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molecules
Review
Saponins from Quillaja saponaria and Quillaja
brasiliensis: Particular Chemical Characteristics and
Biological Activities
Juliane Deise Fleck 1 , Andresa Heemann Betti 2 , Francini Pereira da Silva 1 ,
Eduardo Artur Troian 1 , Cristina Olivaro 3 , Fernando Ferreira 4 and Simone Gasparin Verza 1, *
1
2
3
4
*
Molecular Microbiology Laboratory, Institute of Health Sciences, Feevale University,
Novo Hamburgo 93525-075, RS, Brazil; [email protected] (J.D.F.);
[email protected] (F.P.d.S.); [email protected] (E.A.T.)
Bioanalysis Laboratory, Institute of Health Sciences, Feevale University, Novo Hamburgo 93525-075, RS,
Brazil; [email protected]
Science and Chemical Technology Department, University Center of Tacuarembó, Udelar, Tacuarembó 45000,
Uruguay; [email protected]
Organic Chemistry Department, Carbohydrates and Glycoconjugates Laboratory, Udelar,
Mondevideo 11600, Uruguay; [email protected]
Correspondence: [email protected]; Tel.: +55-51-3586-8800
Academic Editors: Vassilios Roussis and Efstathia Ioannou
Received: 25 October 2018; Accepted: 28 December 2018; Published: 4 January 2019
Abstract: Quillaja saponaria Molina represents the main source of saponins for industrial applications.
Q. saponaria triterpenoids have been studied for more than four decades and their relevance is due to
their biological activities, especially as a vaccine adjuvant and immunostimulant, which have led to
important research in the field of vaccine development. These saponins, alone or incorporated
into immunostimulating complexes (ISCOMs), are able to modulate immunity by increasing
antigen uptake, stimulating cytotoxic T lymphocyte production (Th1) and cytokines (Th2) in
response to different antigens. Furthermore, antiviral, antifungal, antibacterial, antiparasitic,
and antitumor activities are also reported as important biological properties of Quillaja triterpenoids.
Recently, other saponins from Q. brasiliensis (A. St.-Hill. & Tul.) Mart. were successfully tested
and showed similar chemical and biological properties to those of Q. saponaria barks. The aim
of this manuscript is to summarize the current advances in phytochemical and pharmacological
knowledge of saponins from Quillaja plants, including the particular chemical characteristics of these
triterpenoids. The potential applications of Quillaja saponins to stimulate further drug discovery
research will be provided.
Keywords: triterpenoids; Quillaja saponaria; Quillaja brasiliensis;
antimicrobial; antiviral
vaccine;
immunoadjuvant;
1. Introduction
Quillaja saponaria Molina is commonly found in Peru, Chile, and Bolivia. It was first described in
1782 due to the saponin content in the bark. The expression ‘saponin’ is Latin in origin: the word sapo
that means ‘soap’, since saponins form foams when mixed with water [1,2]. The amphiphilic structure
of saponins, due to their lipophilic aglycones and hydrophilic saccharide side chains, is responsible
for the foam formation and chemical properties [1,2]. The most commonly reported aglycone from
Q. saponaria, i.e., quilaic acid [3,4], is characterized by an aldehyde group at the C-23 position. The acyl
side chain, another characteristic of Quillaja saponins, seems to also play an important role in some
biological activities [5].
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Saponins from Q. saponaria have a wide range of applications. Historically, Quillaja bark saponins
have been used as a detergent. Nowadays they are approved for use as food additives in 187 signatory
countries of the Codex Alimentarius, including the European Union, the United Kingdom, the United
States, China, and Japan [6], the latter of which also allows its use in cosmetics [7]. The European
Commission of Cosmetic Ingredients (CosIng) database has listed some Quillaja products as cosmetic
ingredients, such as the bark, bark extract, root extract, and wood extract. Depending on the material
used, different functions are attributed, such as antidandruff, cleansing, emulsifying, foaming, masking,
moisturizing, skin conditioning, and surfactant [8].
Nowadays, there is increasing interest in using saponins as natural emulsifiers, alone or blended
with others. Numerous studies have reported that Quillaja saponins are good emulsifiers for O/W
emulsions [7,9,10] and nanoemulsions [10]. Recently, a food ingredient containing Quillaja bark
saponins (Q-Naturale® ) was approved by the FDA as an effective emulsifier for beverages [6,10].
In addition to their detergent and emulsifying properties, other pharmacological activities,
including antiviral [11–13], antifungal [14], antibacterial [15–17], and antiparasitic ones, have been
reported for Quillaja saponins. Regarding its biological properties, Q. saponaria has been included in
the European and Brazilian Pharmacopoeias. The raw material is recommended as a cough reliever
and expectorant, anti-inflammatory, hypocholesterolemic, and hemolytic agent. Topically, it has been
indicated to treat scalp diseases (dandruff and hair loss) and other dermatological disorders. In the
African Pharmacopoeia, Quillaja bark has been indicated as an emulsifying agent only.
Meanwhile, the strong immunoadjuvant activity reported for Quil-A® , a crude extract of
Q. saponaria bark, and its derivatives, makes them ideal substances for vaccine development [5,18–21].
Since Quil-A® is a heterogeneous mixture of saponins, its various components may exhibit different
biological properties, including toxicological effects [5]. Thus, more purified saponin fractions have
been obtained, especially QS-21, which has been applied in preclinical studies and clinical trials [5,21].
An improvement to reduce saponin toxicity and enhance immunoadjuvant activity has been
reported with the development of immunostimulating complexes (ISCOMs). ISCOMS are cage-like
particles (40 nm), composed of saponins derived from Q. saponaria, phospholipids, cholesterol, and the
targeted antigen [22–24]. Recently, a purified fraction of Quillaja brasiliensis saponins was also tested
in ISCOMs, and the typical cage-like structures were viewed [25]. It is important to note that these
structures are described only for Quillaja species.
Q. brasiliensis is native to Northern Uruguay, Southern Brazil, Northeastern Argentina, and Eastern
Paraguay. It has also been studied as an immunoadjuvant, with crude extracts and purified
saponin fractions being evaluated and shown to stimulate a great immune response to viral antigens,
as observed with saponins from Q. saponaria [25–28]. Saponins are also abundant in the leaves of
Q. brasiliensis, which represents a renewable source of these substances, since in Q. saponaria the main
source of saponins is the barks of the tree. Nowadays, the overexploitation of native forests has been
replaced and other alternative sources are available such as synthetic analogues of QS-21 saponin.
Meanwhile, natural alternative sources of these products are also important [29].
Considering the relevance and biological properties already described for Quillaja species,
a systematic review of published trials was accomplished with respect to their chemical characteristics
and biological activities. To perform the review, the PubMed database was searched using the
keywords Quillaja saponins, chemical characteristics, biological activity, adjuvant, antiviral, antifungal,
antibacterial, and toxicity, either alone or in combination.
2. Chemical Structure/Characterization
Despite their widespread use, the structure of Q. saponaria saponins was not completely elucidated
until 1986, when Higuchi et al. [3] published the structure of two deacetylated saponins from
Q. saponaria bark (Figure 1, Figure 2 and Table 1). Further isolation and structural studies allowed for the
identification of nearly 60 saponins from Q. saponaria, sharing several structural motifs. These saponins
contain a triterpenic aglycone belonging to the β-amyrin series, most frequently quillaic acid (Figure 1).
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Figure
1.
Reported
triterpene
aglycones
in
Quillaja
saponaria
and/or
Figure1.
1.Reported
Reportedtriterpene
triterpeneaglycones
aglyconesin
inQuillaja
Quillajasaponaria
saponariaand/or
and/or Q.
Q. brasiliensis
brasiliensis saponins.
saponins.
Figure
Figure
brasiliensis
saponins.
Figure 2.
2. General
General structure
structure of
of Q.
Q. saponaria
saponaria and
and Q.
Q. brasiliensis
brasiliensissaponins.
saponins.
These
These
saponins
are
bidesmosides,
glycosylated
at
the
C-3
and
C-28
positions
of
the
aglycone
These saponins
saponins are
are bidesmosides,
bidesmosides, glycosylated
glycosylated at
at the
the C-3
C-3 and
and C-28
C-28 positions
positions of
of the
the aglycone
aglycone
(Figure
2).
The
C-3
position
of
the
aglycone
is
most
frequently
glycosylated
by
the
disaccharide
residue
(Figure
(Figure 2).
2). The
The C-3
C-3 position
position of
of the
the aglycone
aglycone is
is most
most frequently
frequently glycosylated
glycosylated by
by the
the disaccharide
disaccharide
βD -Galp-(1
2)-β-D-GlcAp,
most frequently
branchedbranched
at the O-3
the glucuronic
acid unit
residue
β-Galp-(1→2)-βD
most
at
the
position
of
residue
β-D
D→
-Galp-(1→2)-βD-GlcAp,
-GlcAp,
most frequently
frequently
branched
atposition
the O-3
O-3 of
position
of the
the glucuronic
glucuronic
with
a
αL
-Rhap
or
a
βD
-Xylp
residue.
In
a
few
cases,
the
saponin
lacks
the
C-28
linked
oligosaccharide
acid
acid unit
unit with
with aa αα-LL-Rhap
-Rhap or
or aa ββ-D
D-Xylp
-Xylp residue.
residue. In
In aa few
few cases,
cases, the
the saponin
saponin lacks
lacks the
the C-28
C-28 linked
linked
(saponins
3–5, Table
1), but3–5,
normally
position
is glycosylated
through
an acetal-ester
linkage
to a
oligosaccharide
(saponins
Table
1),
normally
this
is
through
an
oligosaccharide
(saponins
3–5,
Table this
1), but
but
normally
this position
position
is glycosylated
glycosylated
through
an acetalacetalcomplex
oligosaccharide.
oligosaccharide
hasoligosaccharide
a conserved moiety
the disaccharide
ester
to
oligosaccharide.
This
has
conserved
composed
ester linkage
linkage
to aa complex
complexThis
oligosaccharide.
This
oligosaccharide
has aacomposed
conservedofmoiety
moiety
composed
αL
-Rhap-(1
→
2)-βD
-Fucp
with
different
decorations
[30].
More
often
this
disaccharide
is
extended
by
of
of the
the disaccharide
disaccharide αα-LL-Rhap-(1→2)-β-Rhap-(1→2)-β-D
D-Fucp
-Fucp with
with different
different decorations
decorations [30].
[30]. More
More often
often this
this
adisaccharide
β-D-Xyl-(1→is
residue, which
bear a second
β-D-Xylp
→3)bear
residue
or a β→3) residue.
by
D
residue,
which
aa second
β-Xylp
(1→3)
residue
disaccharide
is4)extended
extended
by aa ββ-can
D-Xyl-(1→4)
-Xyl-(1→4)
residue,
which(1can
can
bear
second
β-DD
D-Apif
-Xylp(1
(1→3)
residue
The
βD
-Fucp
residue
of
the
constant
disaccharide
can
present
either
a
αL
-Rhap
or
a
βD
-Glcp
at O-3,
or
aa
or aa ββ-D
D-Apif
-Apif (1→3)
(1→3) residue.
residue. The
The ββ-D
D-Fucp
-Fucp residue
residue of
of the
the constant
constant disaccharide
disaccharide can
can present
present either
either
and
can
be
acetylated
or
not.
The
βD
-Fucp
residue
can
be
acylated
at
O-4,
simply
with
an
acetate
αα-LL-Rhap
-Rhap or
or aa ββ-D
D-Glcp
-Glcp at
at O-3,
O-3, and
and can
can be
be acetylated
acetylated or
or not.
not. The
The ββ-D
D-Fucp
-Fucp residue
residue can
can be
be acylated
acylated at
at
residue
or a branched
ester
dimer
composed
of two units
3,5-dihidroxy-octanoic
acid,
called
Fa,
O-4,
with
residue
or
ester
composed
units
of
O-4, simply
simply
with an
an acetate
acetate
residue
or aa branched
branched
esterofdimer
dimer
composed of
of two
two
units
of 3,53,5-
dihidroxy-octanoic
dihidroxy-octanoic acid,
acid, called
called Fa,
Fa, which
which can
can be
be glycosylated
glycosylated in
in the
the terminal
terminal 5-hidroxy
5-hidroxy group
group by
by aa ββD
D-Araf
-Araf residue
residue (Figure
(Figure 2)
2) [30].
[30]. This
This terminal
terminal ββ-D
D-Araf
-Araf residue
residue can
can also
also bear
bear aa (1→2)-β(1→2)-β-D
D-Glcp
-Glcp residue.
residue.
Molecules 2019, 24, 171
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which can be glycosylated in the terminal 5-hidroxy group by a β-D-Araf residue (Figure 2) [30].
This terminal β-D-Araf residue can also bear a (1→2)-β-D-Glcp residue.
The β-D-Fucp residue linked to C-28 can be acylated at O-4 (R4 in Figure 1, see Table 1).
However, the corresponding regioisomers acylated at the O-3 position can be found in solution due
to a transesterification reaction between these two cis-vicinal hydroxyl groups, as has been observed
in the adjuvant saponin QS-21. The rate of this isomerization is higher at pH > 5.5, leading to an
equilibrium mixture of both isomers [31].
Phytochemical studies of the less known Quillaja brasiliensis tree, even though it is a well-known
rich source of saponins, started relatively later. In a pioneering study, a prosapogenin composed of
quillaic acid glycosylated at C-3 with a β-D-GlcAp residue was isolated from the products of partial
hydrolysis, suggesting that these saponins might also present structures like those from Q. saponaria [32].
Recently, Wallace et al. [29] presented the first structural studies of complete Q. brasiliensis saponins
using electrospray ionization ion trap-multiple stage mass spectrometry (ESI-IT-MSn). These authors
identified 27 bidesmosidic triterpenic saponins, with either quillaic acid, gypsogenin, phytolaccinic
acid, or 23-O-acetyl-phytolaccinic acid as aglycones (Figure 1). The Q. brasiliensis saponins described
present glycosylation patterns at positions C-3 and C-28 of the aglycone analogous to those of
Q. saponaria saponins with the same conserved motifs (Figure 2). Some saponins presented different
degrees of acylation, and regioisomers with the Fa acyl substituent at the O-4 or the O-3 positions of
the β-D-Fucp residue were found, as well as the presence of the QS-21 Xyl and QS-21 Api saponins
(S4/S6, Table 1).
Table 1. Structures of Quillaja saponaria saponins.
Nº
Saponin
Structure
Mi
Aglycone
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
DS-1
DS-2
*
*
*
4
5
6
7
8
9
10
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
1512.0
1674.0
824.0
970.0
956.0
1436.6
1582.7
1568.7
1714.7
1700.7
1714.7
1700.7
1598.7
1584.7
1628.7
1614.7
1730.7
1716.7
1760.8
1746.7
1640.7
1626.7
1802.8
1788.7
1744.8
1730.7
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Ref.
R0
R1
R2
R3
Xyl
Xyl
H
Rha
Xyl
H
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Api-Xyl
Api-Xyl
Xyl
Xyl
Xyl
Xyl-Api
Xyl-Api
Xyl-Xyl
Xyl-Xyl
Xyl
Xyl
H
H
Xyl-Api
Xyl-Api
Xyl
Xyl
Xyl
Xyl
Xyl
Xyl
Xyl
Xyl
H
H
H
H
H
H
H
H
H
H
H
Glc
Glc
Glc
Glc
Glc
Glc
H
H
Glc
Glc
Glc
Glc
H
Glc
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Glc
Glc
Glc
Glc
H
H
Glc
Glc
Glc-Ac
Glc-Ac
Glc-Ac
Glc-Ac
Rha
Rha
R4
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
[3]
[3]
[33]
[33]
[34]
[34]
[34]
[34]
[34]
[34]
[34]
[34]
[35]
[35]
[35]
[35]
[35]
[35]
[35]
[35]
[35]
[35]
[35]
[35]
[35]
[35]
Molecules 2019, 24, 171
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Table 1. Cont.
Nº
Saponin
Structure
Mi
Aglycone
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
18a
18b
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
B1
B2
B3
B4
B5
B6
B7
B8
QS-III
20a
20b
21a
21b
22a
22b
19
23
S13
1876.8
1862.8
1870.9
1856.9
2002.9
1988.9
2002.9
1988.9
1912.9
1898.9
2045.0
2030.9
2045.0
2030.9
2033.0
2018.9
2165.0
2151.0
2165.0
2151.0
1886.9
2018.9
2297.0
1656.7
1642.7
1788.8
1774.8
1978.9
1964.9
1392.7
1732.8
1560.7
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q-OH
Q-OH
Q-OH
Q-OH
Q-OH
Q-OH
P
E
P-Ac
R0
R1
R2
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
H
H
Xyl
Rha
Xyl
Rha
Xyl
Rha
Xyl
H
Xyl
H
Xyl-Api
Xyl-Api
Xyl
Xyl
Xyl-Xyl
Xyl-Xyl
Xyl-Api
Xyl-Api
Xyl
Xyl
Xyl-Xyl
Xyl-Xyl
Xyl-Api
Xyl-Api
Xyl
Xyl
Xyl-Api
Xyl-Api
Xyl-Xyl
Xyl-Xyl
Xyl
Xyl-Api
Xyl-Api
Xyl
Xyl
Xyl-Api
Xyl-Api
Xyl-Api
Xyl-Api
H
Xyl
H
Glc
Glc
H
H
H
H
H
H
H
H
H
H
H
H
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
Glc
H
Glc
MeBu
Ref.
R3
R4
Rha
Ac
Rha
Ac
H
Fa-Ara
H
Fa-Ara
H
Fa-Ara
H
Fa-Ara
H
Fa-Ara
H
Fa-Ara
Ac
Fa-Ara
Ac
Fa-Ara
Ac
Fa-Ara
Ac
Fa-Ara
Ac
Fa-Ara
Ac
Fa-Ara
H
Fa-Ara
H
Fa-Ara
H
Fa-Ara
H
Fa-Ara
H
Fa-Ara
H
Fa-Ara
H
Fa-Ara
H
Fa-Ara
Fa-Ara-Rha
H
H
MeBu
H
MeBu
H
MeBu
H
MeBu
Rha
OHMeHex
Rha
OHMeHex
Glc
MeBu
Glc
Ac
Glc
MeBu
[35]
[35]
[36]
[36]
[36]
[36]
[36]
[36]
[4]
[4]
[4]
[4]
[4]
[4]
[36]
[36]
[36]
[36]
[37]
[37]
[37]
[37]
[3]
[34]
[34]
[34]
[34]
[34]
[34]
[34]
[34]
[4]
*: Monodesmosidic C-3 saponins. DS: deacylateded saponins, Q: quillaic acid, Q-OH: quillaic acid, 22β-OH, P:
phytolaccinic acid, P-Ac: phytolaccinic acid, 23-O-Ac, E: echynocystic acid. Fa-Ara: see Figure 2, QS_III: quillaja
saponin with highest molecular mass, MeBu: 2-methylbutanoyl, OHMeHex: 3-hydroxy-4-methylhexanoyl. Adapted
from Kite et al. [30].
3. Biological Activities
Regarding the biological activities, Quillaja saponins have been widely studied. The main activities
described are reviewed below, especially the antibacterial, antiviral, antifungal, antiparasitic, antitumor,
hepatoprotective, and immunoadjuvant ones.
3.1. Antimicrobial Activities of Quillaja Saponins
The antimicrobial activities of saponins from Quillaja are described in several studies, especially
their antibacterial, antifungal, and antiviral ones [11,12,14,15,38–40]. The main reason for these
activities is the affinity of the aglycone with cell membrane cholesterol, leading to changes in the
membrane and, consequently, preventing microorganism infection of the cell [41,42].
3.1.1. Antibacterial Activity
Saponins present detergent properties by destroying membrane lipids and, for this reason,
they can make the bacterial cell membranes permeable facilitating the influx of molecules through
the bacterial wall, such as antibiotics [43–45]. On the other hand, a study conducted with clinical
Molecules 2019, 24, 171
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strains of multiresistant Escherichia coli isolated from human urine demonstrated that commercial
saponin from Q. saponaria barks (12 µg/mL) significantly increased the growth of the six strains tested.
In susceptibility tests of E. coli strains against ampicillin, streptomycin, and ciprofloxacin, saponins
increased the colony forming units per mL (CFU/mL), even in the presence of antibiotics [46].
Saponin extracts from Q. saponaria are sold in the market and produced by different brands, and their
composition can vary according to the process used to obtain them. Therefore, the antimicrobial activity
may be altered since the constitution and nature of the saponins differs. Sen et al. [16] evaluated
the anti E. coli action of Q. saponaria saponins from three different commercial companies (Carl Roth
GmbH® , Karlsruhe, Germany; Sigma® , Saint Louis, MO, USA; Quillaja 300 Powder® , Nor-feed, Hvidovre,
Denmark). The study showed different results between the three brands used, in which the CFU increased
with the different concentrations of saponins used. The percentage of bacterial growth was 151%, 31%,
and 97% for the Roth, Sigma, and Nor-feed brands, respectively. The activity of Q. saponaria saponins,
in the induction of bacterial growth, indicates that they may act by facilitating the transport of nutrients
from the bacterial growth media into the microorganism by increasing bacterial permeability though
the cell membranes, thus stimulating the colonies’ growth [16,46].
In contrast, Hassan et al. [38] showed the antibacterial activity of a commercial extract rich
in Quillaja saponins (Sigma® ) against Salmonella typhimurium, Staphylococcus aureus, and E. coli.
Similarly, Sewlikar and D’Souza [47] observed the strong antibacterial activity of the aqueous extract
of Q. saponaria against four strains of E. coli producers of Shiga toxin (STEC) O157:H7 and six non-O157
STEC serotypes. At the time of treatment with the extracts, the 4 O157:H7 strains had an initial CFU
of approximately 7.5 log and after 16 h of treatment, the remaining counts were reduced to 6.79 and
3.5 log CFU at room temperature. The non-O157 STECs treated with Q. saponaria showed reductions of
6.81 to 4.55 log CFU after 16h at room temperature. Additional tests showed that incubation at 37 ◦ C
reduced the CFU to undetectable levels (control without any significant reduction) after 1h of treatment
for all O157:H7 strains and 30 min post-treatment for non-O157. Scanning electron microscopy analysis
revealed the damage to bacteria cell membranes subjected to the treatment.
The commercial extract of Q. saponaria saponins (Sigma® ) was shown to be a relevant agent
for increasing the efficiency of the disinfection process of the bacteria genus Asaia. Pretreatment
with 1% saponin solution resulted in increased sensitivity of the bacterial cultures tested. Cultures
pre-exposed to saponin solution and submitted to N-ethyl-N,N-dimethylhexadecylammonium bromide
(QAC) showed half of the minimum inhibitory concentration (MIC) compared to non-saponin-treated
bacteria. In tests performed with peracetic acid, the difference in MICs was 4 to 8-times lower than
the bacterial strains without saponin pre-treatment. With these findings, the authors suggest that
treatment with saponin solution prior to disinfection may reduce the need for high concentrations of
the antimicrobials tested. Concerning the inhibition of bacterial biofilms, experiments conducted in
this same study revealed that the extract reduced the live cells number in biofilms by approximately
4 to 5 log [48]. Different studies on the antibacterial activity of Q. saponaria saponins suggest that
saponins’ biological activity is related to their chemical constitution, thus different microorganisms
have different susceptibilities to saponin action [49,50].
3.1.2. Antiviral Activity
Two extracts of Q. saponaria, Ultra Dry 100 Q (a spray-dried purified aqueous extract, consisting of
65% saponins) and Vax sap (purified medical grade material, obtained by an additional purification of
Ultra Dry 100 Q, with saponin content greater than 90%), showed antiviral activity in vitro and in vivo.
Ultra Dry 100 Q showed strong antiviral action against herpes simplex virus type 1, vaccinia virus,
human immunodeficiency viruses 1 and 2, varicella zoster virus, rhesus rotavirus (RRV), and reovirus.
Very low concentrations of the extract were able to prevent the virus from infecting their host cells
and were still able to maintain their blocking activity and stop the virus binding to the cells for up
to 16 h after their removal from the culture medium. Both extracts studied prevented the binding
of RRV and reovirus to host cells pretreated with them, also inhibiting the propagation of infectious
Molecules 2019, 24, 171
7 of 29
particles to the uninfected neighboring cells. This cell protecting effect persisted for up to 16 h after the
extracts were removed from the cell monolayers [11,13]. The action of saponins is possibly due to the
cell membrane and the affinity of the aglycone portion with the membrane cholesterol. Consequently,
without the cholesterol, the fluidity of the cell membrane increases, causing it to be beyond the control
of the enzymatic activities. Thus, saponins may act in cellular proteins by preventing specific binding
of viral receptors [41,51].
Confirming the evidence shown in the in vitro tests, in which the saponin-rich extracts of
Q. saponaria Molina are able to inhibit RRV infection in host cells by some modification in the cell
membrane proteins preventing binding of viral receptors, in vivo experiments with Vax sap extract
presented strong anti-RRV action. Newborn mice were exposed to the aqueous extract for two days
and, afterwards, RRV was administered for another five consecutive days. Doses of 0.015 mg/mouse
reduced the diarrhea caused by the virus by 68% in mice exposed to 500 plaque forming units (PFU).
The experiments revealed that lower concentrations of the extract do not have a strong antiviral effect
when administered in higher viral titers (5000 PFU). In addition, in the case of a higher inoculant
(50,000 PFU), an extract concentration of 0.03 mg/mouse blocked virus infection, also reducing diarrhea
in the animals [12].
3.1.3. Antifungal Activity
Membranes of regenerated cellulose nanofibers supplemented with Q. saponaria saponins are
described as having antifungal activity. Dixit et al. [14] verified that, after 24 h, the nanofiber
membranes containing dead spores of Penicillium roguefortii Thom caused an 80.4% grown reduction
and Aspergillus ochraceus anamorph resulted in a 53.6% reduction. In addition, Chapagain et al. [52]
described moderate activity of Q. saponaria bark saponins against different fungal species: Alternaria
solani (47.5%), Pythium ultimum (59.12%), Fusarium oxysporum (56.11%), and Verticillium dahliae (35.92%),
but not for Colletotrichum coccodes species.
3.2. Antiparasitic Activity
The saponins from Q. saponaria barks showed antiparasitic activity, in vitro, in a ruminal
fermentation system [15,39,40]. A commercial extract of Q. saponaria revealed an antiparasitic effect
inversely proportional to the extract concentrations tested [17].
Quillaja saponins were also evaluated against cloned xenic cultures of different isolates of
Blastocystis sp., Tetratrichomonas gallinarum, and Histomonas meleagridis. The in vitro assays for
antiprotozoal activity showed that the effect of this plant’s saponins are directed towards the
protozoa, although more studies are necessary to elucidate the mode of action of saponins though the
parasites [53].
A study performed to evaluate the effect of commercial saponins obtained from Q. saponaria
reduced the viability of the protozoan Trichomonas vaginalis by 100% at a 0.025% concentration of the
extract in culture medium. The aqueous extract of Q. brasiliensis leaves also showed anti-T. vaginalis
activity (0.1% inhibited 98% of the microorganism’s growth). Further experiments demonstrated that
saponins induced damage to the parasite membrane, suggesting that the extracts acted by inhibiting
the viability of T. vaginalis [54]. This antiparasitic effect may be due to the detergent action of the
saponins from the genus Quillaja in the cholesterol of the protozoa membranes, resulting in cell lysis.
However, the activity may not occur in all protozoa species, since some species may not be susceptible
to the toxic effects of saponins or the parasite may adapt to saponin effects [42,50,55].
3.3. Antitumor Activity of Quillaja Saponins
Fractions rich in Quillaja saponins are also described as therapeutic agents for treating and
preventing cancerous diseases [56]. One of the first studies to demonstrate the saponins’ antitumor
potential was conducted with Quil-A® , where the fraction prolonged the survival of mice with
leukemia [57]. Since saponins interact with the cholesterol of cell membranes and, consequently,
Molecules 2019, 24, 171
8 of 29
cause membrane damage, cell lysis, and necrosis, they present high toxicity for clinical practice.
These toxic effects have been reversed by converting Q. saponaria fractions into stable nanoparticles,
binding them to cholesterol. The QS fraction 21 (QS-21) (a more hydrophobic fraction with an acyl
chain-ASAP) formulated in killing and growth inhibiting (KGI) particles selectively killed nine of the
ten tumor cell lines at a concentration 30 times lower than the concentration required to kill normal
monocytes. In addition, the ASAP’s lytic effect on red blood cells was inhibited by the formulation
with KGI particles; cell death occurred through the apoptosis mechanism [58].
3.4. Hepatoprotective Activity of Quillaja Saponins
Thought-provokingly, Abdel-Reheim et al. [59] demonstrated that bark saponins from Q. saponaria
are interesting hepatoprotective agents. In this study, the authors verified that saponin significantly
decreased the elevation of ALT (achieving 57% hepatotoxicity control), AST (66%), ALP (76%),
GGT (60%), NOx (77%), TC (70%), MDA (65%), LDH (54%), TG (54%), and total (54%), direct (54%),
and indirect (54%) bilirubin, coupled with increased GSH (219%) and albumin (159%) levels.
Furthermore, saponin from Quillaja bark significantly decreased liver MDA and NOx production,
reinforcing that saponin has antioxidant properties, since it is able to scavenge excessive radicals
and thus suppress oxidative stress [60,61]. The hepatoprotective effect of Quillaja bark saponin is
probably due to the amelioration of oxidative stress and suppression of NOS expression, maintaining
hepatocyte integrity and functioning. The decrease in TG and TC serum levels can be explained
by the decrease in intestinal cholesterol absorption and by the reduction in cholesterol levels,
accompanied by an increase in the HMG-CoA reductase activity and LDL receptor levels in the
liver [62]. In addition, the immunohistochemical results help to explain Quillaja bark saponins’
protective activity, since saponins suppressed NOS expression, attenuating oxidative and nitrosative
stress [59].
3.5. Immunoadjuvant Activity of Quillaja Saponins
One remarkable biological activity is their ability to modulate immunity towards a more cell
mediated response or to enhance antibody production [18,21,63]. This property is mainly correlated
with vaccinology, since saponins can act as immunoadjuvants. Despite both steroid and triterpenoid
saponins from different plant species demonstrating immunogenic activity, Quillaja triterpenoid
saponins are the most extensively studied [18]. Because of their amphiphilic structure, both aglycone
and branched sugar radicals play important roles in stimulating the immune system. In contrast to
other saponin structures, Quillaja saponins feature a fatty acid chained to β-D-Fucp residue (C-28) and,
at position 4, an aldehyde group, which increases their immunogenic activity leading to a more intense
stimulation of the cytotoxic T lymphocytes (CTL) [20,64]. Although the immunogenic mechanism is
not yet clearly understood, it is known that they can activate different pathways, including Nod-like
Receptors (NLR) and Toll-like Receptors (TLR) [18,20].
Saponins can stimulate antigen-presenting cells (APC) by inducing inflammasome formation [21,65].
The inflammasome is a set of receptors and sensors that induce inflammation by activating caspase-1.
Saponins can stimulate the secretion of IL-6 and IL-1β in vitro despite requiring the activation of TLR4,
leading to active forms of caspase-1. Also, saponins can stimulate the expression of TLR2 interferon-α
leading to active forms of IL-18 and IL-1β [66,67]. Taken together, Marty-Roix et al. (2016) found that
saponins can cause an enlargement of the lymph node, indicating cell recruitment or cell proliferation
through cytokine signaling [68]. The fatty acid in their structure boosts the cytotoxicity of the CD8
lymphocyte by increasing the interaction of the saponins with the cell membrane through cholesterol,
inducing the formation of cell pores and, possibly, signaling to CTLs [18–20,69].
As for the humoral response, saponins can suppress secretion of IL-4, thus lowering IgE levels.
On the other hand, they can shift a Th2 response to a Th1 response, inducing the secretion of IgG2a
rather than IgG1, indicating a more pronounced Th1 response than Th2 response through changes
in cytokine secretion (in this context IL-2 and IFNγ) [20,70–72]. This mechanism may occur in
Molecules 2019, 24, 171
9 of 29
two different ways: first, through the deacylated aldehyde that binds to T cell surface receptors
(mostly CD2), leading the cell to Th1 immunity; and second, through endocytosis of the saponin with
an antigen, disrupting the endosomal membrane of the processed antigen and facilitating the antigen
Molecules 2019, 24, x
presentation process (Figure 3) [20,69,73].
9 of 30
Figure 3. The endocytosis of the saponin with an antigen can cause disruption to the endosomal
membrane of the processed antigen, facilitating the antigen presentation process by extravasation of the
antigen and its transportation to the endoplasmic reticulum with posterior complexation with MHCI.
Figure 3.
Thedeacylated
endocytosis
of the
saponin
with
anCD4
antigen
can cause
to the endosomal
Also,
saponins
can interact
with
naïve
T-cells through
CD2disruption
receptors, stimulating
T
membrane
the processed
antigen,
theconsequent
antigen presentation
by extravasation of
cell of
activation
biased towards
a Th1facilitating
response with
secretion of IL-2process
and IFNγ.
the antigen and its transportation to the endoplasmic reticulum with posterior complexation with
Despite the immunogenic activity, the considerable toxicity restricts its use in humans.
MHCI. Also, deacylated saponins can interact with naïve CD4 T-cells through CD2 receptors,
An alternative for reducing saponin toxicity and increasing its immunoadjuvancy is to formulate
stimulating
T cell activation
biased towards
a Th1structures
responsecalled
with Immune
consequent
secretion
of IL-2 and
it with cholesterol
and phospholipids
into cage-like
Stimulating
Complexes
IFNγ.
(ISCOMs) [74]. These structures were first described in 1971 and shown to enhance antibody production
when compared to saponins, mostly because they can be easily transported to lymph nodes due to
their nanoscale
size (up to 80
nanometers)
[75–77]. ISCOMs
can modulate
immune
response
in
Despite
the immunogenic
activity,
the considerable
toxicity
restricts
its use
in humans.
An
different
ways
according
to
the
antigen.
It
is
known
that
some
antigens
can
increase
or
decrease
the
alternative for reducing saponin toxicity and increasing its immunoadjuvancy is to formulate it with
production of IL-10, pushing the immune response towards a Th2 or Th1 response, respectively [78,79].
cholesterol and phospholipids into cage-like structures called Immune Stimulating Complexes
ISCOMs can also stimulate CTLs through cytokine modulation [77]. The immunogenic mechanism
(ISCOMs)
[74].
These
structures
first related
described
in 1971
to enhance
is not
clearly
understood,
but it were
is probably
to saponins,
sinceand
they shown
cause similar
changes inantibody
production
when
compared
to to
saponins,
because
they[77,78].
can be easily transported to lymph
cytokine
expression,
leading
different Tmostly
lymphocyte
activation
In their
vaccine
systems, saponins
be nanometers)
used alone or [75–77].
complexed
with different
molecules immune
nodes due to
nanoscale
size (up can
to 80
ISCOMs
can modulate
oil/water,
in different
structures
(liposomes)
in ISCOMs
[81,82]).
semisynthetic
response(i.e.,
in in
different
ways
according
to the
antigen.[80],
It isorknown
that
someAlso,
antigens
can increase or
saponins can be used as adjuvants in order to reduce toxicity and maintain their immunoadjuvant
decrease the production of IL-10, pushing the immune response towards a Th2 or Th1 response,
properties [83]. Considering their effects when used as adjuvants in vaccines, they are promising
respectively
[78,79].
ISCOMs
can
also stimulate
CTLs
through
cytokine
modulation
[77]. The
candidates
and have
featured
in plenty
of pre-clinical
studies
for vaccines,
as mentioned
in Table 2.
immunogenic
mechanism
not clearly
understood,
but
it is probably related to saponins, since they
The studies
reported inisTable
2 are arranged
by year of
publication.
cause similar changes in cytokine expression, leading to different T lymphocyte activation [77,78].
In vaccine systems, saponins can be used alone or complexed with different molecules (i.e., in
oil/water, in different structures (liposomes) [80], or in ISCOMs [81,82]). Also, semisynthetic saponins
can be used as adjuvants in order to reduce toxicity and maintain their immunoadjuvant properties
[83]. Considering their effects when used as adjuvants in vaccines, they are promising candidates and
have featured in plenty of pre-clinical studies for vaccines, as mentioned in Table 2. The studies
reported in Table 2 are arranged by year of publication.
Molecules 2019, 24, 171
10 of 29
Table 2. Pre-clinical studies using Quillaja saponins as an adjuvant in vaccine formulations targeting different antigens.
Antigen
Adjuvant
PorA P1.6 (membrane
porin of Neisseria
meningitidis)
Quil-A®
S. bovis and Lactobacillus
spp.
Quil-A®
Model
Findings
Ref.
Mice
PorA supplemented with Quil-A® resulted in four to seven times
higher antibody titers (IgG1, IgG2a, and IgG2b) when compared with
lipopolysaccharide as an adjuvant.
[84]
Cattle
Immunization of feedlot steers against S bovis and Lactobacillus spp
with vaccines incorporating Freund adjuvant, Quil-A® , dextran, or
alum as an adjuvant effectively induced high IgG concentrations
when dextran was used as an adjuvant.
[85]
[86]
38-kDa mycobacterial
protein
ISCOMs
Mice
An increase in antibody titers was observed after a booster injection,
with significant levels of IgG2a. The ISCOM formulations induced
this in the same way. Regarding the induction of CTL responses, the
differences shown comparing various ISCOMS formulations were
minimal.
Tetanus toxoid
QS-21
Mice
Administration of QS-21 p.o. as an adjuvant elicits strong serum IgM
and IgG Ab responses.
[87]
Babesian equi immunogen
Quil-A®
Donkeys
A mixture of Quil A® and Babesia equi immunogen was optimal in
generating a significant immune response and reducing the lethality.
[88]
OVA
QS-21 and deacylated QS-21 (DS-1)
Mice
Despite DS-1 requiring a higher dose to induce IgG1 responses, it did
not induce IgG2a or CTL responses. Lower doses of QS-21 induced
higher IgG titers, including IgG2a and CTL responses.
[89]
FML *
Quil-A®
Dogs
The formulation protected dogs against canine kala-azar in the field
with robust immunogenicity.
[90]
Measles virus
ISCOMs QA-22
Rats and Macaques
Vaccines induced high levels of antibodies, which showed no
decrease during a 2-year follow up.
[91]
Herpes virus
Quillaja saponins
Mice
Saponins’ deacylation significantly reduced antibody production and
increased mortality rates during viral challenge test.
[92]
Opa J from Neisseria
meningitidis
Quil-A®
Mice
Quil-A® formulated with OpaJ was highly immunogenic, inducing
the production of Opa-specific antibodies.
[93]
Non-fimbrial adhesin
hemagglutinin B (HagB)
Semi-synthetic saponin of Quillaja
GPI-0100
Mice
GPI-0100 showed better immunoadjuvancy than monophosphoryl
lipid A and alum, as a mucosal and systemic adjuvant, in inducing
serum anti-HagB.
[94]
FML *
Quil-A®
Dogs
The formulation was shown be effective in immunotherapy against
visceral leishmaniasis of asymptomatic infected dogs. All animals
showed significantly increased CD8 lymphocyte percentages.
[95]
Molecules 2019, 24, 171
11 of 29
Table 2. Cont.
Antigen
Adjuvant
Model
Findings
Ref.
Human respiratory
syncytial virus (HRSV)
HRSV-ISCOMs prepared with
Quillaja saponin fractions: QH-A;
QH-C; QH-A + QH-C (QH-AC)
Mice
In general, the ISCOMs tested were well tolerated. However, the
combination of QH-A + QH-C ISCOMs was lethal in neonates
despite the QH-A or QH-C fractions alone being well tolerated.
[96]
FML *
Quillaja saponaria sapogenins
containing aldehyde
Mice
Vaccines elicited high levels of antibodies and cellular specific
response to FML and IFNγ sera levels and protection against L.
donovani murine infection was shown.
[97]
[98]
Ovalbumin (OVA)
Quil-A®
Mice
Increasing amounts of Quil-A® (20% to 70%) were tested in
liposomes. Higher doses of Quil A reduced the particle sizes formed,
thus decreasing antigen incorporation and uptake by DC. Liposomes
containing 20% Quil-A® were more effective as immunostimulants,
and more toxic in cell cultures too, when compared with those
containing 70% Quil-A® .
Mtb72F (Mycobacterium
tuberculosis)
AS02A
Mice
Increase in both Th1 and Th2 response, with Th1 response being
more pronounced.
[99]
LSP PbCS 242–310 **
QS-21
Mice
The use of LSP PbCS 242–310 combined with QS-21 induced a
satisfactory immune response similar to the one generated with the
injection of radiation-attenuated sporozoites.
[100]
HRSV
ISCOMs formulated with Quillaja
saponin fractions
Mice
All three formulations favor Th1 responses to different degrees with
IFN-c being produced up to 50 times more than Il-4 and IL-5. The
HRSV 703 ISCOMs induced the most pronounced innate and
acquired response with the most prominent Th1 profile.
[101]
HIV-1 DNA
prime/protein–Env and
Gag ***
QS-21
Rhesus macaques and
mice
Cellular and humoral responses were observed when Polyvalent
DNA prime/protein boost vaccine was administered. CD8+ CTL,
CD4+ T-helper cells and Th1 cytokines were involved in cellular
immune response.
[102]
FML *
Fractions of the Riedel de Haen
saponin mixture (QS-21 saponin
fraction; two deacylsaponins mixture;
a mixture of glucose, rutin, quercetin,
and sucrose).
Mice
QS-21 and the deacylsaponins induced the most significant reduction
of parasite burden in the liver, demonstrating a promising adjuvant
potential in the Riedel de Haen saponin mixture, which contains
deacylated saponins that are not toxic and induce robust immunity.
[103]
Plant-made measles virus
hemagglutinin (MV-H)
protein
Cholera toxin (CTB/CT); LT(R192G)
****; Saponin extracted from Quillaja
bark (Sigma; S-4521)
Mice
Despite both LT(R192G) and the crude saponins showing strong
adjuvant activity, the crude extract had superior immunostimulatory
properties.
[104]
Molecules 2019, 24, 171
12 of 29
Table 2. Cont.
Antigen
Molecules 2019, 24, x
Adjuvant
Model
Findings
Ref.
12 of 30
Mice immunized with ADAD system with Qs had a survival rate of
Fh15 (recombinant 15 kDa
50–62.5% while animals without Qs had a survival rate of 40–50%.
ADAD System *****
Mice and sheep
[105]
Fasciola hepatica protein)
Sheep immunized with ADAD system with Qs showed less hepatic
Table 2. Cont.
damage compared to control group.
Fh15 (recombinant 15
Mice immunized with ADAD system with Qs had a survival rate of 50–62.5% while
and
Molecules
Molecules2019,
2019,
Molecules
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24,
24,xx 2019,
2019,
2019,
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24,
24,
xxMice
x
12
12ofof30
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1
Despite nonspecific reactions being observed in the immunized
kDa Fasciola hepatica
ADAD System *****
animals without Qs had a survival rate of 40–50%. Sheep immunized with ADAD
[105]
FML *
QS-21
Dogs
animals (similar to other veterinary vaccines), a significant decrease
[106]
sheep
protein)
system with Qs showed less hepatic
damage
to control
group.
was
showncompared
with subsequent
doses.
Table
Table2.2.
Cont.
Table
Table
Table
2.2.2.
Cont.
Cont.
Cont. animals (similar to other
Despite nonspecific reactions being observed
inCont.
the
immunized
FML *
QS-21
Dogs
Mice vaccinated with a combination of plant-made LTB-ESAT-6[106]
veterinary vaccines), a significant decrease was shown with subsequent doses.
Fh15
Fh15(recombinant
(recombinant
Fh15
Fh15
Fh15
(recombinant
(recombinant
(recombinant
15
15
15
15
15
Mice
Mice
immunized
immunized
Mice
Mice
Mice
immunized
immunized
with
immunized
with
ADAD
ADAD
with
with
with
system
system
ADAD
ADAD
ADAD
with
with
system
system
system
Qs
Qs
had
had
with
with
with
aasurvival
survival
Qs
Qs
Qs
had
had
had
rate
arate
asurvival
asurvival
survival
ofof50–62.5%
50–62.5%
rate
rate
rate
ofofof
while
50–62.5%
while
50–62.5%
50–62.5%
while
while
while
fusion BCG
oral
adjuvant
had
significantly
more
IL-10
production
Miceaand
and
Mice
Mice
Mice
and
and
and
Mice vaccinated Mice
with
combination
of plant-made LTB-ESAT-6 fusion BCG oral
kDa
kDaFasciola
Fasciola
kDa
kDa
kDa
hepatica
hepatica
Fasciola
Fasciola
Fasciola
hepatica
hepatica
hepatica
ADAD
ADADSystem
System
ADAD
ADAD
ADAD
*****
*****
System
System
System
*****
*****
***** when compared
animals
animals
without
without
animals
animals
animals
Qs
Qs
without
without
had
without
hadaasurvival
survival
Qs
Qs
Qs
had
had
had
arate
arate
survival
asurvival
survival
ofof40–50%.
40–50%.
rate
rate
rate
of
Sheep
of
Sheep
of
40–50%.
40–50%.
40–50%.
immunized
immunized
Sheep
Sheep
Sheep
immunized
immunized
with
immunized
withADAD
ADAD
with
with
with
ADAD
ADAD
ADAD
[105]
[105]
LTB-ESAT-6 ;; and
with
mice
vaccinated
without
adjuvant.
However,
LTB-ESAT-6
and Bacillus
Quillaja extract
[107]
sheep
sheep
sheep
sheep
sheep
adjuvantMice
had significantly
more
production
when during
compared
with
mice test
vaccinated
Calmette-Guérin
noIL-10
protection
was shown
the
challenge
with
M.
protein)
protein)
protein) Mice
system
system
with
with
system
system
system
Qs
Qsshowed
showed
with
with
with
Qs
Qs
less
Qs
less
showed
showed
showed
hepatic
hepatic
less
less
damage
less
damage
hepatic
hepatic
hepatic
compared
compared
damage
damage
damage
to
compared
to
compared
control
controlgroup.
to
group.
toto
control
control
control
group.
group.
group.
Bacillus
Calmette- (BCG)
Quillajaprotein)
extract protein)
[107] compared
without adjuvant. However,tuberculosis,
no protectioninwas
shown
during
the
challenge
test
with
M.
groups
treated
withbeing
oral
BCG
(with
or
without
Despite
Despitenonspecific
nonspecific
Despite
Despite
Despite
nonspecific
nonspecific
reactions
nonspecific
reactions
reactions
being
reactions
reactions
observed
observed
being
being
being
in
observed
in
observed
the
observed
the
immunized
immunized
ininin
the
the
the
immunized
immunized
animals
immunized
animals(similar
(similar
animals
animals
animals
toto(similar
other
(similar
other
(similar
tototo
other
other
other
Guérin (BCG)
FML
FML** FML
FML
FML
***
QS-21
QS-21 tuberculosis,
QS-21
QS-21
QS-21
Dogs
Dogs
Dogs
Dogswith oral BCG (with
[106]
[106]
inDogs
groups treated
or without saponin).
saponin).
veterinary
veterinaryveterinary
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extract
extract
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[111]
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inducing
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improved
improved
improved
improved
the
the
the
the
have
the
same
efficacy
in
reducing
H.
pylori
colonization
as
the
gold
toxin; andepitopes
aluminium
hydroxide
Prophylactic
Prophylactic
Prophylactic
Prophylactic
Prophylactic
vaccination
vaccination
vaccination
vaccination
with
vaccination
with
adjuvanted
adjuvanted
with
with
with
adjuvanted
adjuvanted
TA-CIN
adjuvanted
TA-CIN
protected
protected
TA-CIN
TA-CIN
TA-CIN
the
protected
the
protected
protected
mice
mice
from
from
the
the
the
mice
viral
mice
viral
mice
from
from
from
viral
viral
viral [109]
epitopes
epitopes
epitopes
epitopes
from
from
from
from
Mice
Mice
Mice
Mice
Mice
inducing
inducing
inducing
inducing
inducing
predominantly
predominantly
predominantly
predominantly
predominantly
IgG2c.
IgG2c.
IgG2c.
IgG2c.
IgG2c.
QS-21
QS-21
QS-21
QS-21
QS-21
also
also
also
also
also
induced
induced
induced
induced
induced
the
the
the
the
highest
the
highest
highest
highest
highest
levels
levels
levels
levels
levels
of
of
of
IL-4
of
IL-4
of
IL-4
IL-4
IL-4
compared
compared
compared
compared
compared
[109
[10
[1[1
AD-472
AD-472

from
or
or
HSV-2
HSV-2
Guinea
Guinea
Both
Both
formulations
formulations
reduced
reduced
clinical
clinical
disease;
disease;
however,
however,
GPI-0100
GPI-0100
improved
improved
the
the
GPI-0100
GPI-0100
GPI-0100
GPI-0100




[111]
[111]
[111]
[111]
ISA-720,
ISA-720,
ISA-720,
ISA-720,
or
or
or
CRL-1005
or
CRL-1005
or
CRL-1005
CRL-1005
CRL-1005
standard
cholera
toxin
(CT)
adjuvant
GPI-0100
GPI-0100


[111]
[111
glycoprotein
glycoprotein
glycoprotein
glycoprotein
DDDD
pigs
pigs
pigs
pigs ISA-720,
glycoprotein-D
glycoprotein-D
glycoprotein-D
glycoprotein-D
formulation
formulation
formulation
formulation
only.
only.
only.
only.
Mice
Mice
and
and
Mice
Mice
Mice
challenge,
challenge,
and
and
and
challenge,
whereas
challenge,
whereas
challenge,
vaccination
vaccination
whereas
whereas
whereas
vaccination
vaccination
without
vaccination
without
adjuvant
adjuvant
without
without
without
was
was
adjuvant
adjuvant
adjuvant
almost
almost
was
was
ineffective.
was
ineffective.
almost
almost
almost
ineffective.
ineffective.
Moreover,
ineffective.
Moreover,
Moreover,
GPIMoreover,
GPIMoreover,
GPIGPIGPIPlasmodium
Plasmodium
Plasmodium
Plasmodium
Plasmodium
falciparum)
falciparum)
falciparum)
falciparum)
falciparum)
to
to
to
the
to
the
to
the
the
other
the
other
other
other
other
adjuvants,
adjuvants,
adjuvants,
adjuvants,
adjuvants,
indicating
indicating
indicating
indicating
indicating
Th1
Th1
Th1
Th1
Th1
and
and
and
and
and
Th2
Th2
Th2
Th2
Th2
responses.
responses.
responses.
responses.
responses.
glycoprotein
glycoprotein
D
D
pigs
pigs
glycoprotein-D
glycoprotein-D
formulation
formulation
only.
only.
##
##
##
##
##
TA-CIN
TA-CIN TA-CIN
TA-CIN
TA-CIN
GPI-0100
GPI-0100

GPI-0100

GPI-0100
GPI-0100



[114]
[114]
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
shifted
shifted
shifted
shifted
the
the
the
the
immune
immune
immune
immune
response
response
response
response
to
to
to
an
to
an
an
an
IgG2a
IgG2a
IgG2a
IgG2a
response
response
response
response
(towards
(towards
(towards
aresponse
aTh1
a+Th1
a+Th1
+(towards
+T
monkeys
monkeys
monkeys
monkeys
monkeys
0100
0100
boosted
boosted
0100
0100
0100
IFN-γ
IFN-γ
boosted
boosted
boosted
secreting
secreting
IFN-γ
IFN-γ
IFN-γ
CD8
secreting
CD8
secreting
secreting
Timmunogenicity
cell
cell
CD8
CD8
response
CD8
T+Th1
TT
cell
cell
cell
in
in
response
response
mice
response
mice
compared
compared
in
in
in
mice
mice
mice
to
compared
to
compared
the
the
formulation
formulation
tototo
the
the
formulation
formulation
formulation
The
The
The
The
The
vaccine
vaccine
vaccine
vaccine
vaccine
induced
induced
induced
induced
induced
robust
robust
robust
robust
robust
immunogenicity
immunogenicity
immunogenicity
immunogenicity
with
with
with
with
with
high
high
high
high
high
levels
levels
levels
levels
levels
ofofcompared
of
FML-seroconversion,
of
FML-seroconversion,
of
FML-seroconversion,
FML-seroconversion,
FML-seroconversion,
as
as
as
as
as
MATRIX-M
MATRIX-M
shifted
shifted
the
the
immune
immune
response
response
to
to
an
an
IgG2a
IgG2a
response
response
(towards
(towards
athe
aTh1
Th1
Aluminium
Aluminium
Aluminium
Aluminium
phosphate;
phosphate;
phosphate;
phosphate;
+ +T-cell
+T-cell
+T-cell
FML
FML
FML
FML
FML
*****
QS-21
QS-21
QS-21
QS-21
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Dogs
Dogs
Dogs
Dogs
Dogs
[110]
[110
[11
[1[1
Aluminium
Aluminium
phosphate;
phosphate;
response).
response).
response).
response).
On
On
On
On
the
the
the
the
other
other
other
other
hand,
hand,
hand,
hand,
the
the
the
the
CD8
CD8
CD8
CD8
T-cell
response
response
response
response
could
could
could
could
be
be
be
be
improved
improved
improved
improved
using
using
using
using
without
without
adjuvant.
adjuvant.
without
without
without
Vaccination
adjuvant.
Vaccination
adjuvant.
adjuvant.
Vaccination
Vaccination
Vaccination
ofof
macaques
macaques
of
of
of
macaques
induced
macaques
induced
macaques
induced
induced
induced
T
T
cell
specific
cell
specific
specific
responses.
responses.
TTT
cell
cell
cell
responses.
responses.
responses.
+and
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demonstrated
demonstrated
demonstrated
demonstrated
by
by
by
by
CD8+
by
CD8+
CD8+
CD8+
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and
and
and
CD4+
CD4+
CD4+
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CD4+
Tspecific
Tspecific
T
cell
T
cell
T
cell
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populations.
populations.
populations.
populations.
populations.
response).
response).
On
Ondemonstrated
the
the
other
otherhand,
hand,
the
the
CD8
CD8
T-cell
T-cell
response
response
could
could
be
be
improved
improved
using
using
H9N2
H9N2
H9N2
H9N2
aluminium
aluminium
aluminium
aluminium
hydroxide;
hydroxide;
hydroxide;
hydroxide;
MF59
MF59
MF59
MF59
Mice
Mice
Mice
Mice
[112]
[112]
[112]
[112]
H9N2
H9N2
aluminium
aluminium
hydroxide;
hydroxide;
MF59
MF59
Mice
Mice
[112]
[112
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
or
or
or
MF59.
or
MF59.
MF59.
MF59.
Among
Among
Among
Among
all
all
all
all
tested
tested
tested
tested
molecules,
molecules,
molecules,
molecules,
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
was
was
was
was
the
the
the
the
most
most
most
most
effective
effective
effective
effective
in
in
in
in
# # or
# or
# or
AD-472
AD-472
AD-472
AD-472
AD-472





HSV-2
or
HSV-2
or
HSV-2
HSV-2
HSV-2
Guinea
Guinea
Guinea
Guinea
Guinea MATRIX-M
Both
Both
Both
Both
Both
formulations
formulations
formulations
formulations
formulations
reduced
reduced
reduced
reduced
reduced
clinical
clinical
clinical
clinical
clinical
disease;
disease;
disease;
disease;
disease;
however,
however,
however,
however,
however,
GPI-0100
GPI-0100
GPI-0100
GPI-0100
GPI-0100
improved
improved
improved
improved
improved
the
the
the
the
theinin
MATRIX-M
or
or
MF59.
MF59.Among
Among
all
alltested
tested
molecules,
molecules,
MATRIX-M
MATRIX-M
was
wasthe
the
most
most
effective
effective
;
;
;
;
and
and
and
and
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
##
GPI-0100
GPI-0100
GPI-0100
GPI-0100
GPI-0100





[111]
[111
[11
[1[1
;
;
and
and
MATRIX-M
MATRIX-M
inducing
inducing
inducing
inducing
the
the
the
the
immune
immune
immune
immune
response,
response,
response,
response,
followed
followed
followed
followed
by
by
by
by
MF59
MF59
MF59
MF59
and
and
and
and
aluminium-based
aluminium-based
aluminium-based
aluminium-based
adjuvants.
adjuvants.
adjuvants.
adjuvants.
glycoprotein
glycoprotein
glycoprotein
glycoprotein
glycoprotein
DDDDD
pigs
pigs
pigs
pigs
pigs
glycoprotein-D
glycoprotein-D
glycoprotein-D
glycoprotein-D
glycoprotein-D
formulation
formulation
formulation
formulation
only.
only.
only.
only.
only.
inducing
inducingthe
theimmune
immuneresponse,
response,
followed
followedformulation
by
by
MF59
MF59and
and
aluminium-based
aluminium-based
adjuvants.
adjuvants.
TM
TM
TM
TM
TM
TM
ISCOMATRIX
ISCOMATRIX
ISCOMATRIX
ISCOMATRIX
; TM
;ISCOM
;ISCOM
;ISCOM
ISCOM
; TM
;;;
ISCOMATRIXTM
ISCOMATRIXTM
ISCOMATRIXTM
ISCOMATRIXTM
and
and
and
and
ISCOMTM
ISCOMTM
ISCOMTM
ISCOMTM
vaccines,
vaccines,
vaccines,
vaccines,
using
using
using
using
two
two
two
two
different
different
different
different
antigens
antigens
antigens
antigens
and
and
and
and
different
different
different
different
TM
TM
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
shifted
shifted
shifted
shifted
shifted
the
the
the
the
immune
the
immune
immune
immune
immune
response
response
response
response
response
tototo
an
to
an
to
an
an
IgG2a
an
IgG2a
IgG2a
IgG2a
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response
response
response
response
response
(towards
(towards
(towards
(towards
(towards
adifferent
Th1
aTh1
aTh1
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ISCOMATRIX
ISCOMATRIXTM
; ;ISCOM
ISCOMTM
;;
ISCOMATRIXTM
ISCOMATRIXTM
and
and
ISCOMTM
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vaccines,
vaccines,
using
using
two
two
different
different
antigens
antigens
and
andadifferent
Aluminium
Aluminium
Aluminium
Aluminium
phosphate;
phosphate;
phosphate;
phosphate;
phosphate;
HpaA
HpaA
HpaA
HpaA
and
and
and
and
catalase
catalase
catalase
catalase Cholera
Cholera
Cholera
Cholera
toxin;
toxin;
toxin;
toxin;
and
and
and
and
aluminium
aluminium
aluminium
aluminium Mice
Mice
Mice
Mice Aluminium
delivery
delivery
delivery
delivery
systems
systems
systems
systems
(intranasal
(intranasal
(intranasal
(intranasal
or
or
or
subcutaneous),
or
subcutaneous),
subcutaneous),
subcutaneous),
have
have
have
have
the
the
the
the
same
same
same
same
efficacy
efficacy
efficacy
efficacy
ininin
reducing
in
reducing
reducing
reducing
H.
H.
H.
[113]
[113]
[113]
[113]
+H.
+T-cell
+T-cell
+T-cell
+T-cell
response).
response).
response).
response).
response).
On
On
On
On
the
On
the
the
the
other
the
other
other
other
other
hand,
hand,
hand,
hand,
hand,
the
the
the
the
CD8
the
CD8
CD8
CD8
CD8
T-cell
response
response
response
response
response
could
could
could
could
could
be
be
be
be
improved
be
improved
improved
improved
improved
using
using
using
using
using
HpaA
HpaAand
andcatalase
catalase
Cholera
Choleratoxin;
toxin;and
andaluminium
aluminium
Mice
Mice
delivery
delivery
systems
systems
(intranasal
(intranasal
or
or
subcutaneous),
subcutaneous),
have
have
the
thesame
same
efficacy
efficacy
in
inreducing
reducing
H.
H.
[113]
[113
H9N2
H9N2
H9N2
H9N2
aluminium
aluminium
aluminium
aluminium
aluminium
hydroxide;
hydroxide;
hydroxide;
hydroxide;
hydroxide;
MF59
MF59
MF59
MF59
MF59 as
Mice
Mice
Mice
Mice
Mice
[112]
[112
[11
[1[1
hydroxide
hydroxide
hydroxide
hydroxide H9N2
pylori
pylori
pylori
pylori
colonization
colonization
colonization
colonization
as
as
the
as
the
the
the
gold
gold
gold
gold
standard
standard
standard
standard
cholera
cholera
cholera
cholera
toxin
toxin
toxin
toxin
(CT)
(CT)
(CT)
(CT)
adjuvant
adjuvant
adjuvant
adjuvant
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
or
or
or
MF59.
or
MF59.
or
MF59.
MF59.
MF59.
Among
Among
Among
Among
Among
all
all
all
all
tested
all
tested
tested
tested
tested
molecules,
molecules,
molecules,
molecules,
molecules,
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
was
was
was
was
was
the
the
the
the
most
the
most
most
most
most
effective
effective
effective
effective
effective
in
in
in
in
in
hydroxide
hydroxide
pylori
pylori
colonization
colonization
as
as
the
the
gold
gold
standard
standard
cholera
cholera
toxin
toxin
(CT)
(CT)
adjuvant
adjuvant
#####
;
;
;
;
;
and
and
and
and
and
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
MATRIX-M
Prophylactic
Prophylactic
Prophylactic
Prophylactic
vaccination
vaccination
vaccination
vaccination
with
with
with
with
adjuvanted
adjuvanted
adjuvanted
adjuvanted
TA-CIN
TA-CIN
TA-CIN
TA-CIN
protected
protected
protected
protected
the
the
the
the
mice
mice
mice
mice
from
from
from
from
viral
viral
viral
viral
inducing
inducing
inducing
inducing
inducing
the
the
the
the
immune
the
immune
immune
immune
immune
response,
response,
response,
response,
response,
followed
followed
followed
followed
followed
by
by
by
by
MF59
by
MF59
MF59
MF59
MF59
and
and
and
and
and
aluminium-based
aluminium-based
aluminium-based
aluminium-based
aluminium-based
adjuvants.
adjuvants.
adjuvants.
adjuvants.
adjuvants.
Prophylactic
Prophylactic
vaccination
vaccination
with
withadjuvanted
adjuvanted
TA-CIN
TA-CIN
protected
protected
the
themice
micefrom
from
viral
viral
Mice
Mice
Mice
Mice
and
and
and
and challenge,
challenge,
challenge,
challenge,
whereas
whereas
whereas
whereas
vaccination
vaccination
vaccination
vaccination
without
without
without
without
adjuvant
adjuvant
adjuvant
adjuvant
was
was
was
was
almost
almost
almost
almost
ineffective.
ineffective.
ineffective.
ineffective.
Moreover,
Moreover,
Moreover,
Moreover,
GPIGPIGPIGPITM
TM
TM
TM
TM
TM
TM
TM
TM
TM
########
Molecules
Molecules2019,
2019,24,
24,xx
12
12ofof3
Molecules 2019, 24, 171
13 of 29
Table
Table2.2.Cont.
Cont.
Antigen
TA-CIN ##
Inactivated
Chlamydophila abortus
sLEa -KLH
(glycolipid/glycoprotein
expressed on cancer cell
surface)
Tetanus toxoid
Fh15
Fh15(recombinant
(recombinant15
15
kDa
kDaFasciola
Fasciolahepatica
hepatica
protein)
protein)
Adjuvant
ADAD
ADADSystem
System*****
*****
FML
FML**
QS-21
QS-21
GPI-0100 ;;and
LTB-ESAT-6
LTB-ESAT-6
and
Bacillus
BacillusCalmetteCalmetteGuérin
Guérin(BCG)
(BCG)
Quillaja
Quillajaextract
extract
Mice
Miceimmunized
immunizedwith
withADAD
ADADsystem
systemwith
withQs
Qshad
hadaasurvival
survivalrate
rateofof50–62.5%
50–62.5%while
while
animals
animalswithout
withoutQs
Qshad
hadaasurvival
survivalrate
rateofof40–50%.
40–50%.Sheep
Sheepimmunized
immunizedwith
withADAD
ADAD
sheep
sheep
system
systemwith
withQs
Qsshowed
showedless
lesshepatic
hepaticdamage
damagecompared
compared
controlgroup.
group.
Findings
Ref.totocontrol
Despite
Despitenonspecific
nonspecificreactions
reactionsbeing
beingobserved
observedininthe
theimmunized
immunizedanimals
animals(similar
(similartotoother
other
Dogs
Dogs
Prophylactic
vaccination with adjuvanted TA-CIN protected the mice
veterinary
veterinaryvaccines),
vaccines),aasignificant
significantdecrease
decreasewas
wasshown
shownwith
withsubsequent
subsequentdoses.
doses.
from viral challenge, whereas vaccination without adjuvant was
Mice
Micevaccinated
vaccinatedwith
withaacombination
combinationofofplant-made
plant-madeLTB-ESAT-6
LTB-ESAT-6fusion
fusionBCG
BCGoral
oral
almost ineffective. Moreover, GPI-0100 boosted IFN-γ secreting CD8+
[114]
adjuvant
adjuvant
had
had
significantly
significantly
more
more
IL-10
IL-10
production
production
when
when
compared
compared
with
with
mice
mice
vaccinated
vaccinated
T cell response in mice compared to the formulation without
Mice
Mice
without
withoutof
adjuvant.
adjuvant.
However,
However,
noprotection
protection
wasshown
shownduring
duringthe
thechallenge
challengetest
testwith
withM.
M.
adjuvant. Vaccination
macaques
inducedno
specific
T cellwas
responses.
tuberculosis,
tuberculosis,iningroups
groupstreated
treatedwith
withoral
oralBCG
BCG(with
(withor
orwithout
withoutsaponin).
saponin).
The formulation induced the proliferation of B cells, which is
Mice
Mice
Both
Bothsynthetic
synthetic
Quillaja
saponin
saponin
molecules
molecules
produced
produced
antibodies
antibodiesininmice.
mice.
important
to confer immunity
to theQuillaja
bacteria
rather than
cellular
[115]
QS-21
QS-21was
wasthe
the
second
second
adjuvant
adjuvant
that
that
induced
induced
the
the
most
most
significant
significant
levels
levels
of
of
antibodies,
antibodies,
response.
Mice
Mice
inducing
inducingpredominantly
predominantlyIgG2c.
IgG2c.QS-21
QS-21also
alsoinduced
inducedthe
thehighest
highestlevels
levelsofofIL-4
IL-4compared
compared
totothe
theother
otheradjuvants,
adjuvants,indicating
indicatingTh1
Th1and
andTh2
Th2responses.
responses.
Immunized animals produced high titers of highly reactive IgM and
[116]
The
Thevaccine
vaccine
inducedantibodies.
robust
robustimmunogenicity
immunogenicitywith
withhigh
highlevels
levels
ofofFML-seroconversion,
FML-seroconversion,as
as
IgG induced
specific
Dogs
Dogs
demonstrated
demonstratedby
byCD8+
CD8+and
andCD4+
CD4+TTcell
cellpopulations.
populations.
Guinea
Guinea
Both
Bothformulations
formulationsreduced
reducedclinical
clinicaldisease;
disease;however,
however,GPI-0100
GPI-0100improved
improvedthe
the
Formulations with Quillaja saponins did not increase mucosal IgA.
pigs
pigs
glycoprotein-D
glycoprotein-D
formulation
formulation
only.
only.
On the other hand, the saponins induced the highest systemic IgG
[117]
MATRIX-M
MATRIX-Mshifted
shiftedthe
theimmune
immuneresponse
responsetotoan
anIgG2a
IgG2aresponse
response(towards
(towardsaaTh1
Th1
titers.
response).
response).On
Onthe
theother
otherhand,
hand,the
theCD8
CD8+ +T-cell
T-cellresponse
responsecould
couldbe
beimproved
improvedusing
using
The
Mice
Miceformulation with ISCOMs induced high specificity CTLs
[118]was
MATRIX-M
MATRIX-Mor
orMF59.
MF59.Among
Amongall
alltested
testedmolecules,
molecules,MATRIX-M
MATRIX-M
wasthe
themost
mosteffective
effectiveinin
targeting different tumoral cells.
inducing
inducingthe
theimmune
immuneresponse,
response,followed
followedby
byMF59
MF59and
andaluminium-based
aluminium-basedadjuvants.
adjuvants.
The combination
of Quillaja and
saponins
with CDM
in PLGA
ISCOMATRIXTM
ISCOMATRIXTM
andISCOMTM
ISCOMTM
vaccines,
vaccines,
using
usingtwo
twodifferent
differentantigens
antigensand
anddifferent
different
nanospheres loaded with antigens improved IgA production,
Mice
Mice
delivery
deliverysystems
systems(intranasal
(intranasalor
orsubcutaneous),
subcutaneous),have
havethe
thesame
same
efficacyininreducing
reducingH.
H.
[119]efficacy
suggesting better mucosal protection. The same was observed with
pylori
pylori
colonization
colonization
as
as
the
the
gold
gold
standard
standard
cholera
cholera
toxin
toxin
(CT)
(CT)
adjuvant
adjuvant
systemic IgG.
Prophylactic
Prophylacticvaccination
vaccinationwith
withadjuvanted
adjuvantedTA-CIN
TA-CINprotected
protectedthe
themice
micefrom
fromviral
viral
®
Quil-A
and QB-90
induced
similar
immunitywithout
regarding
humoral
oralmost
Mice
Miceand
and
challenge,
challenge,
whereas
whereas
vaccination
vaccination
without
adjuvant
adjuvant
was
was
almost
ineffective.
ineffective.Moreover,
Moreover,GPIGPI[27]
cellular responses.
monkeys
monkeys
0100
0100boosted
boostedIFN-γ
IFN-γsecreting
secretingCD8
CD8+ +TTcell
cellresponse
responseininmice
micecompared
comparedtotothe
theformulation
formulation
without
without
adjuvant.
adjuvant.
Vaccination
Vaccination
ofofmacaques
macaquesinduced
inducedspecific
specific
cellresponses.
responses.
Increase
in both
Th1 andTh2
response.
[120] TTcell
Miceand
and
Table 2. Cont. Mice
Model
Mice and monkeys
GPI-0100
GPI-0100;
;Mice
sQS-21
sQS-21

GD3-KLH
GD3-KLH
QS-21
FALVAC-1A
FALVAC-1A(different
(different
epitopes
epitopesfrom
from
Plasmodium
Plasmodiumfalciparum)
falciparum)
GP1-0100
FML
FML**
AIPO4,
AIPO4,QS-21,
QS-21,Montanide
Montanide
ISA-720,
ISA-720,or
orCRL-1005
CRL-1005
Mice
QS-21
QS-21
AD-472
AD-472
or
orHSV-2
HSV-2
Quillaja saponins
or cross-linked
glycoprotein
glycoprotein
DD
dextran microspheres (CDM)
GPI-0100
GPI-0100

Rabbits
Ovalbumin
H9N2
H9N2
ISCOM matrices
Aluminium
Aluminiumphosphate;
phosphate;
aluminium
aluminiumhydroxide;
hydroxide;MF59
MF59
Mice
;
;and
andMATRIX-M
MATRIX-M# #
Tetanus toxoid loaded
onto PLGA nanospheres
HpaA
HpaA
and
andcatalase
catalase
Quillaja
saponins,
CDM
TM
TM
ISCOMATRIX
ISCOMATRIXTM
; ;ISCOM
ISCOMTM
;;
Cholera
Choleratoxin;
toxin;and
andaluminium
aluminium
Rabbits
hydroxide
hydroxide
Inactivated Bovine
herpesvirus-5
Aluminum hydroxide, Quil-A® , or
##
TA-CIN
QB-90 (fromTA-CIN
Quillaja##brasiliensis)
ALM (Leishmania major)
PLGA nanoparticles containing
purified saponin extract
Mice
HIV-1 gp120 with Q105N
mutation
Quil-A® or MPL
Mice
Despite both adjuvants producing specific antibodies, there was no
significant neutralizing activity against HIV-1.
[121]
Tetanus toxoid
Quillaja saponins
Rabbits
Quillaja formulation was more efficient in conferring mucosal
protection due to increased levels of IgA.
[122]
No antigen was used
Alhydrogel or MATRIX-MTM
formulated with fractions of Q.
saponaria
Mice
Besides stimuli caused by MATRIX-MTM during the immune
response (increase in cell recruitment and cytokine secretion), it
induced the expression of co-stimulatory molecule CD86,
participating in early events of the immune response.
[123]
Rhoptry Toxoplasma
gondii protein
Quil-A®
Swine
The formulation induced high humoral, local, and systemic immune
response, partially preventing brains from forming cysts.
[124]
Mice
GPI-0100
GPI-0100

[105]
[105
[106]
[106
[107]
[107
[108]
[108
[109]
[109
[110]
[110
[111]
[111
[112]
[112
[113]
[113
[114]
[114
Molecules 2019, 24, 171
14 of 29
Table 2. Cont.
Antigen
Adjuvant
Model
Findings
Ref.
Tetanus toxoid loaded
onto chitosan
functionalized gold
nanoparticles
Quillaja saponins
Mice
When administered orally, the nanoparticles can better deliver the
formulation, increasing immune response up to 28-fold compared to
control.
[125]
HIV-1 gp120
QS-21, aluminum hydroxide, MPLA,
or ISCOMATRIXTM
Mice
Each adjuvant stimulated a different cytokine profile secretion,
having different properties in the induction of the inflammation.
[126]
MUC1-KLH (prostate
cancer marker) and/or
ovalbumin
QS-21, synthetic QS-21, or other
conjugated saponins
Mice
Despite QS-21 inducing robust Th1 and Th2 response, conjugated
saponins had improved activity and toxicity profiles relative to
QS-21.
[127]
Varicella zoster
glycoprotein E and
ovalbumin
AS01
Mice
AS01 can improve adaptive humoral response through the
generation of a great number of antigen-presenting cells.
[128]
Inactivated Poliovirus
Quil-A® , QB-90, and Aqueous Extract
from Quillaja brasiliensis
Mice
The humoral enhancements caused by Quil-A® and QB-90 were
statistically similar. However, the mucosal immune response was
increased with QB-90 by increases in IgA.
[26]
Tau antigen (associated
with Alzheimer’s disease)
Quil-A®
Mice
Animals that received the formulations with Quil-A® had reduced
neuroinflammation and tau pathogenesis.
[129]
MARV-VLP (Marburg virus
virus-like particle
containing glycoprotein,
matrix vp40, and
nucleoprotein)
QS-21 or polyI:C
Cynomolgus macaques
Formulations prepared with either of the adjuvants provided full
protection in the challenge test. QS-21 produced a lower response to
antigens compared to polyI:C.
[130]
EBOVgp-Fc (Ebola virus
glycoprotein fused to Fc
fragment of human IgG1)
QS-21, aluminum hydroxide, or
poly-ICLC
Guinea pigs
All formulations induced antibody formation, however, despite
QS-21 inducing a strong humoral response, in the challenge test only
poly-ICLC induced robust protection.
[131]
Intanza 2013 (Trivalent
Influenza Vaccine, Sanofi
Pasteur)
QS-21
Mice
When administered in the skin as a Nanopatch vaccine, a lower dose
of QS-21 and adjuvant was required to enhance humoral response
compared to intramuscular administration.
[132]
Ovalbumin or HBsAg
(Hepatitis B virus surface
antigen)
QS-21 liposome
Mice
The immunoadjuvancy of QS-21 relies on macrophages to induce
activation of dendritic cells and stimulate the immune system.
[133]
FhSAP2-IBs (Fasciola
hepatica protein)
QS-21 or Montanide ISA720
Mice
Increase in both Th1 and Th2 responses with significant increase in
Th1 response.
[134]
Molecules 2019, 24, 171
15 of 29
Table 2. Cont.
Antigen
Adjuvant
Model
Findings
Ref.
QS-21 or QS-21
formulated with HIV-1
gp120
QS-21
Mice
QS-21 activates NLRP3 inflammasome.
[21]
Inactivated bovine viral
diarrhea virus
Saponins of Q. brasiliensis (QB-90) and
Q. brasiliensis aqueous extract (AE)
Mice
In vaccines adjuvanted with QB-90 and AE higher levels of antibody
were detected. Animals that received QB-90 adjuvanted vaccine had
enhanced cytokines and IFN-γ production by CD4+andCD8+T
lymphocytes whereas AE-adjuvanted preparation stimulated
humoral response only.
[135]
Ovalbumin
ISCOMs of Q. brasiliensis saponin
fraction (IQB-90) and Quil-A®
ISCOMs
Mice
IQB-90 inoculated subcutaneously induced strong antibody response
(IgG1and IgG2a) and increased Th1 response. IQ-90 delivered
intranasally induced secretion of serum IgG and IgG1 and mucosal
IgA.
[25]
No antigen was used
MATRIX-MTM
Swine
MATRIX-MTM formulation induced pro-inflammatory cytokines,
suggesting enhancement of innate immune response of specific
pathogen free pigs exposed to reared pigs.
[68]
Aβ42 (Alzheimer’s
disease pathological
hallmark)
QS-21
Rhesus monkeys
The formulation induced a good humoral response with high titers of
IgG and IgA. No inflammatory cellular immune response was
observed.
[136]
Ovalbumin and HBsAg
(Hepatitis B virus surface
antigen)
QS-21
In vitro (THP-1 cell) and
mice
Human monocyte-derived dendritic cells were directly activated by
QS-21, requiring cathepsin B to induce high CD4 and CD8 T cell
response.
[137]
FMP013 (Falsiparum
Malaria Protein-013 from
Plasmodium falciparum)
Molecules
Molecules
Molecules
Molecules
2019,
2019,
2019,
2019,
24,
24,
24,
24,
xxxx
HBsAg
ALF, ALF + aluminum hydroxide,
Mice
ALFQ + QS-21, or Montanide
ISA720
Molecules
2019, 24, x
Molecules
Molecules2019,
2019,24,
24,xx
Molecules
Molecules
Molecules
Molecules
Molecules
2019,
2019,
2019,
2019,
2019,
24,
24,
24,
24,
x24,
xxxx
QS-21 liposome
Swine
The ALF adjuvant conjugated with QS-21 induced the highest
antibody titer with the highest IgG2c titers. Also, it augmented the
number of activated B-cells compared to Montanide.
Molecules
Molecules
Molecules
2019,
2019,
2019,
24,
24,
24,
xxx
The study demonstrated that the HBsAg formulated with QS-21
liposomes in dissolvable microneedle arraypatches
induced
similar
Table
2. Cont.
immunization to the commercial Table
HBsAg
Table2.formulation.
2.Cont.
Cont.
[138]
12
12
12
12
ofofof30
of30
30
30
[139]
Table
Table
Table
2.2.2.
Con
Co
C
Table
Table
Table
Table
2.2.2.
Cont.
2.
Cont.
Cont.
Cont.
Table
Table
Table
Table
Table
2.2.
2.
Cont.
2.
Cont.
2.
Cont.
Cont.
Cont.
Fh15 (recombinant 15
Mice
immunized
with
ADAD
system
with
Qs
had
a
surviv
The use of Q. brasiliensis saponins
in vaccine formulations either
Mice15
and
Inactivated Bovine Viral
QB-90 and IMXQB-90Fh15
(ISCOMs
and
Fh15
(recombinant
(recombinant
15
Fh15
Fh15
(recombinant
(recombinant
(recombinant
15
15 Mice
Miceimmunized
immunized
with
withADAD
ADAD
system
with
with
Qs
Qshad
had
aMice
aMice
survival
Mice
survival
immuni
immun
immu
rate
rat
kDa
Fasciola 15
hepatica
ADAD
SystemFh15
*****
animals
without
Qs hadsystem
a survival
rate
of
40–50%.
Sheep
Mice
formulated
in
ISCOM
structures
or not promoted
a satisfactory
and
[140]
Mice
Mice
and
and
Mice
Mice
Mice
and
and
and
sheep
Fh15
Fh15
Fh15
Fh15
(recombinant
(recombinant
(recombinant
(recombinant
15
15
15
Fh15
Fh15
Fh15
Fh15
Fh15
(recombinant
(recombinant
(recombinant
(recombinant
(recombinant
Mice
Mice
Mice
Mice
15
15
immunized
15
immunized
15
immunized
15
immunized
with
with
with
with
ADAD
ADAD
ADAD
ADAD
system
system
system
system
with
with
with
with
Qs
Qs
Qs
Qs
had
had
had
had
aaADAD
survival
aADAD
survival
aADAD
survival
survival
Mice
Mice
rate
Mice
rate
Mice
rate
Mice
rate
immunized
of
immunized
of
immunized
of
immunized
50–62.5%
of
immunized
50–62.5%
50–62.5%
50–62.5%
with
while
with
while
with
while
with
while
with
ADAD
ADAD
ADAD
ADAD
ADAD
system
system
system
system
system
with
with
with
with
with
Qs
Qs
Qs
Qs
had
Qs
had
ha
h
Diarrhea
Virus 15
Q. brasiliensis saponins
kDa
kDaFasciola
Fasciolaprotein)
hepatica
hepatica
ADAD
ADAD
System
System
*****
*****
kDa
kDa
kDa
Fasciola
Fasciola
Fasciola
hepatica
hepatica
hepatica
animals
animals
without
without
System
System
System
Qs
Qs
had
had
*****
*****
*****
a
a
survival
survival
rate
rate
of
of
40–50%.
40–50%.
Sheep
animals
Sheep
animals
animals
immun
immu
with
wit
w
promising immune
response,
similar
to
Quil-A®.
system with Qs showed less
hepatic damage compar
Mice
Mice
Mice
Mice
and
and
and
and
Mice
Mice
Mice
Mice
Mice
and
and
and
and
and
sheep
sheep
sheep
sheep
sheep
kDa
kDa
kDa
kDa
Fasciola
Fasciola
Fasciola
Fasciola
hepatica
hepatica
hepatica
hepatica
ADAD
ADAD
ADAD
ADAD
System
System
System
System
*****
*****
*****
*****
kDa
kDa
kDa
kDa
kDa
Fasciola
Fasciola
Fasciola
Fasciola
Fasciola
hepatica
hepatica
hepatica
hepatica
animals
hepatica
animals
animals
animals
without
without
without
without
ADAD
ADAD
ADAD
Qs
ADAD
Qs
ADAD
Qs
Qs
had
had
System
had
System
had
System
a
System
a
System
survival
a
survival
a
survival
survival
*****
*****
*****
*****
*****
rate
rate
rate
rate
of
of
of
40–50%.
of
40–50%.
40–50%.
40–50%.
Sheep
Sheep
Sheep
Sheep
immunized
immunized
immunized
animals
immunized
animals
animals
animals
animals
without
without
with
without
with
without
with
without
with
ADAD
ADAD
ADAD
Qs
ADAD
Qs
Qs
Qs
had
Qs
had
had
had
had
a
a
survival
a
survival
a
[105]
survival
a
[105]
survival
[105]
survival
[105]
rate
rate
rate
rate
rate
of
of
of
40–50%
of
40–50%
of
40–50
40–5
40–
protein)
protein) sheep
protein)
protein)
protein)
system
system
with
with
Qs
Qsshowed
showed
less
lessbeing
hepatic
hepatic
damage
damagecompared
compared
system
system
system
totoc
Despite
nonspecific
sheep
sheep
sheep
sheep
sheep
sheep
sheep
sheep
* FML: Fucose–Mannose-ligand antigen obtained from Leishmania (L.) donovani
Sudan (LD 1S/MHOM/SD/00-strain 1S) antigen. ** LSP PbCS
242–310:
long
synthetic reactions
polypeptide
(LSP)observed in the immuniz
FML * protein)
QS-21
Dogsdamage
protein)
protein)
protein)
protein)
protein)
protein)
protein)
protein) system
system
system
system
with
with
with
with
Qs
Qs
Qs
Qs
showed
showed
showed
showed
less
less
less
less
hepatic
hepatic
hepatic
hepatic
damage
damage
damage
compared
compared
compared
compared
toto
to
control
to
control
system
control
system
control
system
system
system
group.
group.
with
group.
with
group.
with
with
with
Qs
Qs
Qs
Qs
showed
Qs
showed
showed
showed
showed
less
less
less
less
less
hepatic
hepatic
hepatic
hepatic
hepatic
damage
damag
damag
dama
dam
Despite
Despite
nonspecific
nonspecific
reactions
reactions
being
being
observed
observed
in
in
the
the
Despite
immunized
Despite
immunized
Despite
nonspe
nonspe
nonsp
ani
an
PbCS 242–310, which represents C-terminus of circumsporozoite protein of Plasmodium berghei. *** HIV-1 DNA prime/protein–Env and Gag:
polyvalent
DNA
prime/protein
boost
vaccine;
veterinary
vaccines), a significant
decrease was shown
w
FML
FML**
QS-21
QS-21
FML
FML
FML
Dogs
Dogs
*observed
*observed
*observed
QS-21
QS-21
QS-21
Dogs
Dogs
Dogs
Despite
Despite
Despite
Despite
nonspecific
nonspecific
nonspecific
reactions
reactions
reactions
reactions
being
being
being
being
observed
ininin
the
in
the
the
the
immunized
immunized
immunized
immunized
Despite
Despite
Despite
Despite
Despite
animals
animals
animals
animals
nonspecific
nonspecific
nonspecific
nonspecific
nonspecific
(similar
(similar
(similar
(similar
reactions
to
reactions
to
reactions
to
reactions
other
to
reactions
other
other
other
being
being
being
being
being
observed
observed
observed
observed
observed
inwith
inin
the
in
the
in
the
th
**** LT(R192G): Escherichia coli heat-labile enterotoxin trypsin-cleavage site mutant; *****
ADADnonspecific
System:
includes
Q saponaria
saponins
and/or
the
hydroalcoholic
extract
of
P. leucotomos
veterinary
veterinary
vaccines),
vaccines),
a
a
significant
significant
decrease
decrease
was
was
shown
shown
veterinary
veterinary
veterinar
with
su
sit
Mice vaccinated with a combination[106]
of
plant-made LTB-E
FML
FML
FML
FML
****
QS-21
QS-21
QS-21
QS-21
Dogs
Dogs
Dogs
Dogs
FML
FML
FML
FML
FML
*****
QS-21
QS-21
QS-21
QS-21
QS-21
Dogs
Dogs
Dogs
Dogs
Dogs
[106]
[106]
[106]
(PAL), emulsified with Montanide ISA763A as a water/oil (30/70)
emulsion ;LTB-ESAT-6:
Plant-made
antigen
[transgenic
Arabidopsis
thaliana
plants
expressing
the
immuno-dominant
LTB-ESAT-6
and
veterinary
veterinary
veterinary
veterinary
vaccines),
vaccines),
vaccines),
vaccines),
aasignificant
asignificant
asignificant
significant
decrease
decrease
decrease
decrease
was
was
was
was
shown
shown
shown
shown
with
with
with
with
veterinary
subsequent
veterinary
subsequent
veterinary
subsequent
veterinary
subsequent
veterinary
vaccines),
vaccines),
doses.
vaccines),
doses.
vaccines),
doses.
vaccines),
doses.of
aof
asignificant
aplant-made
significant
aplant-made
significant
asignificant
significant
decrease
decrease
decrease
decrease
decrease
was
was
wa
ww
Mice
Mice
vaccinated
vaccinated
with
with
aacombination
combination
Mice
LTB-ESAT
Mice
Mice
vaccin
vacci
vacc
adjuvant
hadmixture
significantly
more and
IL-10
productionLTB-ESAT-6
when
com
tuberculosis antigen ESAT-6 fused to the B subunit of the
Escherichia. ;;and
GPI-0100:
semi synthetic analogue of Quillaja
Saponin; ;;;
sQS-21
(synthetic
QS-21):
of QS-21-Api
LTB-ESAT-6
LTB-ESAT-6
and
LTB-ESAT-6
LTB-ESAT-6
LTB-ESAT-6
and
and
and
Bacillus CalmetteQuillaja
extract
Mice
Mice
Mice
Mice
Mice
vaccinated
vaccinated
vaccinated
vaccinated
with
with
with
with
aacombination
acombination
acombination
combination
ofofof
plant-made
of
plant-made
plant-made
plant-made
LTB-ESAT-6
LTB-ESAT-6
LTB-ESAT-6
LTB-ESAT-6
Mice
Mice
Mice
Mice
Mice
fusion
vaccinated
fusion
vaccinated
fusion
vaccinated
fusion
vaccinated
vaccinated
BCG
BCG
BCG
BCG
with
oral
with
oral
with
oral
with
oral
with
aacombination
acombination
acombination
acombination
combination
ofof
of
plant-ma
of
plant-ma
of
plant-m
plant-m
plant#significantly
adjuvant
adjuvant
had
had
significantly
more
more
IL-10
production
production
when
when
adjuvant
adjuvant
adjuvant
compared
compared
had
had
had
sis
QS-21-Xyl
(65:35); ;;;
AD-472
(HSV) typeLTB-ESAT-6
2LTB-ESAT-6
attenuated;
MF59:
consisting of squalene–oil–water
adjuvant;
MATRIX-M:
mix
of IL-10
two
without
adjuvant.
However,
nomono
protection
was
shown
durin
LTB-ESAT-6
LTB-ESAT-6
LTB-ESAT-6
LTB-ESAT-6
and
;
and
and
and human herpes simplex virus
LTB-ESAT-6
LTB-ESAT-6
LTB-ESAT-6
;;;
and
;
and
;
and
and
and emulsion
Bacillus
Bacillus
CalmetteCalmetteQuillaja
Quillaja
extract
extract
Bacillus
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CalmetteMice
CalmetteMice
CalmetteQuillaja
Quillaja
Quillaja
extract
extract
extract
Mice
Mice
Mice
Guérin
(BCG)
adjuvant
adjuvant
adjuvant
adjuvant
had
had
had
significantly
significantly
significantly
significantly
more
more
more
IL-10
IL-10
production
production
production
production
when
when
when
when
compared
compared
compared
compared
adjuvant
adjuvant
adjuvant
adjuvant
adjuvant
with
with
with
had
with
had
had
had
mice
had
mice
significantly
mice
significantly
mice
significantly
significantly
significantly
vaccinated
vaccinated
vaccinated
vaccinated
more
more
more
more
more
IL-10
IL-10
IL-10
IL-10
IL-10
production
production
production
production
production
wh
w
saponin cages to which an antigen is added; ## TA-CIN: recombinant
Human
papillomavirus
typehad
16
(HPV16)
L2, E6, more
and
E7IL-10
inIL-10
a unique
protein.
without
without
adjuvant.
adjuvant.
However,
However,
no
no
protection
protection
was
was
shown
shown
without
without
without
during
during
adjuva
adjuv
adjuv
the
the
cw
oral BCG (with o
Bacillus
Bacillus
Bacillus
Bacillus
CalmetteCalmetteCalmetteCalmetteQuillaja
Quillaja
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extract
extract
extract
extract
Bacillus
Mice
Bacillus
Mice
Bacillus
Mice
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Bacillus
CalmetteCalmetteCalmetteCalmetteCalmetteQuillaja
Quillaja
Quillaja
Quillaja
Quillaja
extract
extract
extract
extract
extract
Mice
Mice
Mice
Mice
Mice tuberculosis, in groups treated with
[107]
[107]
[107]
[107]
Guérin
Guérin(BCG)
(BCG)
Guérin
Guérin
Guérin
(BCG)
(BCG)
(BCG)was
without
without
without
without
adjuvant.
adjuvant.
adjuvant.
adjuvant.
However,
However,
However,
However,
no
no
no
no
protection
protection
protection
protection
was
was
was
shown
shown
shown
shown
during
during
during
during
without
without
without
the
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the
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the
the
challenge
challenge
challenge
adjuvant.
challenge
adjuvant.
adjuvant.
adjuvant.
adjuvant.
test
test
test
However,
test
However,
with
However,
with
However,
with
However,
with
M.
M.
M.
M.
no
no
no
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protection
no
protection
protection
protection
protection
was
was
was
was
was
show
show
sho
sh
s
tuberculosis,
tuberculosis,
in
in
groups
groups
treated
treated
with
with
oral
oral
BCG
BCG
(with
(with
tubercu
or
tubercu
or
tuberc
with
wit
GD3-KLH
GPI-0100 ; sQS-21 
Mice
Both synthetic Quillaja saponin molecules produced
Guérin
Guérin
Guérin
Guérin
(BCG)
(BCG)
(BCG)
(BCG)
Guérin
Guérin
Guérin
Guérin
Guérin
(BCG)
(BCG)
(BCG)
(BCG)
(BCG)
tuberculosis,
tuberculosis,
tuberculosis,
tuberculosis,
in
in
groups
in
groups
groups
groups
treated
treated
treated
treated
with
with
with
oral
oral
oral
oral
BCG
BCG
BCG
BCG
(with
(with
(with
(with
or
or
or
without
or
without
without
without
tuberculosis,
tuberculosis,
tuberculosis,
tuberculosis,
saponin).
tuberculosis,
saponin).
saponin).
saponin).
ininin
groups
in
groups
in
groups
groups
groups
treated
treated
treated
treated
treated
with
with
with
with
with
oral
oral
oral
oral
oral
BCG
BC
BC
B
GD3-KLH
GD3-KLH (differentGPI-0100
GPI-0100
;
;sQS-21
sQS-21in


GD3-KLH
GD3-KLH
GD3-KLH
Mice
Micewith
GPI-0100
GPI-0100
Both
GPI-0100
Both
synthetic
synthetic
;
;
;
sQS-21
sQS-21
sQS-21
Quillaja
Quillaja



saponin
saponin
molecules
Mice
molecules
Mice
Mice
produced
produced
Both
Both
antibo
Both
antib
sy
s
FALVAC-1A
QS-21
was
the
second
adjuvant
that
induced
the most
signi
AIPO4,
QS-21,
Montanide
GD3-KLH
GD3-KLH
GD3-KLH
GD3-KLH
GPI-0100
GPI-0100
GPI-0100
GPI-0100
;
;
;
;
sQS-21
sQS-21
sQS-21
sQS-21




Mice
Mice
Mice
Mice
GD3-KLH
GD3-KLH
GD3-KLH
GD3-KLH
GD3-KLH
Both
Both
Both
Both
synthetic
synthetic
GPI-0100
synthetic
GPI-0100
synthetic
GPI-0100
GPI-0100
GPI-0100
Quillaja
Quillaja
;
Quillaja
;
Quillaja
;
;
sQS-21
;
sQS-21
sQS-21
sQS-21
saponin
sQS-21
saponin
saponin
saponin





molecules
molecules
molecules
molecules
Mice
Mice
Mice
produced
Mice
produced
Mice
produced
produced
antibodies
antibodies
antibodies
antibodies
Both
Both
Both
Both
in
Both
in
synthetic
in
synthetic
mice.
in
synthetic
mice.
synthetic
mice.
synthetic
mice.
Quillaja
Quillaja
Quillaja
Quillaja
Quillaja
[108]
saponin
[108]
saponin
[108]
saponin
[108]
saponin
saponin
molecules
molecules
molecules
molecules
molecule
pr
FALVAC-1A
FALVAC-1A
(different
(different
FALVAC-1A
FALVAC-1A
FALVAC-1A
(different
(different
(different
QS-21
QS-21
was
wasthe
thepredominantly
second
secondadjuvant
adjuvant
that
thatinduced
induced
the
theinduced
most
most
QS-21
QS-21
QS-21
significant
significant
was
was
was
the
thtp
epitopes
from
Mice
inducing
IgG2c.
QS-21
also
the
high
AIPO4,
AIPO4,
QS-21,
QS-21,
Montanide
Montanide
AIPO4,
AIPO4,
AIPO4,
QS-21,
QS-21,
QS-21,
Montanide
Montanide
Montanide
ISA-720,
or
CRL-1005
FALVAC-1A
FALVAC-1A
FALVAC-1A
FALVAC-1A
(different
(different
(different
(different
FALVAC-1A
FALVAC-1A
FALVAC-1A
FALVAC-1A
FALVAC-1A
(different
(different
(different
QS-21
(different
QS-21
(different
QS-21
QS-21
was
was
was
was
the
the
the
the
second
second
second
second
adjuvant
adjuvant
adjuvant
adjuvant
that
that
that
that
induced
induced
induced
induced
the
the
the
the
most
most
most
most
significant
significant
significant
significant
QS-21
QS-21
QS-21
QS-21
QS-21
levels
levels
was
levels
was
levels
was
was
was
the
of
the
of
the
of
the
antibodies,
of
second
the
antibodies,
second
antibodies,
second
antibodies,
second
second
adjuvant
adjuvant
adjuvant
adjuvant
adjuvant
that
that
that
that
that
induced
induced
induced
induced
induced
the
the
the
the
m
th
epitopes
epitopes
from
from
epitopes
epitopes
epitopes
Mice
Mice
from
from
from
inducing
inducing
predominantly
predominantly
IgG2c.
IgG2c.
QS-21
also
also
Mice
Mice
Mice
induced
induced
inducing
the
inducing
the
inducing
highest
highest
predo
pred
pre
lev
lem
Plasmodium
falciparum) ISA-720,
to
the
otherQS-21
adjuvants,
indicating
Th1
and
Th
AIPO4,
AIPO4,
AIPO4,
AIPO4,
QS-21,
QS-21,
QS-21,
QS-21,
Montanide
Montanide
Montanide
Montanide
AIPO4,
AIPO4,
AIPO4,
AIPO4,
AIPO4,
QS-21,
QS-21,
QS-21,
QS-21,
QS-21,
Montanide
Montanide
Montanide
Montanide
Montanide
ISA-720,
or
or
CRL-1005
CRL-1005
ISA-720,
ISA-720,
ISA-720,
or
or
or
CRL-1005
CRL-1005
CRL-1005
epitopes
epitopes
epitopes
epitopes
from
from
from
from
Mice
Mice
epitopes
Mice
epitopes
Mice
epitopes
epitopes
epitopes
from
from
inducing
from
inducing
from
inducing
from
inducing
predominantly
predominantly
predominantly
predominantly
IgG2c.
IgG2c.
IgG2c.
IgG2c.
QS-21
QS-21
QS-21
QS-21
also
also
also
also
induced
induced
induced
induced
Mice
Mice
Mice
Mice
Mice
the
the
the
the
highest
highest
highest
highest
inducing
inducing
inducing
inducing
inducing
levels
levels
levels
levels
predominantly
of
predominantly
of
predominantly
of
predominantly
IL-4
of
predominantly
IL-4
IL-4
IL-4
compared
compared
compared
compared
IgG2c.
IgG2c.
IgG2c.
IgG2c.
IgG2c.
[109]
[109]
QS-21
[109]
QS-21
[109]
QS-21
QS-21
QS-21
also
also
also
also
also
induced
induced
induce
induc
indu
Plasmodium
Plasmodiumfalciparum)
falciparum)
Plasmodium
Plasmodium
Plasmodium
falciparum)
falciparum)
falciparum)
totothe
theother
otherrobust
adjuvants,
adjuvants,
indicating
indicatingTh1
Th1
and
and
Th2
Th2levels
resp
resp
The vaccine
induced
immunogenicity
with
high
ISA-720,
ISA-720,
ISA-720,
ISA-720,
or
or
or
CRL-1005
or
CRL-1005
CRL-1005
CRL-1005
ISA-720,
ISA-720,
ISA-720,
ISA-720,
ISA-720,
or
or
or
CRL-1005
or
CRL-1005
or
CRL-1005
CRL-1005
CRL-1005
FML
*
QS-21
Dogs
Plasmodium
Plasmodium
Plasmodium
Plasmodium
falciparum)
falciparum)
falciparum)
falciparum)
Plasmodium
Plasmodium
Plasmodium
Plasmodium
Plasmodium
falciparum)
falciparum)
falciparum)
falciparum)
falciparum) to
toto
the
to
the
the
the
other
other
other
other
adjuvants,
adjuvants,
adjuvants,
adjuvants,
indicating
indicating
indicating
indicating
Th1
Th1
Th1
Th1
and
and
and
and
Th2
Th2
Th2
Th2
responses.
responses.
responses.
responses.tototo
the
to
the
to
the
the
other
the
other
other
other
other
adjuvants,
adjuvants,
adjuvants,
adjuvants,
adjuvants,
indicating
indicating
indicating
indicating
indicating
Th1
Th
TT
Molecules 2019, 24, 171
16 of 29
According to Table 2, saponins can be formulated with a variety of antigens, either by pathogens
or human proteins, and elicit a strong immune response, generally geared towards a humoral
response [137]. Interestingly, over time there has been an increase in the number of studies
using QS-21 rather than Quil-A® or Quillaja extracts, probably due to its reduced toxicity [141].
Meanwhile, the chemical instability of QS-21 is reflected by acyl side chain hydrolysis, which occurs
spontaneously in aqueous solutions at room temperature [142], so studies of the structure-activity
relationship have been performed to enhance its adjuvant activity and reduce the undesirable
effects [90,103,127]. QS-21 deacylation at Fuc-C4 was critical for Th1-type response but it was not so
important for Th2-type response [90,142].
The combination of Quillaja saponins with other immunostimulants and formulation
with carriers can take advantage of different delivery systems, such as nanoparticles [124],
ISCOMs [25,86,91,96,101,113,126], ISCOM Matrix [88,113,123] and liposomes [133] for intranasal [117]
or subcutaneous injections.
In Table 2 over 60 pre-clinical studies for infectious diseases, degenerative disorders, and cancer are
reported. For human infectious diseases the most tested antigen was Fucose-Mannose-Ligand (FML)
antigen from Leishmania (L.) donovani [90,95,97,103,106,110]. Other vaccines for infectious diseases
have also been well studied, such as hepatitis B [133,137,139], malaria [113,142], and HIV-1 [21,121,126]
For veterinary use, pre-clinical trials have focused on Bovine Herpes Virus-5 [27] and Bovine
Viral Diarrhea Virus [25,143] Fewer studies have been reported for degenerative disorders
(Alzheimer’s disease) [129,136] and oncology [116,127]. In view of the amount of studies and the
promising findings, many clinical trials have been developed, as mentioned in Table 4.
Most clinical trials test the safety and efficacy of the formulations. Although Quil-A® and other
Quillaja saponins have been used in pre-clinical studies, only one study reported Quillaja saponins for
human use [144]. In all other studies, only QS-21 has been evaluated for human use [142,144–148].
QS-21 has been tested in prophylactic vaccines for treatment of malaria. A phase III clinical trial was
conducted to determine the efficacy, safety, and immunogenicity of the RTS/S ASO1 vaccine and
received positive regulatory approval from WHO, which recommended more evaluations before high
level introduction in Africa, where the clinical study was conducted. Prophylactic vaccines have been
tested using gp120 protein from HIV-I [149] and hepatitis B virus surface antigen [150].
QS-21 has been used in therapeutic vaccines to treat cancer patients. In these cases, instead of
being administered in healthy people, the vaccine formulation needs to induce immune responses
against existing tumors [83,145,146,151,152]. A more important experiment using QS-21 in therapeutic
vaccines occurred after a surgical procedure in patients in stage II melanoma. The vaccine was well
tolerated, and robust antibody responses were obtained [142,145].
Molecules 2019, 24, 171
17 of 29
Table 3. Clinical trials using Quillaja saponins as an adjuvant in vaccine formulations.
Antigen
Adjuvant
CHO-derived gp120 protein
from HIV-1
QS-21 or Al(OH)3
Molecules 2019, 24, x
Objective
Findings
Ref.
Evaluate safety and assess kinetics of immune
response
Improved T cell response inducing CTL activation
with T helper cells was observed in formulations with
QS-21.
[149]
12 of 30
Determine the formulation toxicity against
12
12ofof30
30
Increase in specific antibody production and increased
cancerous cells, its immune response, and if
cytotoxicity either from complement system or
[145]
the conjugation of Globo H with KLH would Table 2. Cont.
antibody signaling.
affect the immune response
Table
Table2.2.Cont.
Cont.
Fh15 (recombinant 15
Mice immunized with ADAD system with Qs had a survival rate of 50–62.5% while
Mice and
PolySA and NP-polySA both
Fh15
Fh15
(recombinant
(recombinant
15
15
Mice
immunized
immunized
with
with
ADAD
system
system
with
with
Qs
Qs
had
hadantibody
aasurvival
survival
rate
rateofof50–62.5%
50–62.5%
while
while
kDa
Fasciola hepatica
ADAD
System the
*****immune response after Mice
animals
without
QsADAD
had
a survival
rate
40–50%.
Sheep immunized
with ADAD
[105]
Determine
NP-polySA
vaccination
resulted
in of
higher
Mice
Mice
and
and
Molecules
Molecules
2019,
2019,
2019,
24,
24,
24,
xxx
12
12
12ofofof30
30
3
conjugated with KLH (protein Molecules
sheep
kDa
kDaFasciola
Fasciola
hepatica
hepatica
ADAD
ADAD
System
System
*****
*****
animals
animals
without
without
Qs
Qs
had
had
aasurvival
survival
rate
rate
ofof40–50%.
40–50%.
Sheep
Sheep
immunized
with
with
ADAD
ADAD
[105]
[105]
QS-21
vaccination
and
assess
the
impact
of polySA
titers
with
IgM
response.
The
IgM
was
reactive
to immunized
[146]
protein)
system
with
Qs
showed
less
hepatic
damage
compared
to
control
group.
that in adults is associated with
sheep
sheep
chemical manipulation
small cell
lung
cancer.
protein)
protein)
system
system
with
withQs
Qs
showed
showed
less
less
hepatic
hepatic
damage
damage
compared
totocontrol
controlgroup.
group.
Despite
nonspecific
reactions
being
observed
in thecompared
immunized
animals
(similar to other
small cell lung cancer)
FML *
QS-21
Dogs
[106]
Table
Table
Table
2.2.
2.
Cont.
Cont.
Cont. abeing
Despite
Despiteveterinary
nonspecific
nonspecific
reactions
reactions
beingobserved
observed
ininthe
the
immunized
immunized
animals
animals
(similar
(similar
totoother
other
vaccines),
significant
decrease
was
shown with
subsequent
doses.
FML
FML**
QS-21
QS-21
Dogs
Dogs
[106]
[106]
GPI-0100 (5000 µg) and QS-21 (100 µg) produced
veterinary
vaccines),
vaccines),
aasignificant
significant
decrease
decrease
was
wasshown
shown
with
withsubsequent
subsequent
doses.
doses.
Present the use of GPI-0100 in humans and veterinary
Mice
vaccinated
with
awith
combination
of plant-made
LTB-ESAT-6
fusion
BCG
oral while
Fh15
Fh15
Fh15
(recombinant
(recombinant
(recombinant
15
15
15
Mice
Mice
Mice
immunized
immunized
immunized
with
with
ADAD
ADAD
ADAD
system
system
system
with
with
with
Qs
Qs
Qs
had
had
had
aasurvival
asurvival
survival
rate
rate
rate
ofofof
50–62.5%
50–62.5%
50–62.5%
while
while
GPI-0100
; GPI-0100-P
comparable
antibody
titers.
All
adjuvanted
vaccine
Mice
Mice
Mice
and
and
and
MUC-2G/Globo H-KLH LTB-ESAT-6 ; and
determine the safety and immunogenicity
of
a vaccinated
[83]
Mice
Mice
vaccinated
with
withaacombination
combination
of
ofplant-made
plant-made
LTB-ESAT-6
LTB-ESAT-6
fusion
fusion
BCG
BCGvaccinated
oral
oral
adjuvant
had
significantly
more
IL-10
production
when
compared
with
mice
kDa
kDa
kDa
Fasciola
Fasciola
Fasciola
hepatica
hepatica
hepatica
ADAD
ADAD
ADAD
System
System
System
*****
*****
*****
animals
animals
animals
without
without
Qs
Qs
Qs
had
had
had
aasurvival
asurvival
survival
rate
rate
rate
ofofof
40–50%.
40–50%.
40–50%.
Sheep
Sheep
Sheep
immunized
immunized
immunized
with
with
with
ADAD
ADAD
ADAD[107][105]
[105]
[105]
QS-21
doses without
were
well
tolerated
and antigen-specific
LTB-ESAT-6
LTB-ESAT-6
;;;and
and
Bacillus
CalmetteQuillaja
extract
Mice
vaccine with different doses of GPI-0100.
sheep
sheep
sheep
adjuvant
adjuvant
had
significantly
significantly
more
more
IL-10
IL-10
production
production
when
when
compared
compared
with
mice
micevaccinated
vaccinated
withouthad
adjuvant.
However,
no
protection
was
shown
during
thewith
challenge
test
with M.
antibody
titers
matched
increasing
dose
levels.
protein)
protein)
protein)
system
system
system
with
with
with
Qs
Qs
Qs
showed
showed
showed
less
less
less
hepatic
hepatic
hepatic
damage
damage
damage
compared
compared
compared
tototo
control
control
control
group.
group.
group.
Bacillus
Bacillus
CalmetteCalmetteQuillaja
Quillajaextract
extract
Mice
Mice
[107]
[107]
Guérin
(BCG)
without
withoutadjuvant.
adjuvant.
However,
However,
no
noprotection
protection
was
was
shown
shown
during
during
the
the
challenge
challenge
test
testwith
withM.
M.
tuberculosis,
in groups
treated
with
oral
BCG
(with
or
without
saponin).
Despite
Despite
Despite
nonspecific
nonspecific
nonspecific
reactions
reactions
reactions
being
being
being
observed
observed
observed
ininin
the
the
the
immunized
immunized
immunized
animals
animals
animals
(similar
(similar
(similar
tototo
other
other
other
Guérin
Guérin(BCG)
(BCG)
The adjuvanted
vaccine
induced
more
significative
FML
FML
FML
***
QS-21
QS-21
QS-21
Dogs
Dogs
[106]
[106]
[106]
tuberculosis,
tuberculosis,
iningroups
groups
treated
treated
with
with
oral
oralBCG
BCG
(with
(withor
orwithout
withoutsaponin).
saponin).
GD3-KLH
GPI-0100 ; sQS-21
MiceDogs
Both
synthetic
Quillaja
saponin
molecules
produced
antibodies
in
mice.
veterinary
veterinary
veterinary
vaccines),
vaccines),
aasignificant
asignificant
significant
decrease
decrease
decrease
was
was
was
shown
shown
shown
with
with
with
subsequent
subsequent
subsequent
doses.
doses.
doses. [108]
humoral
and vaccines),
Th1
immune
responses
compared
with
Evaluate
the
potential
of
an
AS02A
adjuvanted
GD3-KLH
GD3-KLH
GPI-0100
GPI-0100;
;sQS-21
sQS-21

Mice
Mice
Both
Both
synthetic
synthetic
Quillaja
Quillaja
saponin
saponin
molecules
molecules
produced
produced
antibodies
antibodies
ininmice.
mice.
[108]
[108]
FALVAC-1A
(different
QS-21
was
the
second
adjuvant
that
induced
the
most
significant
levels
of
antibodies,
the
non-adjuvanted
formulation.
Despite
the
AS02A
Mice
Mice
Mice
vaccinated
vaccinated
vaccinated
with
with
with
aacombination
acombination
combination
ofof
of
plant-made
plant-made
plant-made
LTB-ESAT-6
LTB-ESAT-6
LTB-ESAT-6
fusion
fusion
fusion
BCG
BCG
BCG
oral
oral
oral
QS-21, Montanide
SL *
AS02A
in healthy individuals
[153]of
LTB-ESAT-6
LTB-ESAT-6
LTB-ESAT-6
and
and
and AIPO4,formulation
FALVAC-1A
FALVAC-1A
(different
(different
QS-21
was
was
the
thesecond
second
adjuvant
adjuvant
that
that
induced
induced
the
themost
most
significant
significant
levels
levels
ofofIL-4
antibodies,
antibodies,
epitopes
from ;;;
Mice andQS-21
inducing
predominantly
IgG2c.
QS-21
alsoproduction
induced
the
highest
levels
compared
[109]
recipients
reporting
local
and
general
reactions
more
adjuvant
adjuvant
adjuvant
had
had
had
significantly
significantly
significantly
more
more
more
IL-10
IL-10
IL-10
production
production
when
when
when
compared
compared
compared
with
with
with
mice
mice
mice
vaccinated
vaccinated
vaccinated
AIPO4,
AIPO4,
QS-21,
QS-21,
Montanide
Montanide
ISA-720,
or
CRL-1005
compare
it toextract
aextract
non-adjuvanted
Bacillus
Bacillus
Bacillus
CalmetteCalmetteCalmetteQuillaja
Quillaja
Quillaja
extract
Mice
Mice
Mice
[107]
[107]
epitopes
epitopes
from
from
Mice
Mice vaccine.
inducing
inducingfrequently
predominantly
predominantly
IgG2c.
IgG2c.
QS-21
QS-21also
also
induced
induced
the
theand
highest
highest
levels
levelsofofIL-4
IL-4compared
compared [109]
[109] [107]
Plasmodium
falciparum)
to than
the other
adjuvants,
indicating
Th1
Th2
responses.
the
non-adjuvanted
group,
safety
without
without
without
adjuvant.
adjuvant.
adjuvant.
However,
However,
However,
no
no
no
protection
protection
protection
was
was
was
shown
shown
shown
during
during
during
the
the
the
challenge
challenge
challenge
test
test
test
with
with
with
M.
M.
M.
ISA-720,
ISA-720,or
orCRL-1005
CRL-1005
Guérin
Guérin
Guérin
(BCG)
(BCG)
(BCG)
Plasmodium
Plasmodium
falciparum)
falciparum)
totothe
theother
other
adjuvants,
adjuvants,
indicating
indicating
Th1
Th1
and
andlevels
Th2
Th2responses.
responses.
profile
was
acceptable.
The vaccine tuberculosis,
induced
robust
immunogenicity
with
high
of
FML-seroconversion,
as
tuberculosis,
tuberculosis,
ininin
groups
groups
groups
treated
treated
treated
with
with
with
oral
oral
oral
BCG
BCG
BCG
(with
(with
(with
or
or
or
without
without
without
saponin).
saponin).
saponin).
FML *
QS-21
Dogs
[110]
The
Thevaccine
vaccine
induced
induced
robust
robust
immunogenicity
immunogenicity
with
with
high
high
levels
ofofFML-seroconversion,
FML-seroconversion,as
as
demonstrated
by CD8+
and
CD4+
Tlevels
cell
populations.
AS02A
(oil-in-water
FMP1
formulated
with
AS02A
was
well
tolerated
and
GD3-KLH
GPI-0100
GPI-0100
GPI-0100
;
;
sQS-21
sQS-21
sQS-21


Dogs
Mice
Mice
Both
Both
Both
synthetic
synthetic
synthetic
Quillaja
Quillaja
Quillaja
saponin
saponin
saponin
molecules
molecules
molecules
produced
produced
produced
antibodies
antibodies
antibodies
ininin
mice.
mice.
mice. [110]
[108]
[108]
FML
FML
*GD3-KLH
*GD3-KLH
QS-21
QS-21 ;
Dogs Mice
[110] [108]
demonstrated
demonstrated
by
by
CD8+
CD8+
and
and
CD4+
CD4+
T
T
cell
cell
populations.
populations.
AD-472

or HSV-2
Guinea
Both
formulations
reduced
clinical
disease;
however,
GPI-0100
improved
formulation
with
Evaluate the safety and immunogenicity
of
showed
high
immunogenicity
with
specific
FALVAC-1A
FALVAC-1A
FALVAC-1A
(different
(different
(different
QS-21
QS-21
QS-21
was
was
was
the
the
the
second
second
second
adjuvant
adjuvant
adjuvant
that
that
that
induced
induced
induced
the
the
the
most
most
most
significant
significant
significant
levels
levels
levels
ofofthe
of
antibodies,
antibodies,
antibodies,
[147]
FMP1 **
GPI-0100

[111]
AIPO4,
AIPO4,
AIPO4,
QS-21,
QS-21,
QS-21,
Montanide
Montanide
Montanide
AD-472
AD-472


or
orHSV-2
HSV-2
Guinea
Guinea
Both
Both
formulations
formulations
reduced
reduced
clinical
clinical
disease;
disease;
however,
however,
GPI-0100
GPI-0100improved
improvedthe
the
anti-MSP-142
antibody
titers
boosted
and
prolonged
monophosphoryl
malaria
vaccine
FMP1/AS02A
inMice
adults
glycoprotein
Dfrom
pigs
glycoprotein-D
formulation
only.
epitopes
epitopes
epitopes
from
from
Mice
Mice
inducing
inducing
inducing
predominantly
predominantly
predominantly
IgG2c.
IgG2c.
IgG2c.
QS-21
QS-21
QS-21
also
also
also
induced
induced
induced
the
the
the
highest
highest
highest
levels
levels
levels
ofofof
IL-4
IL-4
IL-4
compared
compared
compared
[109]
[109]
GPI-0100
GPI-0100

[111]
[111] [109]
ISA-720,
ISA-720,
ISA-720,
or
or
or
CRL-1005
CRL-1005
CRL-1005 pigs
due
toimmune
the vaccination.
lipid ADand
glycoprotein
glycoprotein
D QS-21
pigs
glycoprotein-D
glycoprotein-D
formulation
formulation
only.
only.response (towards a Th1
MATRIX-M
shifted
the
response
to
an
IgG2a
Plasmodium
Plasmodium
Plasmodium
falciparum)
falciparum)
falciparum) Aluminium phosphate;
tototo
the
the
the
other
other
other
adjuvants,
adjuvants,
adjuvants,
indicating
indicating
indicating
Th1
Th1
Th1
and
and
and
Th2
Th2
Th2
responses.
responses.
responses.
+ T-cell
MATRIX-M
MATRIX-M
shifted
shifted
the
theimmune
immune
response
response
totoan
anresponse
IgG2a
IgG2a
response
response
(towards
(towards
aaTh1
Th1
response).
On
the
other
hand,immunogenicity
the
CD8
could
be
improved
using
Local
pain
and
post
vaccination
myalgias
were
greater
The
The
The
vaccine
vaccine
vaccine
induced
induced
induced
robust
robust
robust
immunogenicity
immunogenicity
with
with
with
high
high
high
levels
levels
levels
ofofof
FML-seroconversion,
FML-seroconversion,
FML-seroconversion,
as
as
as
Aluminium
Aluminium
phosphate;
phosphate;
H9N2
aluminium
hydroxide;
MF59
MiceDogs
[112]
+ +T-cell
FML
FML
FML
***
QS-21
QS-21
QS-21
Dogs
DogsMATRIX-M
[110]
[110]
[110]
response).
response).
On
OnMF59.
the
theother
other
hand,
hand,
the
the
CD8
CD8
T-cellresponse
response
could
couldbe
beimproved
improved
using
using in
Evaluate QS21
as an adjuvant
and
compare
it
in
individuals
that
received
QS-21.
Despite
increased
or
Among
all
tested
molecules,
MATRIX-M
was
the
most
effective
demonstrated
demonstrated
demonstrated
by
by
by
CD8+
CD8+
CD8+
and
and
and
CD4+
CD4+
CD4+
TTT
cell
cell
cell
populations.
populations.
populations.
#
H9N2
H9N2 QS-21
aluminium
aluminium
hydroxide;
MF59
MF59
Mice
Mice
[112]
[112]
Influenza
[148]
; hydroxide;
and
MATRIX-M
with standard trivalent
inactivated
influenza
serum
antibodies,
the
mean
titers
for
formulations
MATRIX-M
MATRIX-M
or
or
MF59.
MF59.
Among
Among
all
all
tested
tested
molecules,
molecules,
MATRIX-M
MATRIX-M
was
was
the
the
most
most
effective
effective
in
in
the
immune
response,
followed
bydisease;
MF59
and
aluminium-based
adjuvants.
AD-472
AD-472
AD-472



or
or
or
HSV-2
HSV-2
HSV-2
Guinea
Guinea
Guinea inducingBoth
Both
Both
formulations
formulations
formulations
reduced
reduced
reduced
clinical
clinical
clinical
disease;
disease;
however,
however,
however,
GPI-0100
GPI-0100
GPI-0100
improved
improved
improved
the
the
the
;
;and
andMATRIX-M
MATRIX-M# #
(with
or without
QS-21)
were by
not
different.
GPI-0100
GPI-0100
GPI-0100



[111]
[111]
[111]
TM
TM
inducing
inducingthe
the
immune
immune
response,
response,
followed
followed
byMF59
MF59
and
andaluminium-based
aluminium-based
adjuvants.
vaccines,
using
two
different
antigensadjuvants.
and
different
glycoprotein
glycoprotein
glycoprotein
DDD ISCOMATRIX ; ISCOM ;
pigs
pigs
pigs ISCOMATRIXTM and ISCOMTM
glycoprotein-D
glycoprotein-D
glycoprotein-D
formulation
formulation
formulation
only.
only.
only.
TM
TM
TM
ISCOMATRIX
ISCOMATRIX
; ;ISCOM
ISCOM
;;
ISCOMATRIXTM
ISCOMATRIXTM
and
and
ISCOMTM
ISCOMTM
vaccines,
vaccines,
using
using
two
twodifferent
different
antigens
antigens
and
and
different
different
HpaA and catalase
Cholera toxin;TM
and
aluminium
Mice
delivery
systems
(intranasal
or
subcutaneous),
have
the
same
efficacy
in
reducing
H.
[113]
Lyophilized
formulation
is
as
efficient
and
safe
as
the
MATRIX-M
MATRIX-M
MATRIX-M
shifted
shifted
shifted
the
the
the
immune
immune
immune
response
response
response
tototo
an
an
an
IgG2a
IgG2a
IgG2a
response
response
response
(towards
(towards
(towards
aaTh1
aTh1
Th1
RTS,S (recombinant proteins
Evaluate
the immunogenicity
of RTS,S/AS02D
Aluminium
Aluminium
Aluminium
phosphate;
phosphate;
phosphate; Mice
HpaA
HpaAand
andcatalase
catalase
Cholera
Choleratoxin;
toxin;
and
andaluminium
aluminium
Mice
delivery
deliverysystems
systems
(intranasal
(intranasal
or
or
subcutaneous),
have
the
thesame
same
efficacy
efficacy
ininreducing
reducingH.
H.
[113]
[113]
AS02D
liquid
formulation,
promoting
satisfactory
[154]
hydroxide
pylori
colonization
assubcutaneous),
the
gold
standard
cholera
toxin
(CT)
adjuvant
+have
+T-cell
+T-cell
response).
response).
response).
On
On
On
the
the
the
other
other
other
hand,
hand,
hand,
the
the
the
CD8
CD8
CD8
T-cell
response
response
response
could
could
could
be
be
be
improved
improved
improved
using
using
using
from P. falciparum)
formulation (liquid and lyophilized)
H9N2
H9N2
H9N2
aluminium
aluminium
aluminium
hydroxide;
hydroxide;
hydroxide;
MF59
MF59
MF59
Mice
Mice
Mice
[112]
[112]
[112]
immunization.
hydroxide
hydroxide
pylori
pyloricolonization
colonization
as
aswith
the
thegold
gold
standard
standard
cholera
choleraprotected
toxin
toxin(CT)
(CT)
adjuvant
adjuvant
Prophylactic
vaccination
adjuvanted
TA-CIN
the
mice
from
viral
MATRIX-M
MATRIX-M
MATRIX-M
or
or
or
MF59.
MF59.
MF59.
Among
Among
Among
all
all
all
tested
tested
tested
molecules,
molecules,
molecules,
MATRIX-M
MATRIX-M
MATRIX-M
was
was
was
the
the
the
most
most
most
effective
effective
effective
ininin
###
;
;
;
and
and
and
MATRIX-M
MATRIX-M
MATRIX-M
Prophylactic
Prophylactic
vaccination
vaccination
with
withwithout
adjuvanted
adjuvanted
TA-CIN
TA-CIN
protected
protected
the
themice
micefrom
from
viral
viral GPIMice
and
challenge,
whereas
vaccination
adjuvant
was
almost
ineffective.
Moreover,
inducing
inducing
inducing
the
the
the
immune
immune
immune
response,
response,
response,
followed
followed
followed
by
by
by
MF59
MF59
MF59
and
and
and
aluminium-based
aluminium-based
aluminium-based
adjuvants.
adjuvants.
adjuvants.
TA-CIN ##
GPI-0100 
[114]
+ T adjuvant
Mice
Mice
and
and challenge,
challenge,
whereas
whereas
vaccination
vaccination
without
without
adjuvant
was
wasin
almost
almost
ineffective.
ineffective.to
Moreover,
Moreover,
GPIGPI0100 boosted
IFN-γ
secreting
CD8
cell
response
mice
compared
the formulation
TM
TM
TM
TM
TM
ISCOMATRIX
ISCOMATRIX
ISCOMATRIX
; ;ISCOM
;ISCOM
ISCOM
; ;monkeys
;
ISCOMATRIXTM
ISCOMATRIXTM
ISCOMATRIXTM
and
and
and
ISCOMTM
ISCOMTM
ISCOMTM
vaccines,
vaccines,
vaccines,
using
using
using
two
two
two
different
different
different
antigens
antigens
antigens
and
and
and
different
different
different
TA-CIN
TA-CIN####
GPI-0100
GPI-0100
 TM
[114]
[114]
+
+
monkeys
monkeys
0100
0100boosted
boosted
IFN-γ
IFN-γ
secreting
secreting
CD8
CD8 TTcell
cell
response
responsein
inmice
micecompared
compared
to
the
the
formulation
formulation
without
adjuvant.
Vaccination
of
macaques
induced
specific
Ttoefficacy
cell
responses.
HpaA
HpaA
HpaA
and
and
and
catalase
catalase
catalase
Cholera
Cholera
Cholera
toxin;
toxin;
toxin;
and
and
and
aluminium
aluminium
aluminium
Mice
Mice
Mice
delivery
delivery
delivery
systems
systems
systems
(intranasal
(intranasal
(intranasal
or
or
or
subcutaneous),
subcutaneous),
subcutaneous),
have
have
have
the
the
the
same
same
same
efficacy
efficacy
ininin
reducing
reducing
reducing
H.
H.
H. [113]
[113]
[113]
without
withoutadjuvant.
adjuvant.Vaccination
Vaccinationofofmacaques
macaquesinduced
inducedspecific
specificTTcell
cellresponses.
responses.
hydroxide
hydroxide
hydroxide
pylori
pylori
pylori
colonization
colonization
colonization
as
as
as
the
the
the
gold
gold
gold
standard
standard
standard
cholera
cholera
cholera
toxin
toxin
toxin
(CT)
(CT)
(CT)
adjuvant
adjuvant
adjuvant
Prophylactic
Prophylactic
Prophylactic
vaccination
vaccination
vaccination
with
with
with
adjuvanted
adjuvanted
adjuvanted
TA-CIN
TA-CIN
TA-CIN
protected
protected
protected
the
the
the
mice
mice
mice
from
from
from
viral
viral
viral
Mice
Mice
Mice
and
and
and
challenge,
challenge,
challenge,
whereas
whereas
whereas
vaccination
vaccination
vaccination
without
without
without
adjuvant
adjuvant
adjuvant
was
was
was
almost
almost
almost
ineffective.
ineffective.
ineffective.
Moreover,
Moreover,
Moreover,
GPIGPIGPI######
TA-CIN
TA-CIN
TA-CIN
GPI-0100
GPI-0100
GPI-0100



[114]
[114]
[114]
+ +T
+TT
monkeys
monkeys
monkeys 0100
0100
0100
boosted
boosted
boosted
IFN-γ
IFN-γ
IFN-γ
secreting
secreting
secreting
CD8
CD8
CD8
cell
cell
cell
response
response
response
ininin
mice
mice
mice
compared
compared
compared
tototo
the
the
the
formulation
formulation
formulation
without
without
without
adjuvant.
adjuvant.
adjuvant.
Vaccination
Vaccination
Vaccination
ofofof
macaques
macaques
macaques
induced
induced
induced
specific
specific
specific
TTT
cell
cell
cell
responses.
responses.
responses.
Molecules
Molecules2019,
2019,24,
24,xx
Globo H-KLH (carbohydrate
antigen found in most breast
QS-21
cancer cells)
Molecules 2019, 24, 171
18 of 29
Table 4. Clinical trials using Quillaja saponins as an adjuvant in vaccine formulations.
Antigen
Objective
Findings
Ref.
AlhydrogelTM ,
Montanide ISA720 and
AS02 Adjuvant System
Evaluate the immunogenicity and safety of
Pf AMA1 antigen (two different doses with
three different adjuvants)
All formulations caused different reactogenicity with
no serious reported adverse effects. All formulations
tested induced antibody production, with AS02 being
the most pronounced.
[155]
RTS,S ****
AS02A
Determine the safety of the RTS,S antigen
formulated with AS02A adjuvant.
The formulation was well tolerated in children, with a
good safety profile within the number of doses. The
AS02A formulation caused fewer serious adverse
events compared to the control group.
[156]
FMP2.1 *****
AS02A
Evaluate the reactogenicity, safety, and
immunogenicity of the malaria vaccine
FMP2.1/AS02A in adults.
The formulation was well tolerated and showed good
safety. Also, it was highly immunogenic.
[157]
No antigen was used
Quillaja saponins
Determine if dietary QS can modify
macrophage activity and investigate its effects
on liver function and inflammatory response
An increase in chemotactic and phagocytosis activities
were observed. Furthermore, no adverse effects were
seen since no significant changes in immunoglobulin,
transaminase, IL-1α, and TNF- α were observed.
[144]
NP-polySA-KLH (Polysialic
acid conjugated to keyhole
limpet hemocyanin (KLH))
QS-21
Confirm the safety profile and determine the
optimal dose
The lowest optimal immunogenic dose was 10 µg,
which resulted in consistent high-titer antibody
responses.
[151]
MAGE-A3 (tumor-specific
protein usually expressed in
melanoma)
AS02B or AS15
Discover which adjuvant would cause a more
robust and persistent immune response
AS15 provided a more robust immune response by
activating more dendritic and B cells.
[152]
AS02B, AS02V, or
AS01B
Molecules
Molecules
2019,
2019,24,
24,xx
Evaluate the duration of humoral and cellular
responses
Molecules
Molecules
Molecules
2019,
2019,
2019,
24,
24,
24,
xxx
QS-21
Evaluate
safety
and immunogenicity of the
Table
2. Cont.
pentavalent synthetic vaccine
Pf AMA1 ***
HBsAg (Hepatitis B virus surface
antigen)
Molecules 2019, 24, x
Unimolecular conjugated
Globo-H, GM2, sTn, TF, and Tf
(markers usually expressed on
cancer cell-surface)
Fh15 (recombinantovarian
15
Adjuvant
All formulations induced persistent T CD4+ and CD8+
[150]
specific response, as well as B-cell response, indicating
12 of 30
immunological memory.
83% of individuals
responded
to at least three antigens
Table
Table
2.2.Cont.
Cont.
Table
Table
Table
2.2.2.
Cont.
Cont.
Cont.
[158]
with satisfactory immune response.
Fh15
Fh15(recombinant
(recombinant15
15 Mice immunized
Fh15
Fh15
Fh15
(recombinant
(recombinant
(recombinant
15
15
15 system with Qs had aMice
Mice
immunized
immunized
with
withADAD
ADAD
Mice
system
Mice
system
Mice
immunized
immunized
immunized
with
withQs
Qshad
had
with
with
with
aasurvival
ADAD
survival
ADAD
ADAD
system
rate
system
rate
system
ofofwith
50–62.5
with
50–62
with
QQ
with ADAD
survival
rate of 50–62.5%
while
Mice
Miceand
and
Mice
Mice
Mice
and
and
and
Mice and
kDaFasciola
Fasciolahepatica
hepatica animals
kDa
ADAD
kDa
ADAD
kDa
Fasciola
Fasciola
Fasciola
System
System
hepatica
hepatica
hepatica
*****
*****
ADAD
ADAD
ADAD
System
System
System
*****
animals
*****
animals
*****immunized
without
withoutQs
Qs
had
had
aasurvival
survival
animals
animals
animals
rate
rate
without
without
ofwithout
of40–50%.
40–50%.
Qs
Qs
Qs
had
Sheep
had
Sheep
had
aasurvival
aimmunized
survival
immunized
survival
rate
rate
rate
with
of
wit
ofof
40
4
kDa Fasciola hepatica
ADAD
System
***** kDa
without
Qs
had
a survival rate
of 40–50%.
Sheep
with
ADAD
[105]
HBsAg (surface
antigen
of
sheep
sheep
sheep
sheep
sheep
sheep
Investigate
how
combining
AS01
triggers
innate
response,
such
as
NK-cells,
and
protein)
protein)
protein)
protein)
system
system
with
with
Qs
Qs
showed
showed
less
less
hepatic
hepatic
system
system
system
damage
damage
with
with
with
Qs
Qs
Qs
compared
compared
showed
showed
showed
less
less
to
less
tocontrol
hepatic
control
hepatic
hepatic
gro
da
gr
da
d
protein) Hepatitis B virus), ovalbumin,
system protein)
with
Qs
showed less hepatic
damage
compared
to control
group.
CSP (P. falciparum
AS01
immune-stimulants results in innate immune
activates CD8 T-cells in the lymph nodes, depending
[159]
Despitenonspecific
nonspecific
reactionsto
being
Despite
being
Despite
Despite
observed
observed
nonspecific
nonspecific
nonspecific
ininthe
the
reactions
reactions
immunized
reactions
immunized
being
being
being
animals
animals
observed
observed
observed
(simi
(sim
ini
Despite nonspecific reactions being observed
in the Despite
immunized
animalsreactions
(similar
other
and
response
on macrophage, IL-12,Dogs
and
IL-18.
FML
FML
**
FML
QS-21
FML
QS-21
FML
***
Dogs
DogsQS-21
QS-21
QS-21
Dogs
Dogs
FML * circumsporozoite protein),
QS-21
Dogs
[106]
veterinary
veterinary
vaccines),
vaccines),
a
a
significant
significant
veterinary
veterinary
veterinary
decrease
decrease
vaccines),
vaccines),
vaccines),
was
was
shown
shown
a
a
significant
a
significant
significant
with
with
subsequent
subsequent
decrease
decreas
decrea
veterinary
vaccines),
a
significant
decrease
was
shown
with
subsequent
doses.
Varicella zoster glycoprotein E
Mice
Micevaccinated
vaccinated
with
with
aacombination
combination
Mice
Mice
vaccinated
vaccinated
of
vaccinated
ofplant-made
plant-made
with
with
with
LTB-ESAT-6
aLTB-ESAT-6
acombination
acombination
combination
fusion
fusion
ofofof
plan
pla
pl
BC
B
Mice vaccinated with a combination of plant-made LTB-ESAT-6
fusion
BCG
oral Mice
GPI-0100:
Semi synthetic analogue of Quillaja
saponin; ;;and
GPI-0100P:
GPI-0100
purified; ;;;
AS02A:
LTB-ESAT-6
LTB-ESAT-6
and
LTB-ESAT-6
LTB-ESAT-6
LTB-ESAT-6
and
and
and oil-in-water formulation containing monophosphoryl lipid A and QS-21; * SL:
LTB-ESAT-6 ; and
adjuvant
adjuvant
had
had
significantly
significantly
more
more
adjuvant
adjuvant
adjuvant
IL-10
IL-10
production
production
had
had
had
significantly
significantly
significantly
when
when
compared
more
compared
more
more
IL-10
IL-10
IL-10
with
with
producti
product
produc
mice
mice
adjuvant
had
significantly
more
IL-10
production
when
compared
with
mice
vaccinated
recombinant hepatitis B protein containing the small protein (S) and the modified large (L) protein of the hepatitis B viral envelope, containing pre-Sl and pre-S2 sequences in addition to
Bacillus
BacillusCalmetteCalmetteBacillus
Bacillus
Quillaja
Bacillus
Quillaja
CalmetteCalmetteextract
Calmetteextract
Mice
Mice
Quillaja
Quillaja
Quillaja
extract
extract
extract
Mice
Mice
Mice
Bacillus CalmetteQuillaja extract
Mice
[107]
the entire S sequence. ** FMP1 (Falciparum malaria protein 1): recombinant
based
on the carboxy-terminal
merozoite
surface
(MSP-142)
from
3D7
clone
of
P. during
without
without
adjuvant.
adjuvant.
However,
However,
no
no
without
without
without
protection
protection
adjuvant.
adjuvant.
was
wasshown
shown
However,
However,
However,
during
no
no
no
protection
the
protection
the
protection
challenge
challenge
was
wa
wt
without protein
adjuvant.
However,
no protectionend
wasofshown
during
theprotein-1
challenge
test with
M.theadjuvant.
Guérin
(BCG)
(BCG) Antigen; **** RTS,S:
Guérin
Guérin
Guérin
(BCG)
(BCG)
(BCG)
Guérin (BCG)
falciparum; *** Pf AMA1: Plasmodium falciparum Guérin
Apical
Membrane
Plasmodium
falciparum
circumsporozoite
surface
antigen;
*****
FMP2.1:
recombinant
protein
(FMP2.1)
tuberculosis,
iningroups
groupstreated
treated
tuberculosis,
tuberculosis,
with
tuberculosis,
withoral
oralBCG
BCG
ininin
groups
(with
groups
(with
groups
or
or
treated
treated
without
treated
without
with
with
with
sapon
sapon
ora
or
o
tuberculosis, in groups treated with oral BCG (withtuberculosis,
or
without saponin).
based on Apical Membrane Antigen-1 (AMA-1) from the 3D7 clone of P. falciparum.
GD3-KLH
GD3-KLH
GPI-0100
GPI-0100
GD3-KLH
GD3-KLH
GD3-KLH
;
;sQS-21
sQS-21

saponin
GPI-0100
GPI-0100
GPI-0100
Mice
Mice ;
;
;
sQS-21
sQS-21
sQS-21



Both
Both
synthetic
synthetic
Mice
Mice
Mice
Quillaja
Quillajasaponin
saponin
Both
Both
molecules
Both
molecules
synthetic
synthetic
synthetic
produced
produced
Quillaja
Quillaja
Quillaja
antibodies
saponin
antibodies
saponin
saponin
molecu
molec
in
mole
inmi
m
GD3-KLH
GPI-0100 ; sQS-21 
Mice
Both synthetic
Quillaja
molecules
produced
antibodies
in
mice.
[108]
FALVAC-1A
FALVAC-1A(different
(different QS-21 FALVAC-1A
FALVAC-1A
FALVAC-1A
(different
(different
(different
QS-21
QS-21
was
wasthe
the
second
second
adjuvant
QS-21
QS-21
that
QS-21
thatinduced
was
induced
was
was
the
the
the
second
the
second
the
second
most
most
adjuvant
adjuvant
significant
adjuvant
significant
that
that
that
levels
levels
induced
induced
induced
ofofaat
FALVAC-1A (different
was
the second
adjuvant that induced the most
significant
levels
of adjuvant
antibodies,
AIPO4,
AIPO4,QS-21,
QS-21,Montanide
Montanide AIPO4,
AIPO4,
AIPO4,
QS-21,
QS-21,
QS-21,
Montanide
Montanide
Montanide
AIPO4, QS-21, Montanide
epitopes
epitopes
from
from
epitopes
epitopes
epitopes
from
from
from
Mice
Mice
inducing
predominantly
predominantly
Mice
Mice
Mice
IgG2c.
inducing
inducing
inducing
QS-21
QS-21also
also
predominantly
predominantly
predominantly
induced
inducedthe
theIgG2c.
highest
IgG2c.
highest
IgG2c.
QS-21
QS-21
levels
QS-21
levels
also
also
of
also
ofIL-4
ind
ILinin
epitopes from
Mice
inducing predominantly
IgG2c. QS-21 also
inducedinducing
the
highest
levels of
IL-4 IgG2c.
compared
[109]
ISA-720,
ISA-720,or
orCRL-1005
CRL-1005
ISA-720,
ISA-720,
ISA-720,
or
or
or
CRL-1005
CRL-1005
CRL-1005
ISA-720, or CRL-1005
Plasmodium
Plasmodiumfalciparum)
falciparum)
Plasmodium
Plasmodium
Plasmodium
falciparum)
falciparum)
falciparum)
totothe
theother
otheradjuvants,
adjuvants,indicating
indicating
tototo
the
the
Th1
the
Th1
other
other
and
other
andadjuvants,
Th2
adjuvants,
Th2
adjuvants,
responses.
responses.
indicatin
indicati
indicat
Plasmodium falciparum)
to the
other
adjuvants, indicating Th1 and Th2 responses.
The
Thelevels
vaccine
vaccine
inducedrobust
robustimmunogenicity
immunogenicity
The
The
The
vaccine
vaccine
vaccine
induced
induced
induced
with
withhigh
robust
high
robust
robust
levels
levels
immunogenicity
immunogenicity
immunogenicity
ofofFML-serocon
FML-seroco
ww
The vaccine induced robust immunogenicity with high
ofinduced
FML-seroconversion,
as
FML
FML
**
FML
QS-21
FML
QS-21
FML
***
Dogs
DogsQS-21
QS-21
QS-21
Dogs
Dogs
Dogs
FML *
QS-21
Dogs
[110]
Molecules 2019, 24, 171
19 of 29
4. Toxicity of Quillaja Saponins
Regarding the toxicity of Q. saponaria and Q. brasiliensis, there are few studies in the literature.
The studies available focus on the preclinical toxicity evaluation. One of the first studies to address the
toxicity of Q. saponaria and its fractions was published in 1995, by Rönnberg et al. [160]. This study
evaluated the toxicity of crude Q. saponaria extract and QH-A, QH-B, and QH-C purified triterpenoid
components in WEHI cells (immature B-lymphocytes of Mus musculus). The evaluation of the
lytic activities of Q. saponaria, responsible for the local reactions after injection, demonstrated that
QH-A was safer than QH-B and QH-C, causing hemolysis only at a concentration ten-times higher.
In addition, QH-B and QH-C did not affect RNA or protein synthesis, suggesting that they are not
the primary toxicity targets. Similarly, hydrolyzed saponins of Q. saponaria, resulting in deacylated
forms, have significantly reduced the toxicity of the substance. This supports the role of fatty acids
in the toxic activity of saponins [161]. Thought-provokingly, none of the Quillaja saponins presented
hemolytic activity after incorporation into the ISCOM matrix [160].
Although QH-B has a strong adjuvant effect, it represents the most toxic triterpenoid saponin,
thus limiting its application in vitro and in vivo. Rönnberg et al. [162] demonstrated that a 10-fold
higher QH-B concentration treated with sodium periodate reduced the enzymatic activities of the
cells exposed to QH-B without sodium periodate by a factor of ten. The QH-B toxicity reduction is
possibly the result of changes in the sugars’ chemical structure, such as xylose and galactose [160,162].
Similarly, hydrolyzed saponins of Q. saponaria, resulting in deacylated forms, have significantly reduced
the toxicity of the substance. This supports the role of fatty acids in the toxic activity of saponins [161].
Arabski et al. [46] also studied the in vitro toxicity of Q. saponaria, but in the CHO-K1 cell line
(cells from the ovary of Cricetulus griseus). They demonstrated that the saponins significantly increased
death in this cell line by early apoptosis, at doses ranging from 12 to 50 µg/mL. Minor doses were not
able to lyse the exposed erythrocytes, elucidating the minimal concentration required to intersperse
with cell membranes in order to lyse them.
Quillaja saponin toxicity has also been studied in alternative mammalian models and in vivo
models. Abdula et al. [163] evaluated the effects of Q. saponaria saponins regarding lethality and
alterations in Danio rerio morphology during the first 3 days. This invertebrate, also known as zebrafish,
is an important alternative to mammalian tests and is effective for studying toxicity mechanisms.
The results showed that Q. saponaria presented both harmful and beneficial effects on the embryos
of zebrafish, since a 5 µg/mL concentration promoted growth and a 10 µg/mL concentration was
mortal to zebrafish. These findings suggest that the toxic effects of Q. saponaria saponins could affect
the semipermeable protective chorion expressed by the embryo in development. Alternatively, it could
affect cells in the embryo bodies, by permeating the noncellular chorion.
The toxicity of Quillaja extracts and their saponin derivatives was also studied in neonates of
the aquatic crustacean D. magna and in D. rerio zebrafish embryos [164]. The authors demonstrated
that Quillaja extract and Q. saponaria fractions QS-18 (35.1 ± 4.1% of the total Quillaja saponins)
and QS-21 (3.7 ± 0.3%) have similar toxicity; nevertheless, with a toxicity potential three to eight
times higher than non-saponins. Thus, Quillaja saponins contribute to the majority of the toxicity
(85.1–93.6%), with QS-21 being slightly more toxic than QS-18. The non-saponins contribute to only
14 and 6%. The higher lipophilicity of QS-18 and QS-21 is probably responsible for their higher toxicity,
since lipophilic substances can easily cross the embryo chorion, resulting in a higher uptake in zebrafish
embryos [165].
In vivo Quillaja extract acute toxicity was studied by Tam and Roner [8]. They orally treated
newborn mice with 50 µL of saponin extract. The concentrations ranged from 0 to 0.5 mg/mouse.
The animals were treated for seven days and observed for two months. The 0.0375 mg/mouse dose
presented more than 50% mortality, thus the LD50 of the extract was established at 0.0325 mg/mouse.
The weight evaluation demonstrated that there was no short-term health impact in mice that survived
treatment with saponins. On the other hand, the high concentrations of the extract killed the mice
Molecules 2019, 24, 171
20 of 29
within 24–36 h, suggesting an important alteration in the membrane environment of the gastrointestinal
tract, leading cells to burst.
Finally, Vinai et al. [166] studied the safe dose of vaccine adjuvants, such as Quillaja saponins,
for intra-peritoneal vaccination. Flounder fingerlings were acutely treated and lethality and toxicity
signs, such as body color, feeding, and swimming behavior, were evaluated. The group treated with
saponins showed different toxicity levels, according to the concentrations tested. The toxicity signs
observed were fish lethargy, body color alterations, and alterations in feeding behavior; the liver and
intestine presented important hemorrhages, the fish showed pale gills, and the peritoneal cavity was
filled with red ascites, suggesting high toxicity of the highest dose tested. In this study, Quillaja saponins
presented a LD50 of 105 µg/fish (22.4 mg/kg), reinforcing the high toxicity. In addition, they caused
severe histological damage, as well as liver damage, observed by the increase in AST level at doses
above 16 µg/fish and of ALT at 160 µg/fish.
In clinical studies only QS-21 toxicity have been studied. For most patients the dose-limiting
toxicity is about 50 µg/dose, except for cancer patients (100–200 µg per vaccination) [142].
Therefore, the safety of Quillaja saponins is still not well established and more studies are necessary.
5. Conclusions
Quillaja saponins have been employed in the food industry as emulsifiers for beverages and
food additives. In the cosmetic industry, they have been used as antidandruff, cleansing, emulsifying,
foaming, masking, moisturizing, skin conditioning, and surfactant agents. However, these are not the
only applications of Quillaja saponins. The particular chemical characteristics of these metabolites,
along with their biological activities, show potential for drug discovery and vaccines development.
Most of these saponins are bidesmosides and contain a triterpenic aglycone belonging to the b-amyrin
series, most frequently quillaic acid. Vaccine adjuvation is the main propriety studied for Quillaja
saponins, which have shown potent immunogenic activity and the ability to modulate immunity
towards a cell-mediated response or to enhance antibody production. In addition to improving
immunoadjuvant activity and minimizing their toxicity, these saponins are been evaluated in different
formulations, such as ISCOMs, oil-in-water formulations, and liposomes, among others.
Furthermore, studies have demonstrated antiviral (herpes simplex virus type 1,
human immunodeficiency viruses 1 and 2, varicella zoster virus, vaccinia virus, rhesus rotavirus,
and reovirus), antifungal (Penicillium roguefortii, Aspergillus ochraceus, Alternaria solani, Fusarium oxysporum,
Verticillium dahlia, and Pythium ultimum), and antibacterial (Staphylococcus aureus, Salmonella typhimurium,
and Asaia spp.) activities for Quillaja saponins. However, their activity against E. coli is not established,
since different commercial extracts of Quillaja saponins have shown opposing results. Studies have also
demonstrated antiparasitic (in vitro), antitumor (in vitro and in vivo), and hepatoprotective (in vivo)
activities. Regarding toxicity, there are few studies in the literature, which focuses on evaluating
preclinical toxicity and lacks clinical studies.
Author Contributions: All the authors collaborated in the writing of the present work and approved
its submission.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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