![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Institute for OTC Research, Wakunaga Pharmaceutical Company, Hiroshima 739-1195, Japan
2To whom correspondence should be addressed. E-mail: itakura_y{at}wakunaga.co.jp.
ABSTRACT
The establishment of international monographs for herbs is in progress. Here, we propose both a marker compound and a method for its analysis for the identification of garlic bulbs and their products. The constituents in 26 kinds of fresh edible parts of Allium vegetables and three types of garlic preparations were analyzed. Sulfur compounds are the most characteristic constituents in garlic, but manufacturing processes of garlic products dramatically affect these constituents. Thus, no sulfur compound could be specified as a universal marker of identification applicable for any type of garlic. On the other hand, garlic contains other characteristic compounds, namely, saponins. After analyzing Allium vegetables and garlic preparations, we concluded that sapogenins, especially ß-chlorogenin, may be a viable candidate for identifying and distinguishing garlic from other Allium vegetables.
KEY WORDS: • garlic • Allium vegetables • sulfur compounds • saponins
Each country has its own history and culture concerning the use of herbs
as medicines or prophylactics. Garlic has historically been one of the
most common vegetables to serve as a both spice and a medical herb in
many countries, and its usage varies by country, region and history.
Garlic has long been taken as a tonic, a bactericide and a popular
remedy for various ailments (Blackwood and Fulder 1986
).
More recently, however, it has been recognized as a medicinal plant for
the prevention of blood circulatory disorders (Fogarty 1993
, Steiner et al. 1996
), cancer
(Amagase and Milner 1993
, Nishino et al. 1989
, Wargovich 1986
) and memory loss
(Moriguchi et al. 1994
).
Fresh, crushed garlic has a strong odor and is known to irritate the
stomach. To avoid such problems, various methods of preparation have
been developed. Several monographs (British Pharmacopoeia 1998
, European Pharmacopoeia 1998
, U.S. Pharmacopoeia 1998
, WHO 1998
) on garlic have
been established or proposed by different organizations because of its
proven medicinal properties. Combining these monographs into an
international monograph for garlic would be useful.
If an international monograph for garlic were established, it would be
necessary to distinguish garlic from other plants, especially the other
Allium vegetables (Fig. 1
). Further, methods of recognizing garlic with its many different
preparations should be developed. Sulfur-containing compounds
(Fig. 2
), especially alliin and allicin, are the most prevalent constituents of
garlic. However, it is well known that these sulfur compounds are
unstable; thus they decompose easily during the processing of garlic
preparations. Because the sulfur compounds in garlic are altered by
differing preparation methods (Vernin et al. 1986
), we
propose a method of identifying garlic that uses a chemically stable
sapogenin, which is another constituent of garlic (Matuura et al. 1988 and 1989
).
|
|
MATERIALS AND METHODS
Twenty-six different kinds of Allium vegetables were
purchased in markets in Japan and the United States. They included
Allium sativum L. (garlic), A. ampeloprasum
(elephant garlic), A. ascalonicum, A. canadence,
A. cepa (onion, 10 different types), A. chinense
(Rakkyo), A. fistulosum (3 different types), A.
porrum (leek), A. shoenoprasum (2 different types),
A. tricocum, A. tuberosum,
A.victorialis and A. wakegi (Fig. 1)
.
Each 10 g of fresh raw vegetables was homogenized either with 10 mL of water (sample W) and diluted with 40 mL of methanol or with 50 mL of methanol (sample M). The eluates were used for subsequent analysis. Heated garlic was prepared from raw garlic cloves, which were left for 30 min in boiling water and then homogenized with 5 volumes of water. Aged garlic extract was manufactured by Wakunaga Pharmaceutical as follows: garlic cloves (Allium sativum) were sliced and soaked in a water/ethanol mixture and naturally extracted/aged for >10 mo at room temperature.
For TLC analysis, samples W and M of each of the vegetables were spotted onto high performance thin layer chromatography (HPTLC)3 Silica gel 60 plates (Merck, Darmstadt, Germany) and developed using chloroform/methanol/water (6:4:1) as solvent. Plates were sprayed with iodide-platinate reagent for detection of sulfur compounds. For gas chromatography (GC) and GC-mass spectrometry (MS) analyses of sulfur compounds, sample W of each of the vegetables was extracted with ethylacetate, and the ethylacetate extracts were injected. GC was performed on a Shimadzu GC-17A gas chromatograph equipped with a Hewlett-Packard-WAX (i.d. 0.32 mm x 30 m) column; the column temperature was held at 60°C for 3 min, increased at 4°C/min to 200°C and held for 10 min; the carrier gas was He (53 kPa, split ratio 1/3) and detection was by flame photometry. GC-MS was performed on a JEOL GC-mate under exactly the same conditions except for He (1.2 mL/min, splitless) and detection, which was by electron impact mass spectrometry (TIC model).
For the analysis of saponins, each 10 g of vegetables or processed garlic was crushed in 40 mL of methanol. After removal of the solvent by evaporation, a suspension of the resulting extract in 30% aqueous methanol was applied to a column of MCI gel CHP20P (stepwise elution of 30% aqueous methanol and methanol). Each methanol eluate was analyzed by TLC on a HPTLC Silica gel 60 plate (Merck) and developed using chloroform/methanol/water (6:4:1) as solvent; spots were visualized by spraying of anisaldehyde-H2S04 or Ehrlich’s reagent, followed by heating of the saponins.
For the analysis of sapogenins, the methanol eluates obtained for the analysis of saponins were hydrolyzed using a mixture of 8% sulfuric acid/ethanol (1:1) for 5 h at 100°C. The hydrolyzates were added to 20 mL of water and applied to a column of MCI gel, which was then washed with 70% aqueous methanol and eluted with methanol. The sapogenin fraction from each methanol eluate was analyzed by TLC.
TLC was performed on a HPTLC Silica gel 60 plate (Merck) and spots were visualized by spraying of anisaldehyde-H2S04 followed by heating of saponins and sapogenins or spraying of iodide-platinate reagent for sulfur compounds.
RESULTS AND DISCUSSION
GC and GC-MS analysis of sulfur compounds.
Allium vegetables, especially garlic, can be distinguished
by their characteristic smell. Biochemically, garlic is distinguishable
from other allium vegetables in several ways. Garlic’s smell is
derived from volatile sulfur compounds, such as diallyldisulfide. Gas
chromatography is useful for the analysis of these volatile compounds.
The mass spectrometer is one of the universal detectors of GC and can
detect most volatile compounds. On the other hand, the flame
photometric detector has selectivity for sulfur or phosphorous atoms;
thus it can be used to selectively detect sulfur compounds.
Figure 3
shows volatile sulfur compounds of ethylacetate soluble fractions of
water extracts of four kinds of Allium vegetables.
|
. Because of its
instability and reactivity, allicin cannot be detected in most
biological samples. However, peaks that correspond to volatile sulfur
compounds can be seen on the GC chromatogram when samples of freshly
prepared garlic are injected (Fig. 3)
. Allicin is decomposed at the
injection port of GC or in the organic solvent to produce
vinyldithiins, which are detected as the major peaks of garlic. Onions
and leeks provided almost no peaks on their chromatograms. This GC
method seems to be useful for identifying garlic, but elephant garlic
also shows peaks for vinyldithiins. GC chromatograms of garlic and
elephant garlic were different, but the difference arose only from the
difference in quantity of each constituent. Therefore, GC is likely not
suitable for distinguishing garlic from the other Allium
vegetables. TLC analysis of sulfur compounds.
S-Allyl-L-cysteine (SAC),
-glutamyl-S-allylcysteine (G-SAC), and alliin are the
characteristic sulfur compounds in garlic. Using iodoplatinate reagent
as the spray reagent for TLC, these sulfur compounds can be detected as
pale yellow spots on a brown background, which could be useful for
identification of garlic.
Sample M of garlic yielded spots corresponding to alliin and G-SAC,
but sample W showed only G-SAC (Fig. 4
). The spot at Rf = 0.8 of sample W of garlic corresponds to
allicin and/or decomposed compounds from allicin. Elephant garlic
yielded almost the same results. TLC of onion and leek samples had
spots whose Rf values were almost identical to those of alliin. These
spots are not derived from alliin but from
S-1-propenyl-L-cysteine sulfoxide. Therefore, TLC of sulfur
compounds is also not likely suitable for distinguishing garlic from
the other Allium vegetables.
|
). Only aged garlic extract showed the spot corresponding to SAC. SAC
could be a good maker compound for distinguishing aged garlic extract
from the other garlic preparations.
|
Saponins are glycosides of steroids or triterpenoids, and they can be
detected as dark green or brown spots when sprayed with
anisaldehyde-sulfuric acid reagent. Only furostanol-type
saponins (Fig. 6
) can be detected as pink spots when sprayed with Ehrlich’s reagent.
Figure 7
shows many spots corresponding to saponins, derived from all of the
vegetables except onion. A furostanol-type saponin,
proto-erboside B, is the most characteristic in garlic, and it is
detected from the methanol extract of raw garlic and the water extract
of heated garlic. However, it converts to a spirostanol-type
saponin, eruboside B, when raw garlic is extracted with water as in the
case of the disappearance of alliin.
|
|
TLC analysis of sapogenins.
Sapogenin is the aglycone of saponin, obtained by hydrolysis of
saponin. Twenty-eight different kinds of Allium
vegetables were treated as shown in Figure 8
; the sapogenin fractions obtained, corresponding to each vegetable,
were analyzed by TLC as shown in Figures 9
and
10
. Each chromatogram of Allium vegetables is characteristic
and distinguishable.
|
|
|
TLC analysis of sapogenin has an advantage over other identification methods because it can be used for raw garlic and also
for various garlic preparations, except for garlic oil. On the TLC
plate, spots of ß-chlorogenin are detectable for raw garlic, heated
garlic and aged garlic extract (Fig. 11
).
|
Summary
Monographs for garlic and garlic powder are being developed by several organizations that propose to use sulfur compounds as the marker compounds. Although sulfur-containing compounds are typical components of garlic, they may not be appropriate as universal markers to distinguish among different types of garlic because of their instability. Some saponins are also unique compounds in garlic. However, their structures are also known to be influenced by processing. One specific sapogenin in garlic, ß-chlorogenin, is stable and can be separated from the other sapogenins by TLC. Further, it is found in almost no other Allium vegetable. Thus, ß-chlorogenin may be a suitable marker compound for the identification of garlic and garlic preparations.
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. 
3 Abbreviations: GC, gas chromatography; HPTLC,
high performance thin layer chromatography; G-SAC,
-glutamyl-S-allylcysteine; MS, mass spectrometry;
SAC, S-allyl-L-cysteine.
REFERENCES
1.
Amagase H., Milner J. A. Impact of various sources of garlic and their constituents on 7,12-dimethylbenz(a)anthracene binding to mammary cell DNA. Carcinogenesis 1993;14:1627-1631
2. Blackwood J., Fulder S. Garlic; Nature’s Original Remedy 1986 Javelin Books England
3. British Pharmacopoeia (1998) p. 315.
4. European Pharmacopoeia (1998) 3rd ed., Suppl. p. 629.
5. Fogarty M. Garlic’s potential role in reducing heart disease. Br. J. Clin. Pract. 1993;47:64-65[Medline]
6. Matuura H., Ushiroguchi T., Itakura Y., Fuwa T. Further studies on steroidal glycosides from bulbs, roots and leaves of Allium sativum L. Chem. Pharm. Bull. 1989;37:2741-2743
7. Matuura H., Ushiroguchi T., Itakura Y., Hayashi N., Fuwa T. A furostanol glycoside from garlic, bulbs of Allium sativum L. Chem. Pharm. Bull. 1988;36:3659-3663
8. Moriguchi T., Takashina K., Chu P.-J., Saito H., Nishiyama N. Prolongation of life span and improved learning in the senescence accelerated mouse produced by aged garlic extracts. Biol. Pharm. Bull. 1994;17:1589-1594[Medline]
9. Nishino H., Iwashima A., Itakura Y., Matsuura H., Fuwa T. Antitumor-promoting activity of garlic extracts. Oncology 1989;46:277-280[Medline]
10.
Steiner M., Kahn A. H., Holbert D., Lin R.I.S. A double-blind crossover study in moderately hypercholesterolemic men that compared the effect of aged garlic extract and placebo administration on blood lipids. Am. J. Clin. Nutr. 1996;64:866-870
11. U.S. Pharmacopoeia (1998) 23-National Formulary, 188th Supplement, p. 4449.
12. Vernin G., Metzger J., Fraisse D., Scharff C. GC-MS computer analysis of volatile sulfur compounds in garlic essential oils. Planta Med 1986;52:96-101[Medline]
13. Wargovich M. J. Dietary promoters and antipromoters. Antimutat. Anticancer Mech. 1986;:409
14. World Health Organization Monographs on Selected Medicinal Plants 1998 WHO Geneva, Switzerland.
This article has been cited by other articles:
|
|
 |
|
  H. Amagase Clarifying the Real Bioactive Constituents of Garlic J. Nutr., March 1, 2006; 136(3): 716S - 725S. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
