HPLC of S-Alk(en)yl-L-cysteine Derivatives in Garlic including Quantitative Determination of (+)-S-Allyl-L-cysteine Sulfoxide (Alliin)

Methods developed for the separation of S-alk(en)yl- L-cysteines and their corresponding (+/-)-sulfoxide isomers by reversed-phase HPLC were applied to the analysis of various garlic samples including fresh garlic, dried extracts, and garlic preparations. Extracts were chromatographed following extraction with 50:50 methanol/water, sample clean-up using Bond Elut C18 or SCX cartridges, and pre-column derivatization with O-phthaldialdehyde/ TERT.-butylthiol.(+)- S-Methyl- L-cysteine sulfoxide and (+)- S-allyl- L-cysteine sulfoxide (alliin) were the only compounds which were identified with certainty. Other sulfur amino acids reported to occur in garlic were absent or were below detection limits under standard chromatographic conditions. Assays for alliin, which is an antibiotic precursor and may be used for standardization of garlic preparations, resulted in great variation between samples: alliin contents were found to range from < 0.1% to 1.15% fresh weight. The accuracy and precision of the assay method, including external calibration of alliin, were evaluated.     

TLC analysis of Allium sativum constituents

A steadily increasing number of garlic (Allium sativum) preparations during the last years resulted in a high interest in practical analytical methods for its active principles. In the present work, TLC separation of alliin from other compounds was improved over previous methods. A modified ninhydrin detection reagent was used to optimize the differentiation between cysteine sulfoxides and other amino acid derivatives. In addition, a sensitive and specific colour reaction was developed for detection of allicin after TLC.     

Stabilization and pharmaceutical use of alliinase

In recent years, numerous clinical trials were undertaken in order to elucidate the active principle of garlic (Allium sativum L., Alliaceae). The most prominent effect of garlic preparations is a contribution to the prevention of stroke and arteriosclerosis. Allicin[(2-propenyl)-2-propenethiosulfinate] and other sulfur containing compounds were suggested as active compounds. The extremely unstable allicin itself is liberated from the more stable alliin [S-(+)-2-propenyl-L-cysteine sulfoxide] by the enzyme alliinase (EC 4.4.1.4) if fresh garlic is crunched or garlic powder is moistened. Therefore, an active enzyme is required in alliin containing remedies like those prepared from garlic powder. In order to investigate enzyme stability, alliinase was isolated from garlic powder. The partially purified enzyme could be stabilized over several months by addition of sodium chloride, sucrose, and pyridoxal-5'-phosphate. Alliinase may also be freeze-dried. This allows combinations of synthetic alliin and purified alliinase as components of an acid resistant tablet or capsule. In the intestine, the pro-drug alliin would be enzymatically converted to allicin. In clinical trials, highly dosed preparations of this kind should yield a precise information about the physiological effects of allicin. In addition, alliin-homologues substances which bear a modified alkyl side chain and do not occur in nature may be tested.     

大蒜质量标准中商榷内容的研究

目的:参照美国药典、欧盟药典和英国药典对2010版中国药典收载的大蒜薄层鉴别和含量测定等进行研究。方法:采用微波灭酶法处理样品,对大蒜药材进行薄层色谱鉴别;采用阳离子交换色谱柱,以磷酸盐缓冲溶液为流动相,流速0.5 mL.min-1,检测波长214 nm,测定大蒜药材中蒜氨酸含量。结果:不同产地大蒜药材的薄层色谱在与蒜氨酸和精氨酸对照品斑点Rf值相同的位置上,均检出相同的斑点。建立的蒜氨酸含量测定HPLC法,经方法学实验表明,蒜氨酸浓度在31.2～374.4μg.mL-1范围内呈良好线性关系(r=0.9999);低、中、高浓度平均回收率(n=9)分别为102.8%(RSD=4.0%),101.8%(RSD=3.0%),111.0%(RSD=3.8%)。结论:本研究建立了大蒜药材的薄层色谱鉴别和高效液相色谱含量测定方法,可用于大蒜药材的定性与定量分析,建议作为大蒜质量标准进一步研究的参考。     

Formation of allicin from dried garlic (Allium sativum): a simple HPTLC method for simultaneous determination of allicin and ajoene in dried garlic and garlic preparations.]

In garlic (Allium sativum L.) the enzyme alliin lyase catalyzes the cleavage of alliin into allicin which reacts further to furnish ajoene. A simultaneous determination of allicin and ajoene is introduced which, in contrast to the determination of alliin only, allows for the testing of the activity of alliin lyase. It can be demonstrated that at a pH value of less than 3 the enzyme produces only small amounts of allicin. For this reason preparations from garlic should be administered only as enteric-coated formulations.     

Analytical method for appreciation of garlic therapeutic potential and for validation of a new formulation

The consumption of garlic reduces the risk of cardiovascular disease and cancer, S-allylcysteine sulfoxide (alliin), allicin (DATi), diallyl disulfide (DADS), S-allylcysteine (SAC) and several storage dipeptides are the organo-sulphur compounds (OSC) involved in the protective mechanism of garlic against cardiovascular disorders and carcinogenesis. Thus it is very interesting to quantify simultaneously all these compounds in different garlic powders obtained in several cultural conditions. The quantification of OSC by a new ion-pair HPLC method allowed showing the general sulphur-dependence positive effect of garlic on cardiovascular disorder and carcinogenesis and the variable specific activity of each implicated OSC. The screening of 11 garlic tablets proposed on the market showed the variability and particularly the differential instability of each OSC. From these results, a new garlic tablet was realised and each step was controlled by this method. This analytical method proved to be a very powerful tool for the understanding of the garlic protective mechanism against cancer and cardiovascular diseases and the development and quality control of garlic tablets.     

Quantitative Determination of Allicin and Alliin from Garlic by HPLC*

The procedure for allicin synthesis could be improved. The time for HPLC analysis of allicin was shortened by the application of different isocratic elution systems. Calibration was performed by use of an allicin/silica gel adsorbate as external standard and its allicin content could be confirmed by different methods. L-(+)-Alliin was synthesized and applied as external standard for the quantitative determination of alliin by HPLC. Diastereoisomers had been separated by repeated recrystallization. Fresh ALLIUM SATIVUM bulbs from different origins were analyzed with respect to allicin content after complete enzymatic conversion of alliin; allicin contents found were in the range of 0.4%. The corresponding alliin contents were in the range of 0.9%. For a comparative evaluation of the alliin- and the allicin-HPLC determination methods, commercially available garlic preparations were analyzed, demonstrating that both methods are appropriate. However, the application of the HPLC system for allicin determination, in addition to providing information on the alliin-dependent allicin-generating capacity, enables the simultaneous quantification of allicin transformation products, such as ajoenes, dithiins, and alkyl sulfides. It was found that, for quantitative GLC analysis of allicin, allicin has to be used as an external standard.     

大蒜药材的薄层鉴别方法研究

目的建立大蒜药材的薄层鉴别系统。方法以蒜氨酸和精氨酸为对照品,采用灭酶处理,对大蒜药材进行薄层鉴别,并考察不同展开系统、不同点样量、不同薄层板、不同检视方式、不同温度、不同湿度对大蒜药材薄层色谱的影响。结果以蒜氨酸和精氨酸为对照品,正丁醇-冰醋酸-水(4︰1︰1)为展开系统,大蒜药材薄层鉴别特征明显,专属性强。结论该方法可行,重复性好,可作为大蒜药材的薄层鉴别方法。     

用于蒜氨酸含量和有关物质测定的方法

蒜氨酸是大蒜的功效成分大蒜辣素的前体。大蒜辣素极不稳定，无法直接制成制剂应用于临床，故从鲜蒜中分别提取化学性质相对稳定的蒜氨酸和蒜酶，制成蒜氨酸／蒜酶复合制剂的方法是目前大蒜高效制剂开发的主要思路。为配合蒜氨酸的质量研究，综述了近年来蒜氨酸含量和有关物质的测定方法，包括紫外法、薄层色谱扫描法、气相色谱法、高效毛细管电泳法、生物传感器法、液相色谱法，并讨论了各方法的优点和局限性。     

关于大蒜和大蒜素质量标准的商榷

国家药品标准W S-10001-(HD-0050)-2002中规定大蒜素为三硫二丙烯,本文对大蒜中含硫有机化合物的转化途径及药理作用进行综述,说明大蒜素与大蒜虽然名称相似,但并非大蒜中的天然成分,而是蒜氨酸与蒜酶接触降解出的含硫有机化合物,后改由合成得到,因此在中国药典2010年版一部收载的大蒜药材标准中测定三硫二丙烯含量应进行修订。目前国内合成的大蒜素为丙烯基多硫化物的混合物,建议对其质量标准进行修订并取消大蒜素该名称,改为直接采用化学名称三硫二丙烯进行命名。大蒜中很多含硫有机化合物的命名混乱,造成文献中的研究成果无法准确表述,建议对大蒜中含硫有机化合物的命名进行规范。     

Changes in contents of gamma-glutamyl peptides and fructan during growth of Allium sativum

Determination of gamma-glutamyl peptides, alliin, and fructan during growth of Allium sativum L. was carried out by HPLC. The contents of gamma-glutamyl peptides and alliin in garlic bulbs remarkably increased during one month before the garlic leaves withered.     

有关2010年版中国药典大蒜质量标准的几点建议

通过文献综述阐述大蒜的活性成分———蒜氨酸、蒜酶和大蒜辣素及三者之间的关系;介绍国外药典收载大蒜及相关制剂含量测定的指标,简述大蒜素的由来及结构。对2010年版中国药典大蒜质量标准中含量测定的方法提出疑问,并提出修改建议。     

In vitro inhibition of cytochrome P-450 reductases from pig liver microsomes by garlic extracts.

The activity of microsomal NADPH-cytochrome-P-450-reductase and NADH-cytochrome-b5-reductase are inhibited after the addition of an aqueous extract of a pharmaceutical preparation of garlic (Allium sativum, L.) to buffer-suspended microsomes. Incubation of garlic extract with isolated pig liver microsomes also decreases the activity of cytochrome P-450-dependent ethoxycoumarin deethylation. As measured by malondialdehyde release, the effects on the enzyme system are evidently not due to lipid peroxidation. No loss of cytochrome P-450 pigment is observed. Moreover, it could be shown that addition of garlic extract displays no protective effect on microsomal lipids when oxidation occurs spontaneously or is enforced by short-wave UV-irradiation. The above findings were reproduced after applying a HPLC-purified preparation of alliin to the incubation mixtures, suggesting that alliin is the active principle for the inhibitory effects observed in vitro.     

Characterization of an Alliin Lyase Preparation from Garlic (Allium sativum)

An alliin lyase (EC 4.4.1.4) preparation from garlic, ALLIUM SATIVUM L., has been purified to apparent homogeneity. The purification procedure involved liquid chromatography steps on hydroxylapatite, on an anion exchanger, and on a chromatofocussing medium. The enzyme protein was characterized by a relative molecular mass of 108,000, and was found to consist of two equal subunits. Its isoelectric point was determined to be 4.9. The enzyme appeared rather thermolabile. Simulated gastric-intestinal passage by a modified "half change test" revealed a high acid lability of the active alliinase protein. K (m)-values for different substrates were in the mM range, and activating energies for the cleavage of different substrates could be determined. A maximal specific activity for synthetic alliin in the range of 490 micromoles per min and mg protein could be achieved at 33 degrees C. There are some significant differences in the characterization of the purified protein compared to results previously reported by others on this enzyme.     

Alliin Lyase from Garlic, Allium sativum: Investigations on Enzyme/Substrate, Enzyme/Inhibitor Interactions, and on a New Coenzyme

A purified alliin lyase (EC 4.4.1.4) from garlic ( ALLIUM SATIVUM L.) has been characterized by kinetic and spectral data under the addition of different substrates, inhibitors, and reducing agents. Hydroxylamine sulfate (50 microM) inhibited the alliinase activity by nearly 90%. Rotenone revealed a similar effect at a concentration of 10 nano-molar. Examination of L-cysteine, and a series of related compounds, on a competitive inhibitory effect on the alliinase-catalyzed alliin cleavage resulted in a list of essential functional groups for substances with this property. Spectral studies on the purified, yellow appearing alliinase enzyme confirmed the existance of an unknown flavinecoenzyme.     

Metabolism of garlic constituents in the isolated perfused rat liver.

The metabolic and kinetic behaviour of different garlic (Allium sativum L., Alliaceae) constituents were investigated in the isolated perfused rat liver, using aqueous extracts of garlic powder as well as isolated allicin, the main product of the enzymatic degradation of alliin. Allicin (allyl thiosulfinate) showed a remarkable first pass effect and passed the liver unmetabolized only at high concentrations which caused considerable cell injuries. Diallyl disulfide and allyl mercaptan were identified as metabolites of allicin, whereby diallyl disulfide probably is the metabolic precursor of allyl mercaptan as shown by perfusion with diallyl disulfide alone. The metabolites diallyl disulfide and allyl mercaptan could be determined in the perfusion medium as well as in the bile and the liver tissue. Other degradation products of garlic were also investigated in this model. Ajoenes and vinyldithiins were detected in perfusion medium after liver passage but no metabolites of them could be identified up to now.     

Inhibition of cholesterol biosynthesis by a water-soluble garlic extract in primary cultures of rat hepatocytes.

Cultured rat hepatocytes continually synthesize cholesterol form radiolabeled acetate during a 24 h incubation period and export it, presumably as VLDL (very low density lipoprotein) to the culture medium. Mevastatin inhibits cholesterol biosynthesis by 90%. Incubation of the cultures with water-soluble extracts of garlic powder (Kwai, Sapec) diminish cholesterol biosynthesis (20-25%) as well as its export into the medium (30-35%). The IC50-value is 90 micrograms/ml. Between about 0.25 and 10 mg/ml the average maximal inhibition amounts to about 23%. Cytotoxicity of the extracts is apparent at concentrations above 125 mg/ml only. Pure alliin alone, or after incubation with alliinase (conversion to allicin) in concentrations corresponding to its content in the extracts does not exert any inhibition. Replacement of 14C-acetate by 14C-mevalonate omits the inhibitory effect. The activity of HMGCoA (hydroxymethylglutaryl-CoA) reductase is significantly reduced by garlic extracts at 50 micrograms/ml. At higher concentrations fatty acid synthetase, cholesterol 7 alpha-hydroxylase and cholesterol acyltransferase are slightly inhibited. Fatty acid synthetase is the only one of these enzymes which is inhibited by alliin at very high concentrations. These results demonstrate that water-soluble garlic extracts diminish hepatic cholesterol biosynthesis, thus contributing to the reduction of blood cholesterol. The main target site seems to be HMGCoA-reductase. The actual active principle(s) is still unknown. Alliin, however, does not seem to be of major significance.     

Alliin obtained from leaf extract of garlic grown under in situ conditions possess higher therapeutic potency as analyzed in alloxan-induced diabetic rats

Context: Garlic, Allium sativum L. (Liliaceae), possesses high therapeutic and pharmacological properties. Hypoglycemic activity is attributed to alliin (S-allyl cysteine sulfoxide), the main active principle localized in garlic cloves.

Objective: To compare the production and therapeutic efficiency of alliin extracted from garlic leaves of plants grown under ex situ and in situ conditions.

Materials and methods: Alliin content of leaves was quantified and aqueous leaf extracts (from ex situ and in situ grown plants) were given to normal and alloxan-induced diabetic rats for five weeks.

Results: Alliin production noted ~50% enhancement in leaves from plants grown under in situ conditions. Serum glucose, triglycerides, total lipids, total cholesterol, low-density lipoprotein (LDL)-, and very low-density lipoprotein (VLDL)-cholesterol in diabetic rats treated with alliin produced from in situ grown plants noted significant reduction of ~54%, 15%, 14%, 20%, 24%, and 15%, while 35%, 14%, 10%, 12%, 17% and 11% reduction was noted in diabetic rats treated with alliin produced from ex situ grown plants in comparison with those administered with distilled water. High-density lipoprotein (HDL)-cholesterol did not show any significant change. Leaf extract of plants lowered serum enzyme levels (alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase) toward the norm better than glibenclamide. The histopathological alteration in pancreas caused by alloxan was also reduced by leaf extract.

Discussion and conclusion: These findings demonstrate leaf extract obtained from plants grown under in situ condition possess higher therapeutic efficiency in comparison with leaf extract obtained from plants grown under ex situ condition. Studies suggest that environmental factors influence production of alliin and its therapeutic potential.     

Improvement of scopolamine-induced memory impairment by Z-ajoene in the water maze in mice

Z-ajoene, a major compound containing sulfur in oil-macerated garlic products, exhibited inhibitory effects against scopolamine-induced memory impairment in mice using the Morris water maze test. The effects of Z-ajoene were observed dose-dependently (0.25-25 mg/kg). At the highest dosage, the memory performance of mice was improved compared to normal mice. The acetylcholinesterase (AChE) activity in the brain was reduced by administration of Z-ajoene dose-dependently. However, alliin and diallyl disulfide, organosulfur compounds from garlic, did not improve memory performance nor AChE inhibitory effect. These results suggest that Z-ajoene may act on the cholinergic system and on memory impairment caused by excess activity of AChE.     

In vitro virucidal effects of Allium sativum (garlic) extract and compounds.

Garlic (Allium sativum) has been shown to have antiviral activity, but the compounds responsible have not been identified. Using direct pre-infection incubation assays, we determined the in vitro virucidal effects of fresh garlic extract, its polar fraction, and the following garlic associated compounds: diallyl thiosulfinate (allicin), allyl methyl thiosulfinate, methyl allyl thiosulfinate, ajoene, alliin, deoxyalliin, diallyl disulfide, and diallyl trisulfide. Activity was determined against selected viruses including, herpes simplex virus type 1, herpes simplex virus type 2, parainfluenza virus type 3, vaccinia virus, vesicular stomatitis virus, and human rhinovirus type 2. The order for virucidal activity generally was: ajoene > allicin > allyl methyl thiosulfinate > methyl allyl thiosulfinate. Ajoene was found in oil-macerates of garlic but not in fresh garlic extracts. No activity was found for the garlic polar fraction, alliin, deoxyalliin, diallyl disulfide, or diallyl trisulfide. Fresh garlic extract, in which thiosulfinates appeared to be the active components, was virucidal to each virus tested. The predominant thiosulfinate in fresh garlic extract was allicin. Lack of reduction in yields of infectious virus indicated undetectable levels of intracellular antiviral activity for either allicin or fresh garlic extract. Furthermore, concentrations that were virucidal were also toxic to HeLa and Vero cells. Virucidal assay results were not influenced by cytotoxicity since the compounds were diluted below toxic levels prior to assaying for infectious virus. These results indicate that virucidal activity and cytotoxicity may have depended upon the viral envelope and cell membrane, respectively. However, activity against non-enveloped virus may have been due to inhibition of viral adsorption or penetration.(ABSTRACT TRUNCATED AT 250 WORDS)