Camu-Camu Ellagitannins: Antioxidant Activity & Food Science

LWT - Food Science and Technology 69 (2016) 76e81
Contents lists available at ScienceDirect
LWT - Food Science and Technology
journal homepage: www.elsevier.com/locate/lwt
Antioxidant activity of C-Glycosidic ellagitannins from the seeds and
peel of camu-camu (Myrciaria dubia)
Tai Kaneshima, Takao Myoda*, Mayuko Nakata, Takane Fujimori, Kazuki Toeda,
Makoto Nishizawa
Faculty of Bio-industry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 May 2015
Received in revised form
22 December 2015
Accepted 10 January 2016
Available online 13 January 2016
C-Glycosidic ellagitannins grandinin (1), vescalagin (2), castalagin (3), methylvescalagin (4), stachyurin
(5), and casuarinin (6) were isolated from the 50% acetone extract of camu-camu (Myrciaria dubia) seeds
and peel. These tannins exhibited antioxidant activities measured by 1,1-diphenyl-2-picrylhydrazyl
(DPPH) and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, and the oxygen
radical absorbance capacity (ORAC) assay. In the DPPH and ABTS assays, stachyurin exhibited the
strongest antioxidant activity among the tannins, and the activities were stronger for tannins containing
two hexahydroxydiphenoyl (HHDP) groups and a galloyl group than for tannins containing a nonahydroxyterphenoyl group and a HHDP group. The activity of vescalagin was stronger than that of
castalagin, and a similar relationship was observed for stachyurin and casuarinin. Thus, the antioxidant
activities of these tannins may depend on the conformation of the hydroxyl group at C-1 of the open ring
D-glucose. However, in the ORAC assay, casuarinin exhibited the strongest activity, and the respective
activities could not be explained by the structural rigidity or conformation of the C-1 hydroxyl group.
The seeds and peel of camu-camu, waste products of juice production, were found to contain Cglycosidic ellagitannins with potent antioxidant activities, and thus, they could be a useful resource for
functional foods and food additives.
© 2016 Elsevier Ltd. All rights reserved.
Keywords:
C-glycosidic ellagitannin
Myrciaria dubia
Antioxidant activity
Camu-camu
Seeds
Chemical compounds studied in this article:
Grandinin (PubChem CID: 492392)
Vescalagin (PubChem CID: 168165)
Castalagin (PubChem CID: 168165)
Stachyurin (PubChem CID: 157395)
Casuarinin (PubChem CID: 157395)
1. Introduction
The biological activities of polyphenols have attracted attention
as they have proven to be effective in the prevention of lifestylerelated diseases and in the maintenance of human health. Among
polyphenols, flavonols, isoflavones, flavan-3-ols, and anthocyanidins have been extensively studied, and several of them have been
utilized in a variety of functional foods (Del Rio et al., 2013). Tannins
are categorized as polyphenols as they contain many phenolic hydroxyl groups in their structures. Therefore, the number of reports
on the biological activities of tannins has steadily increased as researchers have focused their attention on relationships between
biological activity and the structures of tannins.
Tannins are classified into two categories, condensed tannins
and hydrolysable tannins. Condensed tannins are comprised
* Corresponding author. Department of Food & Cosmetic Science, Tokyo University of Agriculture, Yasaka 196, Abashiri, Hokkaido 099-2493, Japan.
E-mail address: [email protected] (T. Myoda).
http://dx.doi.org/10.1016/j.lwt.2016.01.024
0023-6438/© 2016 Elsevier Ltd. All rights reserved.
of flavan-3-ols, and hydrolysable tannins are esters of
polyols (mostly D-glucose) and phenolic carboxylic acids such as
gallic acid, hexahydroxydiphenoic acid, valoneic acid, and nonahydroxyterphenoic acid. A variety of biological activities of
hydrolysable tannins have been reported including cytotoxicity
(Del Rio et al., 2013), inhibition of enzymes (Ochir et al., 2010), and
antimicrobial activities (Kamijo, Kanazawa, Funaki, Nishizawa, &
Yamagishi, 2008). Among hydrolysable tannins, C-glycosidic ellagitannins have structures characterized by a CeC bond between the
anomeric carbon of an open ring sugar and the unsubstituted carbon of a hexahydroxydiphenoyl (HHDP) or nonahydroxyterphenoyl
(NHTP) group. Recently, C-glycosidic ellagitannins were demonstrated to be sensory-active non-volatiles that migrate from oak
barrels into alcoholic beverages such as whisky, brandy, and wine
(Glabasnia & Hofmann, 2006). Moreover, biological activities of Cglycosidic ellagitannins including anti-herpes virus activity
(Quideau et al., 2004), alleviation of insulin resistance, inhibition of
adipocyte differentiation (Chang & Shen, 2013; Chang, Shen, & Wu,
2013), inhibition of a human breast cancer cell line growth (MCF-7)
T. Kaneshima et al. / LWT - Food Science and Technology 69 (2016) 76e81
and colon cancer cell line growth (Caco-2 and HT-29) (Fernandes
et al., 2009; Fridrich et al., 2008), and inhibition of human DNA
topoisomerase II (Auzanneau et al., 2012) have been reported.
We have been studying camu-camu (Myrciaria dubia) fruit, a
tropical fruit from Peru, because its juice is of interest in Japan and
other developed countries as an ingredient of functional foods.
Because the fruits are not easy to keep fresh, the juice was processed in Peru, and the residual seeds and peel became an industrial waste. Therefore, utilization of the residual seeds and peel
would be beneficial for the camu-camu industry. We have previously reported that the crude extract of camu-camu seeds and peel
contains large amounts of polyphenols (seeds: 369.4 mg/g, peel:
203.8 mg/g); (Myoda et al., 2010) approximately four and ten times
the amounts from the residues of acerola and passion fruit juice
production, respectively (de Oliveira et al., 2009). The extract of
camu-camu seeds and peel showed potent 1,1-diphenyl-2picrylhydrazyl (DPPH) radical scavenging activity (IC50 ¼ 32.2 mg/
mL), and C-glycosidic ellagitannins, vescalagin (2) and castalagin
(3) were shown to be responsible for the DPPH radical scavenging
activity (Kaneshima, Myoda, Toeda, Fujimori, & Nishizawa, 2013).
In this paper, we report the isolation and characterization of four
additional C-glycosidic ellagitannins and the relationship between
their antioxidant activities and structures.
2. Materials and methods
2.1. Materials and chemicals
The dried powder of the seeds and peel from camu-camu
juice production was obtained from Empresa Agroindustrial del
Peru S.A. (Peru). These samples were used after drying at room
temperature. Fluorescein sodium salt and (±)-6-Hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid (trolox) were purchased
from SigmaeAldrich Co. (St. Louis, USA). 2,20 -Azobis (S-methylpropionamidine) dihydrochloride (AAPH) and ascorbic acid were
purchased from Wako Pure Chemical (Osaka, Japan). Gallic acid, 1,1diphenyl-2-picrylhydrazyl (DPPH), and other chemicals were purchased from Kanto Chemicals (Tokyo, Japan).
2.2. General experimental methods
1
H and 13C NMR spectra of samples were measured in acetoned6 or D2O with an Agilent MR-400 NMR spectrometer. Chemical
shifts were determined using residual acetone (dH: 2.04 ppm, dC:
29.8 ppm) as the internal reference. Mass spectra were measured
with a JEOL JMS-700 spectrometer in negative FAB mode. Optical
rotations were measured on a JASCO P-2100 polarimeter. UVeVis
spectra were measured with a Shimadzu UV-1700 spectrophotometer. Gradient HPLC chromatograms were obtained with a
JASCO LC-2000 Plus HPLC system equipped with a MD-2010 Plus
photodiode array detector and an Atlantis T3 column (3 mm, 4.6 mm
i.d. 150 mm, Waters, Milford, MA, USA), using a solvent system of
acetonitrile e H2O - formic acid. Preparative HPLC was performed
with a Shimadzu LC-8A pump, a Hitachi L-4200 detector with a
prep cell (2 mm), and an Inertsil ODS-3 column (20 mm
i.d. 250 mm, GL Sciences, Tokyo, Japan). The mobile phase was
composed of 8e15% acetonitrile containing 0.1% acetic acid (flow
rate 6.0 mL/min), and peaks were detected at 280 nm. Sephadex
LH-20 (GE Healthcare, Sweden) was used for column
chromatography.
2.3. Extraction and isolation
The dried seeds sample (400 g) was defatted with n-hexane and
then extracted three times with 50% aqueous acetone (v/v) at room
77
temperature for 24 h. The combined extracts were concentrated to
dryness under reduced pressure at 40 C to obtain the crude extract
(43.9 g). The crude extract (5.0 g) was dissolved in 50% methanol
(MeOH, 500 mL) and was applied to a Sephadex LH-20 column
(50 mm i.d. 300 mm). The column was eluted with a solvent
system of H2O e MeOH e acetone, and 8 fractions were obtained:
50% MeOH Fr. (1.60 g), 60% MeOH Fr. (727 mg), 70% MeOH Fr.
(403 mg), 80% MeOH Fr. (268 mg), 90% MeOH Fr. (113 mg), 100%
MeOH Fr. (127 mg), 50% acetone Fr. (1.30 g) and 100% acetone Fr.
(232 mg). The 60e90% MeOH fractions were dissolved in 8e15%
acetonitrile, and the soluble portion was subjected to preparative
HPLC. From 1.0 g of crude extract, compounds 1 (2.3 mg), 2
(19.7 mg), 3 (41.9 mg), 4 (2.9 mg), 5 (2.9 mg), and 6 (5.2 mg) were
obtained. The purities of the isolated compounds were confirmed
by HPLC, 1H NMR, 13C NMR, and the molecular extinction coefficient
of their UV spectra.
2.3.1. Grandinin (1)
A light brown amorphous powder. UV (H2O) lmax ¼ 230 nm,
[a]D ¼ 14.8 (c ¼ 0.80, H2O), HR-MS; m/z ¼ 1065.1068 [MH]
(calcd. for C46H33O30, 1065.1044). 1H NMR (400 MHz, D2O): d 6.85 (s,
1H), 6.79 (s, 1H), 6.58 (s, 1H), 5.39 (d, J ¼ 7.0 Hz, 1H, H-5), 5.32 (s, 1H,
H-2), 4.97 (t, J ¼ 7.0 Hz, 1H, H-4), 4.76 (dd, J ¼ 5.2, 12.5 Hz, 1H, H-6),
4.56 (d, J ¼ 7.0 Hz, 1H, H-3), 4.11 (d, J ¼ 2.3 Hz, 1H, H-20 ), 4.03 (d,
J ¼ 5.2, 12.5 Hz, 1H, H-6), 3.84 (d, J ¼ 5.2 Hz, 1H, H-40 ), 3.82 (br.s, 1H,
H-30 ), 3.76 (dd, J ¼ 5.2, 10.0 Hz, 1H, H-50 ), 3.41 (d, J ¼ 1.2 Hz, 1H, H-1),
3.38 (d, J ¼ 10.0 Hz, 1H, H-50 ). 13C NMR (100 MHz, D2O): d 170.0,
167.4, 167.3, 166.7, 165.8, 146.4, 144.9, 144.8, 144.4, 143.7, 143.7,
143.6, 143.5, 142.9, 137.3, 136.7, 136.1, 135.1, 134.2, 126.2, 125.5,
125.3, 123.9, 123.1, 115.1, 114.0, 113.9, 113.5, 113.3, 111.9, 109.2, 109.2,
107.0, 100.5 (C-10 ), 72.1 (C-2), 71.2 (C-20 ), 71.0 (C-30 ), 70.7 (C-5), 70.5
(C-3), 69.5 (C-4), 65.8 (C-40 ), 65.0 (C-6), 62.0 (C-50 ), 45.1 (C-1).
2.3.2. Vescalagin (2) and castalagin (3)
UV (50% MeOH) lmax (log ε) ¼ 230 nm (4.68) and 230 nm (4.74),
respectively. For other characterization data, see the previous paper
(Kaneshima et al., 2013).
2.3.3. Methylvescalagin (4)
A light brown amorphous powder. UV (50% MeOH)
lmax ¼ 230 nm, [a]D ¼ 36.5 (c ¼ 0.09, MeOH), HR-MS; m/
z ¼ 947.0805 [MH] (calcd. for C42H27O26, 947.0780). 1H NMR
(400 MHz, acetone-d6 þ D2O): d 6.71 (s, 1H), 6.67 (s, 1H), 6.56 (s, 1H),
5.54 (d, J ¼ 7.3 Hz, 1H, H-5), 5.32 (br.s, 1H, H-2), 5.12 (t, J ¼ 7.3 Hz, 1H,
H-4), 4.92 (dd, J ¼ 2.5, 12.4 Hz, 1H, H-6), 4.60 (d, J ¼ 2.0 Hz, 1H, H-1),
4.44 (d, J ¼ 7.3 Hz, 1H, H-3), 4.01 (d, J ¼ 12.4 Hz, 1H, H-6), 3.51 (s, 3H,
OMe). 13C NMR (100 MHz, acetone-d6 þ D2O): d 168.8, 166.6, 166.0,
164.8, 164.8, 147.3, 144.7, 144.5, 144.5, 144.1, 144.0, 144.0, 143.9,
143.5, 136.7, 136.3, 135.7, 135.1, 134.4, 126.8, 125.8, 124.3, 124.2,
123.8, 115.6, 114.8, 114.2, 114.0, 113.7, 113.5, 112.4, 107.9, 107.5, 106.4,
73.7 (C-2), 72.4 (C-1), 70.3 (C-5), 68.8 (C-4), 67.5 (C-3), 64.8 (C-6),
56.1 (OMe).
2.3.4. Stachyurin (5)
A light brown amorphous powder. UV (50% MeOH) lmax
(log ε) ¼ 230 nm (4.68), [a]D ¼ 14.8 (c ¼ 0.02, MeOH), HR-MS; m/
z ¼ 935.0816 [MH] (calcd. for C41H27O26, 935.0780). 1H NMR
(400 MHz, acetone-d6 þ D2O): d 7.14 (s, 2H, galloyl-H), 6.95 (s, 1H,
HHDP-H), 6.59 (s, 1H, HHDP-H), 6.54 (s, 1H, HHDP-H), 5.76 (dd,
J ¼ 2.0, 8.6 Hz, 1H, H-4), 5.36 (dd, J ¼ 3.3, 8.6 Hz, 1H, H-5), 5.05 (t,
J ¼ 2.0 Hz, 1H, H-3), 4.97 (d, J ¼ 2.0 Hz, 1H, H-1), 4.94 (dd, J ¼ 3.3,
13.1 Hz, 1H, H-6), 4.88 (t, J ¼ 2.0 Hz, 1H, H-2), 4.10 (d, J ¼ 13.1 Hz, 1H,
H-6). 13C NMR (100 MHz, acetone-d6 þ D2O): d 168.7, 168.6,
168.2166.1, 165.8, 146.2, 145.1, 145.0, 144.4, 144.4, 143.9, 143.9, 143.2,
143.0, 138.8, 137.8, 136.1, 135.3, 134.4, 126.5, 125.7, 123.9, 120.9,
78
T. Kaneshima et al. / LWT - Food Science and Technology 69 (2016) 76e81
119.5, 117.7, 115.6, 115.5, 115.2, 114.5, 109.4, 107.9, 106.4, 104.7, 80.5
(C-2), 72.4 (C-4), 71.2 (C-3), 70.2 (C-5), 63.8 (C-1),63.8 (C-6).
Gallic acid (30e50 mM) and ascorbic acid (100e200 mM) were used
as positive controls. The trolox equivalent values were calculated
using equation (2).
2.3.5. Casuarinin (6)
A light brown amorphous powder. UV (50% MeOH) lmax
(log ε) ¼ 230 nm (4.71), [a]D ¼ 14.8 (c ¼ 0.23, MeOH), HR-MS; m/
z ¼ 935.0808 [MH] (calcd. for C41H27O26, 935.0780). 1H NMR
(400 MHz, acetone-d6 þ D2O): d 7.02 (s, 2H, galloyl-H), 6.77 (s, 1H,
HHDP-H), 6.47 (s, 1H, HHDP-H), 6.42 (s, 1H, HHDP-H), 5.53 (d,
J ¼ 5.1 Hz, 1H, H-1), 5.41 (dd, J ¼ 2.0, 8.7 Hz, 1H, H-4), 5.38 (t,
J ¼ 2.0 Hz, 1H, H-3), 5.26 (dd, J ¼ 3.3, 8.7 Hz, 1H, H-5), 4.82 (dd,
J ¼ 3.3, 13.0 Hz, 1H, H-6), 4.58 (dd, J ¼ 2.0, 5.1 Hz, 1H, H-2), 4.02 (d,
J ¼ 13.0 Hz, 1H, H-6). 13C NMR (100 MHz, acetone-d6 þ D2O):
d 169.2, 168.7, 168.2, 165.7, 165.2, 145.4, 145.2, 144.9, 144.3, 144.1,
144.1, 143.2, 142.8, 138.8, 138.4, 136.3, 135.3, 134.3, 126.3, 125.8,
123.7, 119.5, 118.7, 116.5, 115.7, 115.4, 115.4, 114.5, 109.3, 107.7, 106.2,
104.5, 76.2 (C-2), 73.2 (C-4), 70.3 (C-5), 68.9 (C-3), 66.0 (C-1), 63.7
(C-6).
The ORAC assay was performed as described by Prior et al.
(2003) with slight modifications. Sample solution (25 mL) at concentrations of 6.25 mg/mL (in 75 mM phosphate buffer, pH 7.4) were
added to 8.22 105 mM fluorescein solution (150 mL, 75 mM
phosphate buffer, pH 7.4), and the mixture was incubated at 37 C
for 10 min. Then, 153 mM AAPH solution (25 mL, 75 mM phosphate
buffer, pH 7.4) was added to the mixture. After shaking, the fluorescence intensity (Ex. 485 nm, Em 520 nm) was measured every
minute for 50 min. Final results were calculated using the liner
standard curve of Trolox (6.25e50 mM) and were expressed as
Trolox equivalents. Gallic acid (25 mM) and ascorbic acid (200 mM)
were used as positive controls.
2.4. DPPH radical scavenging assay
2.7. Statistical analysis
DPPH radical scavenging activity was measured using a spectrophotometric method (Blois, 1958) with slight modifications. A
freshly prepared solution of 100 mM DPPH in MeOH was used.
Sample solutions (20 mL) at concentrations of 5.0e100 mg/mL (in
75 mM phosphate buffer, pH 7.4) and 100 mM acetate buffer (80 mL,
pH 5.5) were mixed with 100 mM DPPH solution (100 mL) in a 96well plate. The mixture was shaken well and incubated in the
dark for 30 min at 30 C. The absorbance was measured at 517 nm
using a microplate reader (MTP-310, Corona Electric, Hitachi,
Japan).
The antioxidant activity of each sample was expressed as the
inhibition of DPPH radical scavenging activity as follows:
Results of the antioxidant assays were expressed as
means ± standard error of triplicate assays. Statistical treatment of
the data was performed using JMP 11 software (SAS Institute Inc.,
Cary, NC USA). The results were analyzed using ANOVA followed by
the Tukey test for statistical comparisons among groups, with a
value of p < 0.05 indicating significance. Comparisons of samples
among these compounds were done by Student's t-test.
i
h
.
Inhibition ð%Þ ¼ fAðcontrolÞ AðsampleÞ g AðcontrolÞ 100
The crude extract of camu-camu seeds or peel was fractionated
by chromatography on Sephadex LH-20, and following purification
by preparative reverse-phase HPLC resulted in the isolation of
grandinin (1), methylvescalagin (4), stachyurin (5), and casuarinin
(6) along with the previously reported vescalagin (2) and castalagin
(3). Vescalagin (2) and castalagin (3) were found to be the main Cglycosidic ellagitannins, approximately 5% and 10% of the total
polyphenols, respectively, as shown in Fig. 1. Spectral data indicated
that these compounds were C-glycosidic ellagitannins with a
bridging open ring D-glucose (Fig. 2).
In 1H and 13C NMR spectra, five additional sugar proton and five
additional sugar carbon signals were observed for compound 1
compared with vescalagin (2). 2D NMR experiments showed that
(1)
IC50 values expressed in mM were calculated from inhibition
curves. The assays were carried out in triplicate. Gallic acid
(30e50 mM) and ascorbic acid (100e200 mM) were used as positive
controls. The trolox equivalent values were calculated as follows:
Trolox equivalent ¼ troloxIC50 ðMÞ=sampleIC50 ðMÞ
(2)
2.5. ABTS radical scavenging assay
ABTS radical scavenging activity was measured using a spectrophotometric method (Thaipong, Boonprakob, Crosby, CisnerosZevallos, & Hawkins Byrne, 2006) with slight modifications. The
aqueous stock solution included 7.4 mM ABST and 2.6 mM potassium persulfate. The working solution was prepared by mixing the
two stock solutions in equal quantities and allowing them to react
for 15 h at 25 C in the dark. The solution was then diluted by
mixing 4 mL of reacted solution with 30 mL of MeOH to obtain an
absorbance of 0.6 at 660 nm using the spectrophotometer. Working
solution was prepared for each assay. Sample solutions (50 mL) at
concentrations of 6.25e25 mg/mL (in 75 mM phosphate buffer, pH
7.4) and MeOH (50 mL) were mixed with ABTS working solution
(100 mL) in a 96-well plate. The mixture was shaken well and
incubated in the dark for 10 min at 30 C. The absorbance was
measured at 660 nm using a microplate reader.
The inhibition of ABTS radical scavenging activity was calculated
by equation (1). The IC50 values expressed in mM were calculated
from inhibition curves. The assays were carried out in triplicate.
2.6. ORAC assay
3. Results and discussion
3.1. Isolation and characterization of C-glycosidic ellagitannins
Fig. 1. HPLC chromatogram of the crude extract of camu-camu seeds. 1; grandinin, 2;
vescalagin, 3; castalagin, 4; methylvescalagin, 5; stachyurin, 6; casuarinin. For HPLC
conditions, see materials and methods.
T. Kaneshima et al. / LWT - Food Science and Technology 69 (2016) 76e81
79
Fig. 2. Structures of C-ellagitannin from camu-camu seeds. Compounds 1; grandinin, 2; vescalagin, 3; castalagin, 4; methylvescalagin, 5; stacyurin, 6; casuarinin.
compound 1 had a pentose moiety attached to vescalagin (2). The
pentose was determined to be attached to C-1 of the D-glucose via a
CeC linkage, because the anomeric carbon of D-glucose was
observed at d 45.1 and the H-2 signal of the glucose (d 5.32) showed
cross peaks with the anomeric carbon (d 100.5) of a pentose in the
HMBC spectrum. Comparing the spectral data with literature data
(Fridrich et al., 2008), compound 1 was characterized as grandinin
with a D-lyxose linked to C-1 of the D-glucose as a C-glycoside.
The 1H and 13C NMR spectra of compound 4 closely resembled
those of vescalagin (2), but signals of a methoxy methyl group were
observed at dH 3.51 and dC 56.1, and a cross peak was observed
between the methoxy methyl protons (d 3.51) and the anomeric
carbon (d 72.4) of D-glucose in the HMBC spectrum. Therefore,
compound 4 was characterized as methylvescalagin, and was
confirmed by comparison of the spectral data with literature data
(Yoshida et al., 1991).
The 1H and 13C NMR spectra of compounds 5 and 6 were analogous to those of vescalagin (2) and castalagin (3), respectively, but
signals for two HHDP groups and a galloyl group were observed in
the aromatic region of the 1H NMR spectra. HMBC experiments
showed the presence of a galloyl group at the C-5 position of the
open ring D-glucose, and two HHDP groups were attached between
C-2-C-3 and C-4-C-6. Therefore, compounds 5 and 6 were characterized as stachyurin and casuarinin, respectively, and the structures were confirmed by comparison of their spectral data with
literature data (Bai et al., 2008).
These C-glycosidic ellagitannins have been isolated from many
plants mainly belonging to Fagaceae, Myrtaceae, Juglandaceae,
Casuarinaceae, Stachyuraceae, Betulaceae, Punicaceae, Lythraceae,
(see reference in Nonaka, Sakai, Tanaka, Mihashi, & Nishioka, 1990),
Combretaceae (Lin, Hsu, & Cheng, 1993), Melastomataceae (Yoshida
et al., 1991), and Trapaceae (Hatano, Okonogi, Yazaki, & Okuda,
1990), and in most cases, these tannins were isolated from the
leaves, bark, wood, and branches. Tanaka, Nonaka, and Nishioka
(1986) isolated C-glycosidic ellagitannins from the fruits of Lagerstroemia speciosa (Lythraceae), and Hager, Howard, Liyanage, Lay,
and Prior (2008; 2009) detected these tannins in the seeds of
Rubus sp. (Rosaceae) by LC-MS, but camu-camu is the first example
of these tannins being isolated from the seeds and peel of a plant
belonging to Myrtaceae.
3.2. Antioxidant activities of C-glycosidic ellagitannins
DPPH and ABTS radical scavenging activities, and ORAC activities of compounds 1, 2, 3, 4, 5, and 6 are shown in Table 1. The
activities are expressed as trolox equivalents (mol TE/mol). ABTS
radical scavenging activities and ORAC activities of compounds 1
and 4 were not tested because of the small amounts of these
compounds that were recovered. The antioxidant activities of the
tannins were found to be two times more potent than gallic acid
and ten times more potent than ascorbic acid.
Among these tannins, grandinin (1) and methylvescalagin (4)
have the same conformation as vescalagin (2), but their DPPH
scavenging activities were 7.19, 7.52, and 7.81 (mol TE/mol),
respectively, which were significantly different from each other. As
shown in Fig. 2, the configuration of the moieties of compound 2
and those of compounds 1 and 4 were almost the same, but
replacement of the C-1-hydroxyl group of compound 2 by a
methoxy group or a CeC bonded D-lyxose caused a significant
decrease in activity. This decrease in activity is presumably due to
steric effects from the replaced group on the phenolic hydroxyl
groups of ring-I of the NHTP group.
Vescalagin (2) and castalagin (3), and stachyurin (5) and casuarinin (6) are pairs of diastereomers that have opposite configurations at C-1 of the open ring D-glucose. Compounds 2 and 3 are
hypothetically formed by oxidative coupling of the HHDP group
attached between C-2 and C-3 and the galloyl group at C-5 of stachyurin (5) and casuarinin (6), respectively (Quideau et al., 2004).
The spatial positions of the galloyl moieties for compounds 5 and 6
are apart from the HHDP groups, but for compounds 2 and 3, CeC
bonds between the HHDP and galloyl groups constrain rotation
around C-3, C-4, and C-5. Compounds 5 and 6 exhibited more
potent DPPH and ABTS radical scavenging activities (single electron
transfer assays) than compounds 2 and 3, respectively (Table 1).
This result might be due to flexibility around C-3, C-4, and C-5. The
DPPH radical scavenging activities of ellagitannins with HHDP and
galloyl group(s) were reported to be more potent than those with
only HHDP group(s) (Fukuda, Ito, & Yoshida, 2003), and our results
are in agreement with their findings.
On the other hand, the results of antioxidant activities from the
ORAC assay, a hydrogen atom transfer assay, were different from
80
T. Kaneshima et al. / LWT - Food Science and Technology 69 (2016) 76e81
Table 1
DPPH and ABTS radical scavenging activities and the ORAC of C-glycosidic ellagitannins from camu-camu seeds.
(mol TE/mol)
DPPH radical scavenging activity
±
±
±
±
±
±
0.05e
0.06c
0.06de
0.05d
0.08a
0.05b
Grandinin (1)
Vescalagin (2)
Castalagin (3)
Methylvescalagin (4)
Stachyurin (5)
Casuarinin (6)
7.19
7.81
7.42
7.52
9.87
8.75
Gallic acid
Ascorbic acid
3.08 ± 0.03f
0.65 ± 0.00g
ABTS radical scavenging activity
ORAC
e
6.58
6.43
e
7.61
7.47
e
2.97
3.36
e
2.50
3.71
± 0.12b
± 0.02b
± 0.04a
± 0.02a
3.30 ± 0.04c
0.80 ± 0.00d
± 0.03c
± 0.07b
± 0.02d
± 0.13a
1.43 ± 0.04e
0.11 ± 0.00f
Each value is the mean ± SE, n ¼ 3. Trolox equivalent values with different letters in the same column are statistically different at p < 0.05.
e; Not tested.
those of the single electron transfer assays. Compound 3 was found
to be a more potent antioxidant than compound 2, and this relationship was similar for compounds 5 and 6. Comparing compounds 3 and 6, compound 6 was a more potent antioxidant than
compound 3, but the relationship was not the same for compounds
2 and 5.
Vivas, Laguerre, De Boissel, De Gaulejac, and Nonier (2004)
pointed out that 2 had more polar chromatographic behavior and
was more prone to oxidation and thermal decomposition, and they
concluded that the orientation of the hydroxyl group at C-1 of the
open ring D-glucose affected the molecular lipophilicity and molecular electrostatic potential. Quideau et al. (2004) reported antiherpes virus activities of compounds 2 and 3, and they concluded
that the difference in activities might be due to the orientation of
the hydroxyl group at C-1. The hydroxyl group of compound 3 has
an orientation that allows it to form a hydrogen bond with the
phenolic hydroxyl group of the NHTP I-ring, whereas the hydroxyl
group of compound 2 cannot form a similar H-bond.
The antioxidant activity of the galloyl moiety (three adjacent
phenolic hydroxyl groups) was reported to be responsible for stabilization of oxygen radical through hydrogen bonds to adjacent
hydroxyl groups (Ajitha, Mohanlal, Suresh, & Jayalekshmy, 2012). In
the case of compound 3 (also compound 6), the hydroxyl group at
the C-1 position participates in a hydrogen bond with the phenolic
hydroxyl group in the I-ring of the NHTP group; therefore, stability
of a 4-O-radical would be reduced.
Considering our results and those from previous reports, antioxidant activities of C-glycosidic ellagitannins measured by single
electron transfer assays (DPPH and ABTS radical scavenging) can be
rationalized on the basis of the flexibility of rotation around C-3, C4, and C-5 of the D-glucose and the orientation of the hydroxyl
group at the C-1 position, which causes a difference in the stability
of oxygen radical. However, antioxidant activities of C-glycosidic
ellagitannins measured by the ORAC assay (hydrogen atom transfer
assay) were revealed to be controlled by other mechanism(s). More
studies are needed to clarify this mechanism(s).
The seeds and peel of camu-camu (Myrciaria dubia) are industrial waste products from the production of camu-camu juice, thus,
applications of the seeds and peel for functional foods and food
additives may provide a financial benefit to the camu-camu industry. Moreover, recently, the food additive importance and biological activities of vescalagin (2) and castalagin (3) have been
extensively studied, and camu-camu seeds and peel could become
important sources of these compounds.
4. Conclusion
Six C-glycosidic ellagitannins (compounds 1, 2, 3, 4, 5, and 6)
were isolated from the seeds and peel of camu-camu (Myrciaria
dubia), and this study represents the first time these tannins have
been isolated from fruit seeds and peel. These tannins (compounds
2, 3, 5 and 6) exhibited stronger antioxidant activities measured by
both single electron transfer assays and a hydrogen atom transfer
assay than gallic acid and ascorbic acid. The peel and seeds of camucamu are industrial waste products from the production of camucamu juice, thus, applications of the seeds and peel as functional
foods and food additives may be beneficial for the camu-camu
industry.
Acknowledgment
The authors wish to express their gratitude to Empresa Agroindustrial del Peru S.A. for the donation of camu-camu samples, and
to Professor Koji Wada of the Hokkaido Pharmaceutical University
School of Pharmacy.
References
Ajitha, M. J., Mohanlal, S., Suresh, C. H., & Jayalekshmy, A. (2012). DPPH radical
scavenging activity of tricin and its conjugates isolated from “njavara” rice bran:
a density functional theory study. Journal of Agricultural and Food Chemistry, 60,
3693e3699.
gu, L., Deffieux, D., et al.
Auzanneau, C., Montaudon, D., Jacquet, R., Puyo, S., Pouyse
(2012). The polyphenolic ellagitannin vescalagin acts as a preferential catalytic
inhibitor of the a isoform of human DNA topoisomerase II. Molecular Pharmacology, 82, 134e141.
Bai, N., He, K., Roller, M., Zheng, B., Chen, X., Shao, Z., et al. (2008). Active compounds from lagerstroemia speciosa, insulin-like glucose uptake-stimulatory/
inhibitory and adipocyte differentiation-inhibitory activities in 3T3-L1 cells.
Journal of Agricultural and Food Chemistry, 56, 11668e11674.
Blois, M. S. (1958). Antioxidant determinations by the use of a stable free radical
[10]. Nature, 181(4617), 1199e1200.
Chang, W.-C., & Shen, S.-C. (2013). Effect of water extracts from edible myrtaceae
plants on uptake of 2-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose in TNF-a-treated FL83B mouse hepatocytes. Phytotherapy Research, 27,
236e243.
Chang, W.-C., Shen, S.-C., & Wu, J. S.-B. (2013). Protective effects of vescalagin from
pink wax apple [Syzygium samarangense (blume) merrill and perry] fruit
against methylglyoxal-induced inflammation and carbohydrate metabolic disorder in rats. Journal of Agricultural and Food Chemistry, 61, 7102e7109.
Del Rio, D., Rodriguez-Mateos, A., Spencer, J. P. E., Tognolini, M., Borges, G., &
Crozier, A. (2013). Dietary (poly)phenolics in human health: structures,
bioavailability, and evidence of protective effects against chronic diseases. Antioxidants and Redox Signaling, 18, 1818e1892.
Fernandes, A., Fernandes, I., Cruz, L., Mateus, N., Cabral, M., & De Freitas, V. (2009).
Antioxidant and biological properties of bioactive phenolic compounds from
quercus suber L. Journal of Agricultural and Food Chemistry, 57, 11154e11160.
Fridrich, D., Glabasnia, A., Fritz, J., Esselen, M., Pahlke, G., Hofmann, T., et al. (2008).
Oak ellagitannins suppress the phosphorylation of the epidermal growth factor
receptor in human colon carcinoma cells. Journal of Agricultural and Food
Chemistry, 56, 3010e3015.
Fukuda, T., Ito, H., & Yoshida, T. (2003). Antioxidative polyphenols from walnuts
(Juglans regia L.). Phytochemistry, 63, 795e801.
Glabasnia, A., & Hofmann, T. (2006). Sensory-directed identification of taste-active
ellagitannins in american (Quercus alba L.) and european oak wood (Quercus
robur L.) and quantitative analysis in bourbon whiskey and oak-matured red
wines. Journal of Agricultural and Food Chemistry, 54, 3380e3390.
Hager, T. J., Howard, L. R., Liyanage, R., Lay, J. O., & Prior, R. L. (2008). Ellagitannin
T. Kaneshima et al. / LWT - Food Science and Technology 69 (2016) 76e81
composition of blackberry as determined by HPLC-ESI-MS and MALDI-TOF-MS.
Journal of Agricultural and Food Chemistry, 56, 661e669.
Hager, T. J., Howard, L. R., Liyanage, R., Lay, J. O., & Prior, R. L. (2009). Antioxidant
properties of raspberry seed extracts on micronucleus distribution in peripheral
blood lymphocytes. Food and Chemical Toxicology, 47, 2853e2859.
Hatano, T., Okonogi, A., Yazaki, K., & Okuda, T. (1990). Trapanins A and B, oligomeric
hydrolysable tannins from Trapa japonica FLEROV. Chemical and Pharmaceutical
Bulletin, 38, 2707e2711.
Kamijo, M., Kanazawa, T., Funaki, M., Nishizawa, M., & Yamagishi, T. (2008). Effects
of rosa rugosa petals on intestinal bacteria. Bioscience, Biotechnology and
Biochemistry, 72, 773e777.
Kaneshima, T., Myoda, T., Toeda, K., Fujimori, T., & Nishizawa, M. (2013). Antioxidative constituents in camu-camu fruit juice residue. Food Science and
Technology Research, 19, 223e228.
Lin, T.-C., Hsu, F.-L., & Cheng, J.-T. (1993). Antihypertensive activity of corilagin and
chebulinic acid, tannins from lumnitzera racemosa. Journal of Natural Products,
56, 629e632.
Myoda, T., Fujimura, S., Park, B., Nagashima, T., Nakagawa, J., & Nishizawa, M. (2010).
Antioxidative and antimicrobial potential of residues of camu-camu juice production. Journal of Food, Agriculture and Environment, 8, 304e307.
Nonaka, G., Sakai, T., Tanaka, T., Mihashi, K., & Nishioka, I. (1990). Tannins and
related compounds. XCVII. Structure revision of C-glycosidic ellagitannins,
castalagin, vescalagin, casuarinin and stachyurin, and related hydrolyzable
tannins. Chemical and Pharmaceutical Bulletin, 38, 2151e2156.
Ochir, S., Nishizawa, M., Jae Park, B., Ishii, K., Kanazawa, T., Funaki, M., et al. (2010).
Inhibitory effects of rosa gallica on the digestive enzymes. Journal of Natural
Medicines, 64, 275e280.
81
de Oliveira, A. C., Valentim, I. B., Silva, C. A., Bechara, E. J. H., Barros, M. P.d.,
Mano, C. M., et al. (2009). Total phenolic content and free radical scavenging
activities of methanolic extract powders of tropical fruit residues. Food Chemistry, 115, 469e475.
Prior, R. L., Hoang, H., Gu, L., Wu, X., Bacchiocca, M., Howard, L., et al. (2003). Assays
for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance
capacity (ORACFL)) of plasma and other biological and food samples. Journal of
Agricultural and Food Chemistry, 51, 3273e3279.
Quideau, S., Varadinova, T., Karagiozova, D., Jourdes, M., Pardon, P., Baudry, C., et al.
(2004). Main structural and stereochemical aspects of the antiherpetic activity
of nonahydroxyterphenoyl-containing C-glycosidic ellagitannins. Chemistry and
Biodiversity, 1, 247e258.
Tanaka, T., Nonaka, G., & Nishioka, I. (1986). Tannins and related compounds. XLI.
Isolation and characterization of novel ellagitannins, punicacorteins A, B, C and
D, and punigluconin from the bark of Punica granatum L. Chemical and Pharmaceutical Bulletin, 34, 656e663.
Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., & Hawkins Byrne, D.
(2006). Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating
antioxidant activity from guava fruit extracts. Journal of Food Composition and
Analysis, 19, 669e675.
Vivas, N., Laguerre, M., De Boissel, I. P., De Gaulejac, N. V., & Nonier, M.-F. (2004).
Conformational interpretation of vescalagin and castalagin physicochemical
properties. Journal of Agricultural and Food Chemistry, 52, 2073e2078.
Yoshida, T., Ohbayashi, H., Ishihara, K., Ohwashi, W., Haba, K., Okano, Y., et al. (1991).
Tannins and related polyphenols of melastomataceous plants. I. hydrolyzable
tannins from tibouchina semidecandra COGN. Chemical and Pharmaceutical
Bulletin, 39, 2233e2240.