
Abs
OH
OH
HO
Lysidiside L
OH
O
OH
O
OH
HO
O
O
OH
O
OH HO
O
OH
OH
O
HO
FIG. 3.9.1
Structure of lysidiside C and lysidiside L.
FIG. 3.9.2
Structure of phloroglucinol.
OH
OH
HO
OH
glycoside, which has been isolated from Cannabis sativa, Cornus capitata, and some citrus
fruits. Halogenated phloroglucinols are largely monohalogenated derivatives. Approximately, 15 halogenated phloroglucinol compounds have been recognized from natural
sources and mostly contain chlorine and bromine as halogens. Prenylated phloroglucinols
include mono-, di-, and poly-prenylated/geranylated phloroglucinols. More than 50
prenylated and/or geranylated phloroglucinols have been reported from plant sources.
With respect to dimeric phloroglucinols, this class is characterized by having two
phloroglucinol units linked together a methylene group or by a chroman ring. Many researches have been carried out on this class with many dimeric and higher phloroglucinols
being successfully isolated and synthesized.
Regarding trimeric and tetrameric phloroglucinols synthesis, analogous approaches to
those used in the synthesis of dimeric compounds were used. Finally, phlorotannins have
the highest molecular weight among the phloroglucinols. Its complex structure and poor exploration of biological activity reduced the interest in their synthesis (Singh et al., 2010).
3.9.1.2 Xanthones
Xanthones are secondary metabolites found in some higher plants, fungi, and lichens. A
remarkable interest of their pharmacological properties has been aroused due to their high
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3.9.1 Phytochemistry and classification of the phloroglucinols, xanthones, and anthrones
177
taxonomic values (Bennet et al., 1993). In 1977, xanthone glycosides were described by
Hostettmann and Wagner as an extensive group of natural xanthones (Hostettmann and
Wagner, 1977). Concerning its promising activity indicated for the pharmacological properties, there was a noteworthy concern in xanthone derivatives of plant-extracted xanthones in
recent years (Vieira and Kijjoa, 2005).
The xanthone skeleton (the word “xanthon” is derived from the Greek word xanthos,
meaning yellow) is a planar, conjugated ring system composed of carbon 14 (aromatic ring
A) and carbon 58 (aromatic ring B), combined through a carbonyl group and an oxygen
atom (Fig. 3.9.3). The simplest member of the class, 9H-xanthen-9-one, is a symmetrical
compound with a dibenzo-γ-pyrone skeleton (El-Seedi et al., 2009). The numbering
starts from ring A, while ring B is given prime locants or consecutively numbered
from ring A.
Xanthones are classified as oxygenated xanthones, prenylated xanthones, xanthone glycosides, xanthonolignoids, bis-xanthones, and miscellaneous xanthones, which entail caged
xanthones (Fig. 3.9.3) (Vieira and Kijjoa, 2005; Jensen and Schripsema, 2002; Pouli and
Marakos, 2009). The polyphenolic xanthones are also divided into subclasses depending
upon the degree of oxygenation. These subgroups entail non-, mono-, di-, tri-, tetra-,
penta-, and hexa-oxygenated substances (El-Seedi et al., 2010; Velisek et al., 2008; Jamwal,
2012). The other xanthone subclasses rely more upon the level of oxidation of ring A, which
can occur either as fully aromatic or as dihydro-, tetrahydro-, and hexahydro derivatives, or in
monomeric or dimeric form (Masters and Brase, 2012). Several xanthones could be present as
hydroxylated xanthones with prenyl or geranyl units (El-Seedi et al., 2010). Prenylated xanthones are mainly present in the Clusiaceae family, while the Gentianaceae family has oxygenated xanthones.
Based on the literature, about 650 xanthones are known from natural sources and its abundant remedy found in mangosteen fruit (Fig. 3.9.4). These xanthones have been isolated from
62 families of higher plants, fungi, and lichens (Lannang et al., 2005). Xanthones from higher
plants are distributed in Gentianaceae, Moraceae, Guttiferae, Polygalaceae, and
Leguminosae. Clusiaceae or Guttiferae (55 species in 12 genera) and Gentianaceae (121 species in 21 genera) are the families, which contain the higher proportions of xanthones (Vieira
and Kijjoa, 2005; Jensen and Schripsema, 2002). Mixed shikimate and acetate biogenic origin is
accounted for the structural diversity of xanthones. The biosynthetic pathway defines xanthones as cyclized 2,30-dihydroxybenzophenone derivatives.
O
FIG. 3.9.3
Xanthone backbone chemical structure.
O
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3. Analysis of polyphenolics
α-Mangostin
λ-Mangostin
O
HO
OH
OH
O
OH
O
HO
FIG. 3.9.4
O
OH
O
OH
Chemical structures of the most abundant xanthones in mangosteen fruit.
3.9.1.3 Anthrones
An anthrone is a planar tricyclic aromatic ketone (Fig. 3.9.5). In the basic skeleton of an
anthrone, the two aromatic rings are linked by a keto group and a methylene group (sp3 carbon) in such a way as to form a six-membered cyclic ketone in the middle of the condensedfused ring system. Thus, the simplest anthrone, anthracene-10(9H)-one, is formed by the reduction of the structure of anthracene-9,10-dione (anthraquinone) to form anthrone skeleton,
which has one keto (C5O) group.
Anthrones are largely spread among the Aloe species. The most common aloe anthrones,
aloin A and B, aloinoside A and B, and microdontin A and B, have been found in around 36
species of Aloe (Viljoen et al., 2001). The other genera containing anthrones are Bulbine, Cassia, Frangula, Hypericum, Harungana, Picramnia, Rhamnus, Rhubarb, Rubus, Senna, and
Vismia. From the African plants, around 30 anthrones have been isolated and characterized
from the Guttiferae and Aloe (Xanthorrhoeaceae) families (Fig. 3.9.6).
The biosynthesis of anthrones could take place via the acetate pathway. The cause of C-16
polyketide precursor importance is the condensation of one acetyl-CoA and seven malonylCoA units. This polyketide pathway also yields into formation of anthraquinones, which result from anthrone metabolism. Anthraquinones and dianthrones are created from
anthrones’ ability to compose reactive oxygen types under physiological conditions. The
anthrone derivatives exist as glycosides, oxanthrone mayoside, carboxylic acids, tetrahydroxyanthrone, and polycyclic derivatives. There are prenylated and methylated derivatives of
olyphenolic anthrones (Kouam et al., 2005; Abe, 2008; Shiono et al., 2002; Lu et al., 2002).
FIG. 3.9.5
Anthrone backbone chemical structure.
O
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179
3.9.2 Biological activities of phloroglucinols, xanthones, and anthrones
OH
O
Abs
OH
OH
O
Abs
OH
OH
H
OH
H
H
H
OH
OH
O
O
OH
OH
OH
OH
OH
Aloin A
OH
O
OH
Aloin B
Abs
OH
OH
O
Abs
OH
OH
OH
O
H
O
OH
H
H
OH
O
H
OH
O
OH
OH
OH
OH
Aloinoside A
FIG. 3.9.6
O
O
OH
OH
OH
OH
OH
Aloinoside B
Anthrone C-glycosides from the aloe family (Mazimba et al., 2013).
3.9.2 Biological activities of phloroglucinols, xanthones, and anthrones
3.9.2.1 Phloroglucinols
Phloroglucinols encompass around seven hundred of compounds, which are naturally occurring and with multiple biological activities. Phloroglucinols as a prototype molecule has been
inoculated from natural sources. Importantly, phloroglucinol procedures of synthesis were
reviewed by Bridi et al., (2018) as many syntheses have been declared, patented, and marketed.
Moreover, phloroglucinol is used in medicine, cosmetics, pesticides, paints, cements, and
dyeing. Existence of phloroglucinol derivatives, pharmacological, biosynthetic characteristics,
and particularly synthetic features of naturally occurring polycyclic polyprenylated acylphloroglucinols have been reviewed in these days as well as in the past (Singh et al., 2010).
II. Phenolics
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3. Analysis of polyphenolics
Researchers have checked the structural variety among the naturally occurring
phloroglucinol category. Interestingly, it was noticed that two phloroglucinol terpene adducts, robustadials A and B, lying among monomeric phloroglucinols, show in vivo antimalarial activity against P. berghei. In addition, intense antibacterial and HIV-RTase inhibitory
activity were displayed by macrocarpals. Type III polyketides synthases (PKS) are involved
in biosynthesis of hypericin, which is an antidepressant action. Moreover, chinesins A and B
show strong antiviral activity against Herpes simplex and Vesicular somatitis viruses. Also, a
broad spectrum of biological activities was possessed by dimeric phloroglucinols with a methylene linkage. For example, antiallergic and antiinflammatory effects, which are exerted by
mallotophillipens A and B; antimalarial activity by robustaol A, japonicine A, and sarothralen
A; HIV-RTase activity by mallotojaponin; and cytotoxic activity by mallotojaponin and their
analogues. Jensenone and sideroxylonals within diformylated phloroglucinols group are
antifeedant agents. Trimeric phloroglucinols murtucommulone A and agrimols are considered as very strong antibacterials. The activities, shown by dibenzodioxin-linked compounds, entail tyrosinase inhibitory activity, antioxidant activity, and inhibitory effects on
glycation and R-amylase, which are involved in diabetic complications. These compounds
also exhibit bactericidal and algicidal activities. It’s worthy to say that many of phloroglucinols have been elucidated structurally, but they as a group have not been fully explored
(Singh et al., 2010).
3.9.2.2 Xanthones
Xanthones have diverse pharmacological properties, mainly due to their oxygenation nature and diversity of functional groups. Some previous review articles in the literature
discussed the biological activities of xanthones during 2000–2012. These activities include
antibacterial, antiviral, antioxidant, antiinflammatory, antiproliferative, antihypertensive,
antithrombotic, in vitro and in vivo antitumor, cytotoxic, coagulant, monoamine oxidase
(MAO) inhibition, gastroprotective effects, antiatherosclerotic action, suppression of hypotension, cardioprotection, inhibition of cholinesterase, cyclooxygenase activity, immunosuppression, and binding to transthyretin (TTR) (Farah et al., 1992; Pinto et al., 2005).
The action of xanthones and their sources against diabetes and HIV/AIDS is due to the
α-glucosidase inhibitory activity (Li et al., 2011). Xanthones have exhibited the potential
to avoid disease development by their concerted action of protecting cells from oxidative
stress damage and acting as phytoalexin to impair pathogen growth (Franklin et al.,
2009). Antimalarial property is significant activity for tropical plants in Asia and Africa
(Pontius et al., 2008; Alaribe et al., 2012). Xanthones are rare antioxidants ( Jensen and
Schripsema, 2002) that only occur in exclusive types of fruits such as mangosteen. Xanthones have powerful anticancer components; thus, one compound, α-mangostin, was
found to have a counteractive effect against the progressive growth of tumors (Pinto
et al., 2005). Garcicone E is another congener, which can batter cancer in vital organs in
the body such as liver, lung, and colon. For more various effects, xanthones are commonly
occurring in herbs such as Swertia davidi Franch (Gentianceae), which is useful in treating
inflammation, allergy, or hepatitis (Pouli and Marakos, 2009).
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181
3.9.2.3 Anthrones
There are diverse pharmacological activities of anthrones. This includes insecticidal, immunological adjuvant and wound healing, antimicrobial, antioxidant, photodynamic
antitumor, antiviral, antimetastatic, and laxative activities, and modulation of apoptosis
(El-Seedi et al., 2010; Hostettmann et al., 2000; Kimura et al., 2008; Asamenew et al., 2011).
Their toxicity is due to inappropriate artemisinin from Artemisia annua and a tetrahydroanthracene from Psorospermum febrifugum as antimalarial drugs (Hostettmann et al., 2000). The
ability of phenolic anthrones (such as anthralin) to create free radicals is the basis for their
antipsoriastic, antiinflammatory 5-lipoxygenase, and 12-lipoxygenase activity (Muller and
Gawlik, 1995). In African plants species, the Harungana madagascariensis (Guttiferae), which
contains prenylated anthrones, is used as a treatment for diarrhea and dysentery and is a potent laxative (Kouam et al., 2005). Aloe microdonta (aloe family) is used for jaundice and skin
diseases treatment (Farah et al., 1992) The Cape aloe, which contains anthrone C-glycosides, is
used as a laxative in Africa and is considered to have antioxidant, antiinflammatory, antimicrobial, and anticancer properties (Chen et al., 2012).
3.9.3 Extraction and purification techniques of phloroglucinols, xanthones,
and anthrones
3.9.3.1 Phloroglucinols
Phloroglucinol-based phenolic polymers range from low molecular weight to high molecular weight. Especially those occurring in brown macroalgae have four classes based on the
phloroglucinol units: fucols (phenyl linkage), fucophlorethols (ether and phenyl linkages),
phloroethols and fuhalols (ether linkages), and eckols (dibenzodioxin linkages)
(Magnusson et al., 2017). Different techniques of extraction such as supercritical fluid extraction, solid liquid extraction, microwave-assisted extraction, various other liquid-liquid extractions have been used to extract and purify phloroglucinols. Various factors influence
the extraction of phloroglucinol-based phenolic polymers, including solvent polarity, time
of extraction, temperature, solvent-solid ratio, and particle size. The extraction of
phloroglucinols from a plant is a process and mainly starts with preparing the plant by air
drying and grinding. Then, once the plant material is ready, the liquid extraction can be
performed using the appropriate solvent like petroleum ether, ethyl acetate, hexane,
dichloromethane, and aqueous mixture of ethanol or methanol to obtain the crude extract.
The crude extract can be further extracted using a silica gel column chromatography and
eluted with a gradient mixture of polar and nonpolar solvents, which is mostly a mixture
of petroleum ether/EtOAc mixture (100:0–0:100, v/v) (Liu et al., 2018a) or n-hexane/EtOAc
as gradient (90:10–0:100) (Kabran et al., 2015) or n-hexane-CH2Cl2 mixtures followed by
increasing concentration of EtOAc in CH2Cl2 (Lim et al., 2019) to afford various fractions. Further separation of the newer fractions using Sephadex LH-20 yields different subfractions.
Successive fractionations of each subfraction yield a final pure phloroglucinol compound.
Yet, in some cases, obtaining final phloroglucinols need HPLC (CH3CN-H2O, 70:30,
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3. Analysis of polyphenolics
3 mL/min) or reverse-phase semipreparative HPLC (CH3CN-H2O, 70:30, 3 mL/min). On the
other hand, supercritical extraction of phloroglucinols uses supercritical fluid such as CO2, to
get the initial crude extract, whereas the remaining fractionation and purification process is
similar to other liquid-liquid extraction (Zhang et al., 2018). Preparative TLC has also been
used in the separation of subfractions and purification of the phloroglucinols by using different solvents such as 30% CH2Cl2/hexane as an eluent (Hiranrat et al., 2017). Currently, resin
column adsorption technique has been broadly applied in separation and isolation of bioactive compounds such as flavonoids, polyphenols, alkaloids, and saponins. In this regard, DM130 macroporous adsorption resin is reported to have a good separation and enrichment capabilities in phloroglucinol compounds since DM-130 macroporous adsorption resin has
weak polarity, and its functional group belongs to polystyrene (Zhe et al., 2015).
3.9.3.2 Xanthones
The first step to obtain the xanthones’ rich fraction is the grinding of the dried or lyophilized plant material with subsequent solvent extraction. Significantly, Dorta et al. (2012)
revealed that the freeze-drying is better than the oven-drying treatment, as the former is more
suitable method than the latter one for saving the stability of peel and seed of mango without
reducing their polyphenol content and antioxidant capacity. On the other hand, the latter is
the worse regarding its negative adverses on polyphenol characteristics. As a fraction of biomass status, there may be two types of solvent extraction, liquid-liquid extraction or solidliquid extraction. Xanthones from mangosteen fruit beverages could be isolated using
liquid-liquid extraction. Meanwhile, xanthone could be extracted from different parts of mangosteen fruit and mango fruit (pericarp, peel, seeds, pulp, whole fruit) by solid-liquid extraction, which has been widely employed (Ignat et al., 2011; Dorta et al., 2012, 2013a,b, 2014;
Zhou et al., 2015; Ibrahim et al., 2016). Typically, the first step when applying the procedure
is the use of an organic or aqueous solvent such as methanol, ethanol, and acetone. For example, while Yu et al. (2007) used 70% concentrated methanol to extract the air-dried pericarp
of mangosteen fruit, Chin et al. (2008) and Mohamed et al. (2014) used also methanol to identify and inoculate various xanthones from mangosteen fruit powder and pericarp, respectively. Barreto et al. (2008) also employed methanol in the extraction as a second step of
freeze-dried kernels and seeds of mango fruit after a first extraction using hexane in a soxhlet
to eliminate lipids. Furthermore, acetone was utilized to extract xanthones from mangosteen
pericarp ( Ji et al., 2007) or using mixtures of methanol/ water (60:40, v/v) from mango peel
and seed powder (Ribero et al., 2008) or 70% methanol has been used with air-dried mangosteen pericarp (Yu et al., 2007). Due to methanol toxicity and its avoidance in food industry,
mixtures of ethanol/water or acetone/water (between 50% and 99.5%) are the most widely
used extraction solvents. For example, 95% ethanol used by Zhou et al. (2015) in order to extract xanthones from mangosteen pericarp. Additionally, mixtures of acetone/water (50:50,
v/v and 80:20, v/v) were applied to extract polyphenols, including xanthones from lyophilized mango seeds (Schieber et al., 2003; Chin et al., 2008; Ribero et al., 2008; Dorta et al., 2013b,
2014). Other solvents such as methylene chloride have been used (Wittenauer et al., 2012). The
xanthones to be isolated from plant material are also influenced by the weight of plant material to solvent ratio. The most commonly reported ratios range between 1:1 and 1:10 (Yu
et al., 2007; Chin et al., 2008; Dorta et al., 2014; Zhou et al., 2015). Temperature plays an important role in the extraction of xanthones from plant material. The conditions found in the
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183
literature ranged between room temperature (between 3 h and 3 days; Berardini et al., 2005;
Chin et al., 2008; Mohamed et al., 2014; Zhou et al., 2015) to 50–75°C during several hours (2–
3 h; Yu et al., 2007; Wittenauer et al., 2012; Dorta et al., 2014). The extractions at room temperature could be combined with sonication for a short time (approximately 20 min; Ji et al.,
2007), mechanical stirring for several hours (approximately 3 h; Berardini et al., 2005), or maceration for 1 day (Chin et al., 2008). Microwave-assisted extraction of mango peels and seeds
(one hour at 75°C) has been assayed with exit to obtain xanthone-rich extracts (Dorta et al.,
2014). Interestingly, a slight decrease in mangiferin concentration has been detected when
mango peel is subjected to high temperatures, while the concentration of other xanthone
derivatives increases remarkably. These changes may be accounted for the formation of xanthones from bezophenone derivatives in mango peel, which are considered to be the precursors of xanthone C-glycosides (Berardini et al., 2005). Nowadays, the concern in the use of
supercritical fluid extraction (SFE) is elevated as it can be a better substituent in the extraction
by the conventional organic solvent with respect to its environmental benefits. The SFE of
xanthones from mangosteen pericarp with the use of SC-CO2 (60°C/30 MPa) results in a
much higher product (7.56%) and xanthone content (65.93%) with an augment of xanthones
1.4–3.2 folds than the traditional ethanol extraction using a SoxtecTM (Zarena and Sankar,
2011). The separation of xanthones from the extract was commonly carried out by column
chromatography on silica gel using different solvent mixtures with increasing polarity
(e.g., in order of addition into the column: n-hexane, chloroform, ethyl acetate, and methanol;
Yu et al., 2007; Mohamed et al., 2014). Also, the extract could be partitioned sequentially and
treated with solvents of different polarities (hexane, dichloromethane, ethyl acetate, etc.; Chin
et al., 2008). However, most of xanthone extraction studies have been included in several reviews (Obolskiy et al., 2009; Negi et al., 2013); there was a proof of its time-consuming and
limited application to routine analysis of a larger number of samples. Therefore, it becomes
preferable to assay other procedures such as the purification of polyphenol extract using
solid-phase extraction with polyamide or Sephadex LH20 as solid-phase previous to
HPLC-DAD and HPLC-MS analysis (Schieber et al., 2003; Wittenauer et al., 2012; Dorta
et al., 2014). Xanthones may be also separated and identified using thin-layer chromatography (TLC) and HPLC by comparison with authentic standards (Negi et al., 2013).
3.9.3.3 Anthrones
Regarding color reactions, it is worthy to say they are still useful especially when an investigation begins in which the crude extract is set on a thin-layer chromatography (TLC) plate
and reagents are added to produce valuable information. Before TLC, the powdered plant
material could be filtered after being extracted for 5 min with methanol (1 g of plant in
100 mL). It is necessary to hydrolyze the extract to distinguish the aglycones and for this
1 g of powder plant is heated under reflux with 25 mL 7.5% hydrochloric acid for 15 min. After
cooling, the mixture is extracted by shaking with 50–100 mL of chloroform or ether. The organic phase is then taken and concentrated to about 1 mL, and then used for TLC. Except for
senna, chromatography is performed on precoated plates of silica gel with light petroleumethyl acetate-formic acid (75:25:1) or ethyl acetate-methanol-water (100:13.5:10) for all drug
extracts of anthracene. In the case of senna, n-propanol-ethyl acetate-water-glacial acetic acid
(40:40:29:1) is employed. For the nonlaxative dehydrodianthrones of St John’s wort
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3. Analysis of polyphenolics
(Hypericum perforatum), TLC is carried out with the eluent toluene-ethyl formate-formic acid
(50:40:10) (Isac-garcia et al., 2016).
3.9.4 Identification and quantification techniques of phloroglucinols, xanthones,
and anthrones
3.9.4.1 Phloroglucinols
The presence or absence of phloroglucinol can be examined using solid-state NMR experiments. In such experiments, the spectra are obtained from a spectrometer equipped with an
indirect HR-MAS 1H/31P probe head. Pure phloroglucinol (a reference standard) is used in
the NMR tube to get another spectrum. Hence, by comparing both spectra, phloroglucinol is
easily identified ( Jegou et al., 2015). In both spectra, the phloroglucinol singlet, i.e., at
6.02 ppm with 3 aromatic protons per molecule appears as confirming peaks ( Jegou et al.,
2015). Other techniques of identification include the use of spray reagents for the detection
of phloroglucinols. This includes aqueous 0.1% (w/v) ferric chloride plus 0.1% (w/v) potassium ferricyanide (7); 1% (w/v) vanillin in a mixture of nine volumes 95% ethanol and one
volume concentrated HC1; 0.01% (w/v) aqueous fast Bordeaux salt BD aver sprayed with
saturated aqueous sodium carbonate; 2% (w/v) anisaldehyde in a mixture of nine volumes
95% ethanol and one volume concentrated H2S04 (Ragan and Craigie, 1976). In addition,
newer visualizing reagents such as bromocresol green (M) and neutral red (C) have been applied in the detection of phloroglucinols. The limits of detection of the phloroglucinol using
these spray reagents range from 3.0 to 30 mg (Pyka et al., 2002).
There are few analytical methods for the separation and determination of phloroglucinols,
such as fluorimetry, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and chemiluminescence
(Cui et al., 2003). Chemiluminescence is a detection method known for its high sensitivity
and wide linear working ranges. It has been used as a sensitive detector along with flow injection analysis or HPLC. However, the application of combined HPLC and CL detection
method is quite limited since the separation is conducted by ordinary silica-based C18 column in HPLC, and the silica column performance is good only in the pH range of 2–8,
whereas the CL systems needs basic medium (Wu et al., 2012). Moreover, it was reported that
in Na2CO3 NaHCO3 buffer, phloroglucinol demonstrated strong chemiluminescent enhancement at pH 9.4. Thus, a flow injection method is an established method for the determination
of phloroglucinol. The method is simple, sensitive, rapid, and convenient with a detection
limit of 2.0 109 mol/L (Cui et al., 2003). Additionally, a colorimetric method, which uses
a cinnamaldehyde-HCl reagent and gives a pink color during reaction with PG, is also used
for quantification of PG. In this method, the absorbance of the sample is measured at 550 nm
and PG quantified by comparing the sample to a standard (Kidarsa et al., 2011). Quantification of phloroglucinols using the Folin-Ciocalteu (FC) assay is a classic protocol for
phlorotannin quantification. However, the use of the FC assay on crude extracts has been
reported to cause an overestimation of phenols. In this assay, the phloroglucinol content of
the organic phase is examined using the FC assay procedure. This is done by taking
100 mL of diluted organic phase and mixing it with 50 mL of Folin-Ciocalteu reagent, then
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185
heating the mixture for 20 min at 70°C, and putting on ice for 10 min to quench the reaction.
Then, the absorbance is measured at 750 nm. Standard phloroglucinol solutions are also used
in this FC assay to get a calibration curve. The result is expressed as phloroglucinol content in
% DW ( Jegou et al., 2015).
3.9.4.2 Xanthones
The establishment of structural identification of all known xanthones has been founded on
the basis of ultraviolet visible spectroscopy (UV), infrared spectroscopy (IR), 1H and/or 13C
nuclear magnetic resonance (NMR), X-ray crystallographic, and mass spectroscopy (MS) data
(Chin et al., 2008; Obolskiy et al., 2009; Negi et al., 2013; Mohamed et al., 2014). Basically, highperformance liquid chromatography (HPLC) is the analytical technique used to isolate, identify, and determine the quantity of polyphenol compounds of fruit and derived products. The
chromatographic conditions of the HPLC method entail the use of reverse-phase C18 column
and a binary solvent system containing acidified water solvent (solvent A) and polar organic
solvent (solvent B). HPLC are combined with different detectors, such as UV-Vis diode array
detector (HPLC-DAD), mass, or tandem mass spectrometry (HPLC-MS). Many researchers
have described quick and efficient HPLC-DAD chromatographic systems to separate and
identify xanthones from mangosteen fruit (Walker, 2007; Chaivisuthangkura et al., 2009).
For instance, the use of HPLC-DAD for fingerprinting the main xanthones of mangosteen
fruit uses 14 xanthone standards previously isolated from mangosteen pericarp. The
HPLC-DAD (254 nm) chromatogram of the xanthones standards was perfectly separated
in a run of 60 min using a C18 column and mobile phase in a gradient preparing from acetonitrile (solvent A), 2% (v/v), acetic acid in water (solvent B), and n-butanol (solvent C). This
HPLC-DAD fingerprinting can be readily used as a proper method for evaluation of the quality of mangosteen fruit and its derivatives (Chaivisuthangkura et al., 2009). These days,
HPLC-MS techniques are the best analytical approach to investigate polyphenols from various biological sources, such as xanthones in mangosteen fruit ( Jung et al., 2006; Ji et al., 2007;
Zarena and Sankar, 2011; Wittenauer et al., 2012). For more precise characterization of phenolic compounds occurring in mangosteen fruit, mango fruits, and derived products, it is necessary to use sophisticated and potential techniques such as high-performance liquid
chromatography combined with electrospray ionization and quadrupole-time of flight-mass
spectrometry (HPLC-ESI-QTOF-MS) (Dorta et al., 2014).
3.9.4.3 Anthrones
Vast majority of anthrones are of different colors, sometimes yellow powder or yellow
needles, and in some cases orange or violet, etc. They are characterized by strong absorption
when viewed under a UV lamp. The carbonyl-olefinic chromophore in their structures is the
reason behind this character. The UV spectrum of anthrone is characterized by intense absorption in the region of 240–290 nm (εmaxλ26.00), a medium band λ285 nm (εmaxλ300), characteristic of the conjugated carbonyl moiety and attributed to an electron transfer (ET) transitions,
and much weaker absorptions (n ! π∗) in the visible region. Its IR spectrum exhibits absorption bands for carbonyl group at 1680 cm1 (Chawla et al., 1973). The most common aspects of
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3. Analysis of polyphenolics
the mass spectra of all anthrones are peaks related to the loss of one or two molecules of carbon dioxide. Anthrone compounds eliminate an ethylenic (dC]Cd) and/or acetylenic (O]
CdC]Cd) fragment. The 13C NMR spectra display signals of one conjugated carbonyl carbon, the aromatic carbons, and one methylene carbon (Chawla et al., 2005).
3.9.5 Levels founds of phloroglucinols, xanthones, and anthrones in foods/plants
3.9.5.1 Phloroglucinols
Phloroglucinols are bioactive compounds mainly isolated from marine brown alga (Wan
et al., 2019). Phloroglucinol polymers from brown algae are widely studied, due to their biotechnological potential and their importance in chemical ecology (Bule et al., 2018). The
brown alga, Carpophyllum maschnlocarpum, comprise newer derivatives of PG in the class
of fucophlorethols, which contain 4 to 14 PG units and other species of the Australian brown
alga Zonaria comprise also PG derivatives (Armstrong and Patel, 1994). As a matter of fact, the
phlorotannin content in brown alga differs based on the techniques used for extraction, specifically between “classic” solvent extraction methods and other methods. A typical spectrum
used for the quantification of phloroglucinol content in C. tamariscifolia shows the levels were
in the range of 0.53%–0.83% of dry weight ( Jegou et al., 2015). In addition to brown alga, various plant species have been found to possess a great deal of phloroglucinols. Eugenia
umbelliflora, which is a tree with edible fruits, contains sesquiterpenyl phloroglucinol
(eugenial E) up to 90 mg per 100 g of the fruit (Farias et al., 2018). Furthermore, Myrtus
communis is a sclerophyll shrub that belongs to the Myrtaceae family (Issa and Bule, 2015),
which has been used as a spice, as medicine, and in food preparation, is a rich source of
phloroglucinol derivatives. According to Tanaka et al., Myrtus communis contains up to
321.6 mg per 100 g phloroglucinol (Tanaka et al., 2018). In addition, Rhodomyrtus tomentosa
(Aiton) Hassk. (Myrtaceae), which is used as traditional medicine in China and its berries
are used to prepare traditional jams, wines, and beverage in Asian countries, contain various
phloroglucinol derivatives. Zhang and colleagues investigated Rhodomyrtus tomentosa and
identified seven novel phloroglucinol derivatives and eleven known ones in the range of
2.0–783.7 mg (Zhang et al., 2018). Several dimeric phloroglucinols such as mallotojaponin,
mallotochromene, mallototorin, and mallotophenone have also been isolated from Mallotus
japonicus (Chauthe et al., 2012). Phloroglucinols also occur abundantly in other genera such
as Eucalyptus (Liu et al., 2018b), Garcinia (Bridi et al., 2018), Mallotus (Kabran et al., 2015),
Xanthostemon (Liu et al., 2018a), Ecklonia (Yang et al., 2018), Dryopteris (Chauthe et al.,
2010), Melaleuca (Xie et al., 2019), and Hypericum (Stolz et al., 2014) and families like
Guttiferae, Euphorbiaceae, Aspidiaceae, Compositae, Rutaceae, Rosaceae, Clusiaceae,
Lauraceae, Crassulaceae, Cannabinaceae, and Fagaceae (Singh and Bharate, 2006).
3.9.5.2 Xanthones
Wittenauer et al. (2012) found a similar HPLC xanthone profile for pericarp and aril segments of mangosteen fruit, and also in the functional beverage made from whole mangosteen
fruit. The highest total xanthone content was reported for the pericarp, followed by the aril
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3.9.6 Effects of food processing on phloroglucinols, xanthones, and anthrones
TABLE 3.9.1
Xanthone content in mangosteen pericarp, aril segments, and functional beverage.
Pericarp
S.
no.
187
Aril segments
(mg/100 g
dw)
Beverage
(mg/
100 mL)
Xanthone
(mg/100 g fw)
(mg/100 g dw)
(mg/100 g
fw)
1.
1,7-Dihydroxy-3methoxy-2-(3-methylbut2-enyl)xanthone
35.27 0.85
99.92 2.41
12.52 0.24
65.88 1.28
0.48 0.01
2.
γ-Mangostin
303.64 6.16
860.17 17.5
8.25 0.24
43.39 01.27
3.01 0.06
3.
8-Deoxygartanin
50.09 0.80
141.90 2.26
5.01 0.09
26.39 0.45
0.67 0.01
4.
1,3,7-Trihydroxy2,8-di(3-methylbut-2enyl)xanthone
19.12 0.6
54.17 1.58
25.76 0.68
135.57 3.56
0.53 0.01
5.
Gartanin
70.41 1.2
199.46 3.50
5.49 0.11
28.92 0.58
0.85 0.01
6.
α-Mangostin
1173.33 29
3323.88 8
40.59 1.62
213.63 8.53
12.53 0.4
7.
Garcinone E
48.4 1.9
137.09 0.85
9.25 0.17
48.70 0.88
0.83 0.0
1700.26 40.5
4816.59 114.6
106.87 3.15
562.48 16.56
18.79 0.49
Total amount
segments and the functional beverage (Table 3.9.1). Considering the consumption of the edible part of a mangosteen fruit (30 g fw of aril segments), it results in an intake of 12.2 and
2.5 mg of α-mangostin and γ-mangostin, respectively. Furthermore, 90 mL of functional beverage have the same amount of major xanthones α-mangostin and γ-mangostin as 0.9 g of pericarp, according to data shown in Table 3.9.1 (Wittenauer et al., 2012). Traditional uses of
mangosteen fruit are multivariate. Only the aril segments are eaten when they are used fresh
as a dessert, and then the pericarp is discarded. The arils and the whole fruit could be used in
products such as wine, preserves, jam, and puree. A new important market for puree yielded
from the whole mangosteen fruit is the production of functional beverage (Wittenauer
et al., 2012).
Within the group of polyphenols, the predominant compounds are mangiferin and
quercetin-3-O-galactoside (Table 3.9.2). Mangiferin is a xanthone C-glycoside mainly found
in the peel and seeds of mango fruit. The cultivar and ripeness stage of the fruit is responsible
for the mangiferin content of mango peel and seeds. Therefore, mangiferin may be the abundant polyphenol compound in some cultivars (between 1263.2 mg/kg dw in Tommy Atkins
and 13.9 mg/dw in Kent), whereas the flavonol quercetin 3-O-galactoside may be in the
others (Table 3.9.2).
3.9.6 Effects of food processing on phloroglucinols, xanthones, and anthrones
3.9.6.1 Phloroglucinols
Dimeric phloroglucinols occurring naturally are reported to have a diverse biological activity. Dimeric phloroglucinols are those containing two phloroglucinols linked together via a
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3. Analysis of polyphenolics
TABLE 3.9.2 Xanthones C-glycosides and flavonol content in peel, pulp, and kernel of different mango
cultivars (mg/kg dw).
Tommy Atkins
Jose
Kent
Haden
Ubá
Mangiferin
1263.2 197.2
983.6 50.1
13.9 1.5
11.2 0.1
199 5.3
Isomangiferin
40.3 0.8
45.5 1.9
4.0 0.3
21.0 0.8
16.4 2.9
Mangiferin gallate
87.3 1.5
25.2 2.0
ND
ND
28.0 1.0
Isomangiferin gallate
12.3 0.6
ND
ND
ND
26.9 0.7
Quercetin diglycoside
55.1 0.7
40.3 2.8
ND
ND
ND
Quercetin 3-O-galactoside
1217.3 18.0
1467.3 42.3
944.5 38.3
1309.1 26.0
151 12.3
Quercetin 3-O-glucoside
882.0 4.2
1045.3 41.6
890.0 39.8
912.7 20.5
370 25.6
Quercetin 3-O-xyloxide
239.5 3.8
278.6 8.4
150.7 8.2
179.1 4.5
84.4 6.2
Quercetin 3-O-arabinofuranoside
163.5 2.8
191.8 7.5
91.6 3.4
104.9 5.1
64.8 5.3
Quercetin 3-O-arabinopyranosid
152.4 2.7
119.6 3.5
84.8 3.8
70.5 0.8
35.0 2.5
Quercetin 3-O-rhamnoside
38.2 1.7
116.4 4.3
58.1 3.5
52.7 0.6
15.8 1.2
Kaempferol 3-O-glucoside
77.3 5.3
171.7 8.8
30.6 1.8
43.7 1.1
ND
Rhamnetin 3-O-galactoside
215.6 4.9
374.4 11.1
70.6 3.4
228.6 2.7
35.3 2.7
Quercetin
ND
ND
3.3 0.1
2.8 0.0
64.1 1.6
Total phenolics
4444.0 198.3
4860.2 80.0
2342.0 56.4
2936.4 33.9
1091 67.3
4.6 0.1
19.4 0.2
ND
16.2 2.7
12.4 0.3
Peel
Pulp
Mangiferin
Kernel
46.5 4.7
Mangiferin
methylene linkage or by the formation of a chroman ring. The dimeric and polymeric
phloroglucinol derivatives are prone to chemical and microbial degradations during
processing. Biodegradation of phloroglucinol by microbes occurs either through aerobic or
anaerobic metabolic pathways. Aerobic microbes induce the decomposition of
phloroglucinol by either a reductive pathway, epoxide formation, or a specific hydroxylating
mechanism. On the other hand, anaerobic biodegradation is suggested to occur by the reductive formation of a dihydrophloroglucinol (1,3-dioxo-5-hydroxycyclohexane), which is hydrolyzed by hydrolase enzyme. In a similar manner, the decomposition of other
polyphenols by anaerobic pathway comprises phloroglucinol as a major metabolite
(Armstrong and Patel, 1994). For instance, it was reported that catechin is decomposed by
Pseudomonas solanacearum into phloroglucinol carboxylic acid and protocatechuic acid by catechin oxygenase. The phloroglucinol carboxylic acid decomposition process occurs via the
II. Phenolics
3.9.6 Effects of food processing on phloroglucinols, xanthones, and anthrones
189
formation of phloroglucinol, resorcinol, and hydroxy-hydroquinone (Boominathan and
Mahadevan, 1984). A number of rumen bacterial strains, which are capable of breaking down
the phloroglucinol (1,3,5-trihydroxybenzene) under anaerobic environment, were isolated
from the bovine rumen microflora in a prereduced medium containing 0.02 M phloroglucino
(Tsai and Jones, 1975). The isolated strain of bacteria includes Streptococcus bovis and
Coprococcus sp. (Tsai and Jones, 1975). The main byproducts of the bacterial catabolism are
acetate and carbon dioxide. Coprococcus sp. at also degrades partially the flavonols, quercetin
and rhamnetin (Tsai et al., 1976) (Krumholz and Bryant, 1986). In another experiment in anaerobic environment, resting cell suspensions of Coprococcus sp. Pe15 decomposed 1 molecule
of phloroglucinol to 2 molecules of acetic acid and 2 molecules of carbon dioxide. Under a
similar condition, rhamnetin and quercetin were metabolized anaerobically in 20% rumen
fluid medium. However, the Coprococcus sp. was unable to grow in a similar environment
when tested with 39 other aromatic or flavonoid derivatives (Tsai et al., 1976).
3.9.6.2 Xanthones
Processing of mango fruits leads to the formation of a notable number of byproducts, such
as the peels and seeds that represent approximately 15%–20% and 45%–60% of the fresh fruit
weight, respectively (Masibo and He, 2009). Mango byproducts have been extensively studied in the last few years. High concentrations of various health-promoting bioactive compounds, such as dietary fiber, vitamin C, carotenoids, and polyphenols, have been
detected in the byproducts of mango such as peels and seeds (Ajila et al., 2007; Berardini
et al., 2005; Sogi et al., 2013; Juhurul et al., 2015; Torres-León et al., 2016; Serna-Cock et al.,
2016). Being rich in phytochemicals, the interest is raised regarding the investment of
byproducts resulting from fruit processing that can’t only be used as natural additives with
different activities (antioxidant, antibrowning, antimicrobials, colorants, texturizers, etc.) but
also as a source of bioactive compounds to obtain functional ingredients or functional foods
(Masibo and He, 2009; Ayala-Zavala et al., 2011; Juhurul et al., 2015; Torres-León et al., 2016;
Serna-Cock et al., 2016; Rymbai et al., 2016). It is well documented that mango byproducts,
such as peels and seeds, present high antioxidant capacity and high levels of healthenhancing substances (dietary fiber, vitamin C, carotenoids, and polyphenols). Mango peel
and seeds can be dried and powdered and then used as a food supplement, bakery products
(bread, cookies), ice cream, breakfast cereals, pasta products, beverages, snacks, meat, etc.
( Juhurul et al., 2015; Serna-Cock et al., 2016).
3.9.6.3 Anthrones
McDougall and Jordan-Mahy (2010) have assessed the effects of a variety of processing
methods on the polyphenolic compounds occurring in garden rhubarb Rheum rhapontigen.
Total polyphenolic composition, anthocyanins content, and total antioxidant characteristics
were appraised. The analysis of the products was by using liquid chromatography-mass
spectrometry, and the identification of 40 polyphenol compounds was anticipated. Four
methods for cooking were developed: blanching (boiling water), slow cooking (70–80°C),
fast cooking (100°C), and baking (180°C), from 2 to 30 min. It is worthy to say that total
II. Phenolics
190
3. Analysis of polyphenolics
polyphenol content and overall antioxidant capacity increases with most cooking methods,
except blanching, if compared to the raw material. This leads to an increase in all components, but this was followed by destruction of some anthraquinone aglycones, thereby a
long cooking time. For example, the relative amounts of the anthraquinone aglycones have
been dramatically reduced between 5 and 10 min of baking, and this was accompanied with
a composition reduction of the anthraquinone glycoside derivatives. A destruction of the
anthraquinone dimer derivatives forming anthraquinone monomer glycosides was
suggested.
Yen and Chung (1999) studied the influence of increasing the heat on water extracts from
Cassia tora L. seeds, prepared under several degrees of roasting. It was revealed that the total
content of anthraquinones in water extracts was in the order of unroasted >150°C roasted
>200°C roasted, ensuring that anthraquinones were damaged by temperature-related treatments. Wu and Yen (2004) have investigated the components of chrysophanol, emodin, and
rhein in C. tora seeds, exhibiting also that the unroasted samples contained the largest anthraquinones content.
3.9.7 Pharmaceutical applications of phloroglucinols, xanthones, and anthrones
3.9.7.1 Phloroglucinols
Phloroglucinol-containing extracts have been used as traditional medicine in China, Southern Africa, and Latin America (Wan et al., 2019). Naturally occurring dimeric phloroglucinols
have revealed various biological activities such as anticancer activity (Chauthe et al., 2012). As
a result of its biological activity, phloroglucinol has been incorporated in many medicines,
cosmetics, pesticides as well as paints, cements, and dyes (Kim et al., 2015). Various antispasmodic formulations containing phloroglucinol as active ingredient have been patented. The
therapeutic safety and efficacy of the phloroglucinol derivatives have been proved through
pharmacological evaluations (Singh et al., 2009).
The mechanism of antispasmodic action of phloroglucinol is via inhibiting catecholo-methyltransferase action in smooth muscle cells. It has also been used for renal colic
and irritable bowel syndrome in Korea, China, Italy, and France. The phloroglucinoltrimethoxybenzene combination is used as antispasmolytic agent in urology for the treatment of ureteral lithiasis and also in obstetrics. Moreover, the use of phloroglucinol
trimethyl ether is common in treating biliary and intestinal diseases (Singh and Bharate,
2006). Lately, studies have revealed the potential antioxidant action of phloroglucinol on
experimental models in vivo and in vitro. It is also indicated that phloroglucinol diminishes
cell damage that results from H2O2 in lung fibroblast or cancer cells. Furthermore, it attenuates γ-ray-induced oxidative stress through enhancing the antioxidant defense of the body
and upregulates enzymes like catalase and glutathione. Phloroglucinol’s antioxidant action
in the CNS enhanced motor function in animal model of Parkinson’s disease via increasing
nuclear factor-like 2 (Nrf2) activity, which controls the expression of antioxidant enzymes.
Yet, the exact antioxidant mechanism of action has not been fully elucidated (Yang et al.,
2018). Phloroglucinol has also been reported to have antidepressant action in hepatitis patients and it is also used to treat nephritic colic (Singh et al., 2009). In addition, there are a
II. Phenolics
3.9.7 Pharmaceutical applications of phloroglucinols, xanthones, and anthrones
191
couple of Japanese patents related to its antiherpes virus activity (Singh et al., 2009). The
antitumor activity of phloroglucinol is also widely reported especially in relation to the
eriocitrin and/or eriodictyol, 3,4-dihydroxycinnamic acid, and phloroglucinol formulation,
which is apoptosis inducing agents. The 2,6-diisobutyryl-4,4-diethylcyclohexane-1,3,5trione is one of the derivatives of phloroglucinol manufactured as medication for allergy
(Singh and Bharate, 2006). Another phloroglucinol under the trade name Spasfon is approved and widely marketed as tablet in France for irritable bowel syndrome, biliary/urinary tract spasm, in painful periods and to reduce contraction of uterus during pregnancy.
It is also used widely as oxidant and colorant in hair dyes. Buflomedil hydrochloride is
phloroglucinol’s aminoketone derivative and it is marketed as tablets and injections
(Singh et al., 2009).
3.9.7.2 Xanthones
Naturally occurring xanthones have protruded as a significant phytochemical group based
on their valuable pharmacological and other biological activities. The biological activities of
xanthones are related to their tricyclic scaffold but differ with respect to the nature and/or
position of the various substituents (Pinto et al., 2005). It has been found that many plant
products regularly used in traditional medicine as therapeutic agents contain xanthones as
active constituents (Pinto et al., 2005; El-Seedi et al., 2010; Panda et al., 2013). Regarding their
pharmacological importance, the approach of scientists turned toward synthesis of xanthones
as novel drugs in addition to isolation from natural products (Shagufta and Ahmad, 2016).
High antioxidant activity of the xanthone extracts of G. mangostana L. was indicated by a
wide range of studies in the literature ( Jung et al., 2006; Yu et al., 2007). In addition, xanthones
and extracts from mangosteen could be potent candidates against Alzheimer’s disease (Wang
et al., 2012) along with antiinflammatory, antibacterial, antimalarial, antifungal, antiviral,
anti-HIV, antiallergic, and antitumoral properties (Gutierrez-Orozco and Failla, 2013;
Shagufta and Ahmad, 2016).
Xanthones exhibit a considerable action against depression and tuberculosis; meanwhile,
xanthone glycosides may act as depressant agents. Furthermore, some of xanthones have also
emerged such as choleretic, diuretic, antimicrobial, antiviral, and cardiotonic actions (Peres
and Nagem, 1997). The antifungal activity was also documented on some species of fungi.
Antiviral activity has been documented for some xanthones identified in African plants.
1,7-Dihydroxyxanthone showing an anti-HIV property in cells genetically modified and
infected with HIV-1 (EC50) 1.00 μg/mL (Kang and Xu, 2008), and also inhibited HIV-1 reverse
transcriptase (IC50: 50.1 μg/mL) (Iinuma et al., 1996). The hit compounds involved in cancer
chemotherapy include 5,6-dimethylxanthenone-4-acetic acid (DMXAA), psorospermin,
mangiferin, norathyriol, mangostins, and 6-isopropoxy9-oxoxanthene-2-carboxylic acid
(AH6809), a prostanoid receptor antagonist (Pinto et al., 2005). Gambogic acid was found
to have a remarkable value in the field of antitumor chemotherapy (Anantachoke et al.,
2012). Some xanthones may have a possible role in treating diabetes mellitus through their
strong inhibitory action toward α-glucosidase (E.C.3.2.1.20). Moreover, it was suggested that
enhancing cardiovascular health can be achieved via the inhibitory effect of xanthones on
xanthine oxidase (Dawson and Walters, 2006).
II. Phenolics
192
3. Analysis of polyphenolics
3.9.7.3 Anthrones
The
barbaloin
(10-beta-D-glucopyranosyl-1,8-dihydroxy-3-hydroxymethyl-9(10H)anthracenone) is considered to be the most specific secondary phytochemical product in Aloe
species. Yellow fluorescence is one of the main characteristics of barbaloin. Barbaloin is
C-glucoside of aloe emodin anthrone, occurring in the outer peel of the aloe plant. Along with
its defensive role against herbivores, barbaloin constitutes up to 30% of the dried leaf exudates in aloe plants.
Barbaloin has been reported to have a potential inhibitory action on mast cells that results
in controlling histamine release. Importantly, it is more potent than indomethacin regarding
antiinflammatory effect. These results indicate that barbaloin acts through several active sites
at mast cells. Interestingly, barbaloin is converted into aloe emodin when taken orally as it is
poorly absorbed, while aloe emodin is promptly absorbed. Barbaloin and aloe emodin are
broadly used for its purgative properties and as a bitterish agent in alcoholic beverages
(Patel et al., 2012).
Barbaloin elucidates antiinflammatory and purgative effects in vivo. In vitro studies
indicated that it has preferential toxicity to carcinoma cells and it is a strong inhibitor
of stellate cell transformation (Chang et al., 2006). Moreover, Barbaloin has a notable laxative effect, which was detected in rats. Some methods were developed and validated for
the determination of barbaloin such as colorimetry, fluorometry, and HPLC ( Jun
et al., 2002).
3.9.8 Trends and concluding remarks
Phloroglucinols, xanthones, and anthrones are important plants derivatives with wide
range of pharmacological activities such as antiviral and antiinflammatory activities. Interestingly, among monomeric phloroglucinols, two phloroglucinol terpene adducts, robustadials
A and B, exhibit in vivo antimalarial activity against P. berghei, macrocarpals have strong
antibacterial and HIV-RTase inhibitory activity, cyclic polyketides such as hypericin is distinctive for its antidepressant effect. Naturally occurring xanthones have emerged as an important phytochemical in view of their remarkable pharmacological and other biological
activities. Isolation and fractionation of Phloroglucinols, xanthones, and anthrones is done
via different techniques. Solid-state NMR, TLC, HPLC, GC-MS, IR, UV, MS, HPLC-ESIQTOF-MS, and Folin-Ciocalteu (FC) assays are used to identify and quantify phloroglucinols,
xanthones, and anthrones. High antioxidant activity of the xanthone extracts of G. mangostana
L. was indicated by a large number of studies in the literature. Future research work is encouraged in order to explore further the potential biological activities of these compounds
and to illustrate their applications for curing certain human ailments.
Acknowledgment
All the authors of the manuscript thank and acknowledge their respective universities and institutes.
II. Phenolics
References
193
Conflict of interest
There is no conflict of interest.
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