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Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612
ABSTRACT
Most chemical and biological studies about garlic have been conducted using organosulfur compounds. However, a variety of steroid saponins from garlic and related Allium species are being increasingly recognized for their importance in biological processes. This report demonstrates the isolation and structure determination of steroid saponins from garlic and aged garlic extract (AGE). In addition, the in vitro antifungal antitumor cytotoxicity and blood coagulability effects of steroid saponins from garlic and related Allium species are provided. Animal studies on the cholesterol-lowering effects of the saponin fractions from garlic are also summarized.
KEY WORDS: • garlic • steroid saponins • sapogenins • cholesterol • LDL
The ability of garlic to lower serum cholesterol has been demonstrated
in experimental animals and humans. In the past, it has been reported
that steam-distilled garlic oil (Abo-Doma et al. 1991
, Kamanna and Chandrasekhara 1984
), the
ether fraction of garlic (Bordia et al. 1975
,
Jain and Konar 1978
), alliin (Itokawa et al. 1973
) and its enzymatic transformation products, allicin
(Augusti and Mathew 1974
) and diallyl disulfide
(Adamu et al. 1982
), might be responsible for the
cholesterol-lowering effects of garlic in animal experiments.
However, because of the high doses used in animal studies and the lack
of data on the absorption/metabolites or pharmacokinetics, especially
for allicin, it is not known to what extent and by what mechanism these
organosulfur compounds might contribute to the lowering of serum
cholesterol levels.
Plants of the genus Allium are known for their production of
steroid saponins, as well as organosulfur compounds (Kravets et al. 1990
). Recently, steroid saponins have been found to have
some interesting biological and pharmacologic activities including
antifungal, antibacterial, anti-inflammatory and hypocholesteremic
influences (Lacaille-Dubois and Wagner 1996
).
This paper reviews chemical studies of steroid saponins from garlic and aged garlic extract (AGE)3 and their biological characteristics. In addition, animal studies on the cholesterol-lowering effects of steroid saponins from garlic are summarized.
Steroid saponins from garlic and AGE
In 1988, a furostanol saponin, proto-eruboside-B (Fig. 1
) was first isolated from a crude glycoside fraction prepared from a
methanolic extract of frozen garlic (Matsuura et al. 1988
). Further studies of steroid saponins from frozen garlic
led to the isolation of a new furostanol saponin named sativoside-B1
and to the discovery of proto-desgalactotigonin (Matsuura et al. 1989a
). No spirostanol saponins have been isolated from
frozen garlic. The conversion of furostanol saponins in garlic bulbs to
spirostanol saponins is probably due to the depressed enzymatic
activity of ß-glucosidase caused by the crushing of frozen garlic
bulbs in methanol. A spirostanol saponin corresponding, to
eruboside-B (Fig. 1
, Compound 2) was isolated, as well as several
unidentified steroid saponins using TLC. Raw garlic bulbs were crushed
at room temperature for extraction with methanol. These findings reveal
that the processing of garlic leads to not only variation in the
amounts and types of organosulfur compounds found in garlic but also of
steroid saponins.
|
As a continuation of our studies on steroid saponins in garlic and
related Allium species with medicinal potential, we
investigated the isolation and structure determination of steroid
saponins from AGE. AGE has been shown to have interesting pharmacologic
properties, especially cardioprotective effects, which include the
lowering of serum cholesterol in clinical studies and animal models
(Efendy et al. 1997
, Lau et al. 1987
,
Steiner et al. 1996
, Yeh et al. 1995
). A
glycoside fraction from AGE was subjected to a combination of silica
gel and reversed-phase, highly porous polymer to afford
ß-chlorogenin (Fig. 2
, Compound 3), an unidentified sapogenin, eruboside-B (Fig. 1
,
Compound 2), 10 furostanol saponins and 7 spirostanol saponins. In
recent reviews (Agrawal 1996
), the structure elucidation
of steroid saponins was reported in detail. This report will provide
structural information about a new furostanol saponin and its
corresponding new spirostanol saponin.
|
-furostane-3ß, 6 ß, 22, 26-tetraol, with sugar
units at the 3- and 26-hydroxyl groups (Tables 1
and
2
). Enzymatic hydrolysis of saponin 13 with ß-glucosidase liberated
glucose and a new glycoside, which is identical to
C57H94O28
(Compound 20, Table 1
). Acid hydrolysis of Compound 20 revealed the
presence of Compound 3 and glucose, galactose and rhamnose. Proton
nuclear magnetic resonance (1H NMR) and
13C NMR spectra of Compound 20 revealed the
presence of three ß-glucopyranosyl units, one ß-galactopyranosyl
unit and one -rhamnopyranosyl unit. Negative fast atom bombarding
mass spectrum (FAB-MS) of Compound 20 showed ions at m/z
1225 [M-H]-, 1063
[1225-hexosyl]-, 901
[1063-hexosyl]-, 739
[901-hexosyl]- and 593
[739-methylpentosyl]-, indicating that the
-rhamnopyranosyl unit is linked to the hexosyl unit attached at the
3-hydroxy group of the aglycone.
|
|
). A comparison of the 13C NMR spectrum of the sugar
moiety of Compound 20 was made with Compound 2. An additional set of
signals due to an -rhamnopyranosyl unit appeared in the spectrum
of Compound 20. Thus, the formulation of Compound 20 is likely to be
ß-chlorogenin
3-O-ß-D-glucopyranosyl(1
2)-O-[ß-D-glucopyranosyl(13)]-O-ß-D-glucopyranosyl(1 4)-O-[-L-rhamnopyranosyl
(12)]-O-ß-D-galactopyranoside.
Because Compound 13 was a furostanol saponin corresponding to
Compound 20, it was established to be
(25R)-26-O-ß-D-glucopyranosyl-22-hydroxy-5-furostane-3ß,
6ß, 22, 26-tetraol
3-O-ß-D-glucopyranosyl(12)-O-[ß-D-glucopyranosyl(13)]-O-ß-D-glucopyranosyl(1 4)-O-[-L-rhamnopyranosyl(12)-O-ß-D-galactopyranoside.
A similar approach was taken to determine the structure of the other
steroid saponins (Fig. 2)
. This analysis revealed that a variety of
spirostanol saponins are produced in AGE during the aging process by
the conversion of furostanol saponins originally present. Furostanol
saponins isolated from AGE were also identical to the saponin fraction
obtained from frozen garlic bulbs by liquid chromatography-mass
spectrometry (LC-MS).
|
In vitro biological activities of steroid saponins
Monodesmosidic spirostanol saponins have significant antifungal
activity. Their activity is greater than that of triterpenoid saponins
(Hostettmann and Marston 1995a and 1995b
).
Eruboside-B was found to be active against Candida
albicans with a mean inhibitory concentration of 25
µg/mL (Matsuura et al. 1988
). The antifugal
activity of eruboside-B is comparable to that reported for allicin
or ajoene (Yoshida et al. 1987
). The genuine saponin
proto-eruboside-B was inactive against C. albicans.
Interestingly, when raw garlic bulbs are crushed, the enzymatic
conversion of Compound 1 into Compound 2 by ß-glucosidase is similar
to the conversion of alliin into allicin by alliinase, and both
end-products exhibit antifungal properties.
The tumor-inhibitory effects of garlic have been demonstrated in
various experimental systems. The effects of steroid saponins and their
sapogenins from garlic and related Allium species have been
examined by using an in vitro assay of
12-O-tetradecanoylphorbol-13-acetate–enhanced
32P-incorporation into phospholipids in HeLa
cells (Table 3
). All of the spirostanol saponins and sapogenins, including laxogenin
and agigenin, respectively, isolated from the bulbs of Allium
chinense (Matsuura et al. 1989b
) and A.
ampeloprasum (Morita et al. 1988
), possessed
inhibitory effects and eruboside-B (2) exhibited a
similar inhibition to glycyrrhetinic acid, which has
antitumor-promoting activity in vivo (Nishino et al. 1986
). The furostanol saponins tested in this study, however,
were found to have no effect or to exhibit weak activity.
Eruboside-B (2) was found to promote cytotoxity against
several cell lines, including BC1, Lu1, Col2, KB and KB-V
(Table 4
).
|
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). These saponins had no
effect on platelet aggregation. Nevertheless, isoeruboside-B
inhibited blood coagulation and had a fibrinolysis-promoting
effect. Proto-isoeruboside-B was found to promote only fibrinolysis
(Peng et al. 1996
).
Cholesterol-lowering effect of the steroid saponin fractions from garlic in rats
Several saponins have been shown to inhibit the intestinal
absorption of cholesterol and to reduce plasma cholesterol levels in a
variety of experimental animal models (Harwood et al. 1993
, Hosttetman and Marston 1995b
,
Sauvaire et al. 1991
). Koch (1993)
indicated that the cholesterol-lowering effect of garlic
preparations may be due to its saponin content. However, no
investigation of the cholesterol-lowering effects of steroid
saponins in garlic has been published. We investigated the
cholesterol-lowering effects of the saponin fractions from garlic
with a rat model of experimental hyperlipidemia induced by feeding a
0.5% cholesterol-enriched diet for 16 wk. In this study, two
garlic extract types, frozen garlic extract (FG-EXT) and raw garlic
extract (RG-EXT), were prepared. Two preparations were used because it
has been reported that spirostanol saponins are more active than
furostanol saponins in screening for the ability to remove excess
cholesterol from blood (Kintia 1996
). These extracts
were divided into three fractions (HP20-W, HP20–20 and HP20–100)
after chromatography on a reversed-phase column (Fig. 4
). TLC and LC-MS analysis revealed that RG-HP20–100, a saponin
fraction from raw garlic, contained spirostanol saponins produced by
the conversion of furostanol saponins via ß-glucosidase, whereas
fraction FG-HP20–100 from frozen garlic was rich in furostanol
saponins. Doses of garlic extract [0.3 g/(kg · d] and fractions,
HP20-W [0.3 g/(kg · d)], HP20–20 [3 mg/(kg · d)] and
HP20–100 [10 mg/(kg · d)], equivalent to 
2 g/(kg · d) of
garlic bulbs, were used in this animal study.
|
). The decrease of total cholesterol in the group treated with
RG-HP20–100 was greater than that of FG-HP20–100. The active
fractions, RG-HP20–100 and FG-HP20–100, commonly contain steroid
saponins as major constituents; therefore, the steroid saponins,
especially spirostanol saponins, might be responsible for the decrease
in the plasma total cholesterol level. Interestingly, fraction
RG-HP20–20 was most active at a low dose [3 mg/(kg · d)] of
these fractions. Additional studies will be required to elucidate the
active components.
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Summary
Plant saponins have been shown to inhibit cholesterol absorption
from the intestinal lumen in experimental animals, and consequently to
reduce the concentration of plasma cholesterol. This may be the result
of a complex formation with cholesterol in the digestive tract or a
direct effect of plant saponins on cholesterol metabolism
(Hosttetmann and Marston 1995b
). Saponins may also
account for the cholesterol-lowering effect of garlic. In this
study, we found that the saponin fractions from garlic lowered plasma
total and LDL cholesterol concentrations without changing HDL
cholesterol levels in a hypercholesterolemic animal model. Several
steroid saponins occur in both garlic and AGE. These results suggest
that special consideration should be given to steroid saponins, as well
as organosulfur compounds, in biological and pharmacologic studies of
garlic and its preparations.
ACKNOWLEDGMENTS
The author is especially grateful to N. R. Farnsworth and C.W.W. Beecher, University of Illinois at Chicago, for their advice and encouragement, and K. Slowing and T. Tejerina, Complutense University of Madrid, Spain, for assisting with animal experiments to examine the cholesterol-lowering effects of saponins. Thanks are extended to J. Graham, University of Illinois at Chicago, for assisting with the LC-MS measurement and R. Kasai, Hiroshima University, Japan, for help with the FAB-MS measurement. H.-B. Chai, University of Illinois at Chicago, for assisting with the cytotoxic assay, and K. Ryu, Wakunaga Pharmaceutical, Japan, for the NMR measurement.
FOOTNOTES
1 Presented at the conference "Recent Advances on the Nutritional Benefits Accompanying the Use of Garlic as a Supplement" held November 15–17, 1998 in Newport Beach, CA. The conference was supported by educational grants from Pennsylvania State University, Wakunaga of America, Ltd. and the National Cancer Institute. The proceedings of this conference are published as a supplement to The Journal of Nutrition. Guest editors: John Milner, The Pennsylvania State University, University Park, PA and Richard Rivlin, Weill Medical College of Cornell University and Memorial Sloan-Kettering Cancer Center, New York, NY. 
2 Current address: Wakunaga Pharmaceutical, 1624 Shimokotachi, Koda-cho, Takata-gun, Hiroshima 739-1195, Japan.
3 Abbreviations used: AGE, aged garlic extract; 13C NMR, carbon-13 nuclear magnetic resonance spectroscopy; 2D NMR, two-dimensional nuclear magnetic resonance spectroscopy; FAB-MS, fast atom bombarding mass spectrometry; FG-EXT, frozen garlic extract; 1H-1H COSY, proton-proton chemical shifts correlation spectroscopy; HMBC, heteronuclear multiple-bond correlation; HMQC, heteronuclear multiquantum coherence; 1H NMR, proton nuclear magnetic resonance spectroscopy; LC-MS, liquid chromatography-mass spectrometry; RG-EXT, raw garlic extract.
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