Quality of herbal remedies from Allium sativum: differences between alliinase from garlic powder and fresh garlic

Alliinase (EC 4.4.1.4) has been isolated from commercially available garlic (Allium sativum L., Alliaceae) powder and was investigated with respect to its use as ingredient of herbal remedies. The enzyme was purified to apparent homogeneity and results were compared with those obtained from a sample of fresh A. sativum var. pekinense. The purification of the enzyme involved a gel filtration step as well as affinity chromatography on concanavalin-A agarose. Vmax using L-(+)-alliin as substrate (252 mumol min-1 mg-1) was at the lower range of data given in the literature (214-390 mumol min-1 mg-1). L-(-)-Alliin was also accepted as substrate (54 mumol min-1 mg-1). Vmax for alliinase from A. sativum var. pekinense was at 332 mumol min-1 mg-1 and 90 mumol min-1 mg-1 for L-(+)- and L-(-)-alliin, respectively. The Km values for alliinase from garlic powder were estimated to be 1.6 mM for L-(+)-alliin and 2.8 mM for L-(-)-alliin. In contrast to literature values, both temperature and pH optima were somewhat higher (36 degrees C and pH 7.0 versus 33 degrees C and pH 6.5, respectively). The enzyme was found to be active in a range from pH 5 to pH 10. Gel electrophoresis gave evidence that the alliinase obtained from garlic powder consisted of two slightly different subunits with molecular weights of 53 and 54 kDa whereas alliinase obtained from fresh garlic consists of two identical subunits. It is assumed that the alliinase gets significantly altered during the drying process of garlic powder but is still capable to convert alliin to allicin.     

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.     

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。     

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.     

Allicin release under simulated gastrointestinal conditions from garlic powder tablets employed in clinical trials on serum cholesterol

The failure of five recent clinical trials to show significant reduction in elevated serum cholesterol by a single brand of allicin-standardized garlic powder tablets is in contrast to many prior positive studies with the same brand. The hypocholesterolemic activity of garlic is mainly due to allicin, a compound that is produced by the acid-sensitive garlic enzyme, alliinase, only after tablet consumption. Therefore, the allicin-releasing ability of ten lots of these tablets--manufactured over the same years that the positive and negative clinical trials were conducted (1989-1997)--was determined under simulated gastrointestinal dissolution conditions, as defined by U.S. Pharmacopeia Method 724A. It was found that the older lots were more resistant to acid-disintegration (2.5 h vs. 1.3 h, P < 0.001) and that they released three times as much allicin (44% vs. 15 % of their potential, P < 0.001) as the newer lots. A second brand of tablets employed in a recent negative trial released no detectable amount of allicin, while a third set of tablets with high allicin release was used in a trial that gave positive effects. Hence, the persons involved in the recent negative clinical trials probably received considerably less allicin than did those in the older positive studies, possibly accounting for much of the discrepancy in the outcomes. In conclusion, clinical trials using garlic powder tablets to assess any effect of garlic that might be related to allicin, as most are, cannot be considered valid for garlic when the trial shows no effect, unless the expected allicin release from the tablets has at least been determined under standardized drug release conditions (USP 724A).     

A high-throughput method for the quantitative determination of alliin

The quality of most garlic (Allium sativum L., Alliaceae) preparations made from garlic powder or garlic dry extract is determined by their content of alliin. Therefore, a comprehensive documentation of alliin concentration beginning with the crude material up to the final remedy is required. The newly developed analytical method described in this paper was designed in order to fulfill these demands. In contrast to conventional HPLC methods, neither a pre-column derivatization nor a chromatographic separation are involved in this analytical procedure allowing a high throughput of samples. The currently investigated technique is based on immobilized alliinase (EC 4.4.1.4) which was combined with a two-channel flow injection analyser (FIA) coupled to an ammonia detecting device. A high specificity for alliin could be demonstrated and a variety of garlic samples including garlic powders, dry extracts, and garlic preparations was analysed. The results were in good correlation with those obtained by conventional HPLC methods.     

Conjugates of daidzein-alliinase as a targeted pro-drug enzyme system against ovarian carcinoma

Human ovarian cancer cells specifically bind the isoflavone daidzein. A chemical conjugate between daidzein and the garlic enzyme alliinase was prepared. The conjugate specifically bound to ovarian cancer cells and upon addition of the prodrug alliin, it effectively produced cytotoxic allicin molecules which killed the cancer cells. In vivo targeting and antitumor effect was confirmed by NIR and bioluminescence imaging using daidzein-alliinase-CyTE-777 conjugates and luciferase-expressing ovarian cancer cells. Co-localization of the fluorescent conjugate with bioluminescence was observed for intraperitoneal tumors while nonconjugated alliinase did not accumulate. Biodistribution studies with Europium-labeled conjugate revealed a five fold higher uptake in tumors as compared to other tissues. Treatment of tumor bearing mice with daidzein-alliinase and alliin effectively attenuated tumor progression during the first 12 days while a 5-fold increase in bioluminescence was detected in placebo-treated animals. Autopsy revealed only small individual foci of luminescence at the site of tumor cells inoculation. Histological examination of organs and tissues did not reveal any additional foci of carcinoma or signs of toxicity. These results suggest that the targeted alliinase conjugates in the presence of alliin, generated therapeutically effective levels of allicin which were capable of suppressing tumor progression of intraperitoneal ovarian cancer in an animal model.     

Unusual cystine lyase activity of the enzyme alliinase: direct formation of polysulphides

Garlic ( Allium sativum L.) and related species are highly estimated as foods, spices, and herbal remedies in many parts of the world. Sulphur-containing flavour compounds like allicin (allyl 2-propenethiosulphinate) are responsible for the smell and taste of freshly crunched garlic. These substances are formed by the action of alliinase (EC 4.4.1.4) on cysteine sulphoxides, e. g., alliin [ S-(+)-allyl- L-cysteine sulphoxide]. Additionally, alliinase catalyses the C-S lysis of cystine in the manner of a cystine lyase. Ammonium, pyruvate and elementary sulphur but not cysteine could be detected as reaction products. The ratios between cystine, ammonium and pyruvate are 1 : 1.9 : 1.9 suggesting a new type of reaction mechanism. Thiocysteine and disulphine were assumed as intermediates. The pH optimum of the cystine lyase activity was found at pH 7.5 and the temperature optimum was at 44 degrees C. The KM value for the homogeneous enzyme was at 2.65 mM and Vmax was at 4.12 nkat/mg using cystine as substrate. Moreover, parallel incubation of cystine and alliin gave mainly allyl (poly)sulphides as reaction products instead of allicin. These substances had not been observed as direct enzymatic products until now. Thus, the significance of alliinase and its enzymatic products has to be newly considered in terms of ecological, pharmacological, and biochemical aspects.     

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)     

Characterization of the formation of allicin and other thiosulfinates from garlic

The effects of pH, neutralization after acidification, time, and temperature on the yield of dialkyl thiosulfinates released from garlic powder and garlic cloves were determined. All dipropenyl thiosulfinates (allicin, 1-propenyl allyl, and allyl 1-propenyl) were formed at an optimum pH of 4.5-5.0. The methyl propenyl thiosulfinates (allyl methyl + methyl allyl and 1-propenyl methyl + methyl 1-propenyl) and dimethyl thiosulfinate were optimally formed at pH 6.5-7.0 and pH 5.5, respectively. Below pH 3.6 no thiosulfinates were formed. Neutralization of the pH failed to restore thiosulfinate generation from garlic previously incubated at pH 3 or below. Thus, alliinase is completely and irreversibly inhibited by the acidic conditions found in the stomach. The dipropenyl thiosulfinates were completely formed in 0.3 min at 37 degrees C, while the methyl thiosulfinates were not completely formed until 3.5 min. Allyl 1-propenyl thiosulfinate was the most rapidly formed, and the most unstable, thiosulfinate. The stability of the dipropenyl thiosulfinates was improved at pH 4.5 or lower. Drying garlic at 60 degrees C had no effect on alliin or the rate of formation of the dipropenyl thiosulfinates, but decreased trans-1-propenylcysteine sulfoxide (isoalliin) and the rate of formation of the methyl thiosulfinates. The results demonstrate that there are two alliinase activities in garlic, that a stomach acid-resistant coating on garlic powder tablets is necessary for thiosulfinate release, and that carefully prepared garlic powder can release similar amounts of total thiosulfinates to whole garlic cloves.     

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.     

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.     

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.     

大蒜蒜酶提取纯化及临床应用研究

目的:研究从鲜蒜中分离纯化蒜酶的方法和临床常用剂对酶活性的影响,为今后工艺的进一步放大、优化和合理临床应用提供必要理论参数。方法:以天然蒜氨酸作底物,采用丙酮酸法测定酶浓度;以牛血清白蛋白为标准,采用考马斯亮兰G-250法测定蛋白质浓度。结果:从鲜蒜中提取纯化蒜酶,其粗酶浸提液的最佳组成为Na2HPO4-KH2PO4缓冲溶液;硫酸铵沉淀酶蛋白的最适饱和度范围为0%~35%;选用超滤技术脱盐;蒜酶最佳调节等电点为4.9。在临床常用溶剂中,含氯化钠的注射液(不含葡萄糖)对蒜酶活力有显著的激活作用,而含葡萄糖的注射液则较强地抑制了蒜酶活力;75%和95%乙醇具有强烈的杀酶作用。结论:本研究为今后工艺的进一步放大与优化和蒜酶合理临床应用提供了指导。     

Garlic and its active metabolite allicin produce endothelium- and nitric oxide-dependent relaxation in rat pulmonary arteries

1. The aims of the present study were to investigate the effects of fresh garlic and one of its active metabolites, allicin, on rat isolated pulmonary arteries (RPA). 2. In endothelium-intact and phenylephrine-precontracted RPA, the addition of a water or a 5% ethanol extract of fresh garlic (1-500 microg/mL) resulted in a dose-dependent relaxation reaching a maximum (mean +/- SEM) of -91 +/- 3 and -93 +/- 2%, respectively, with an ED(50) of 113 +/- 12 and 106 +/- 10 microg/mL, respectively. The vasorelaxation was readily reversible upon washing and no tachyphylaxis was noted. 3. An extract of the external garlic storage leaf produced a significantly greater relaxation than the inner stem. Microfiltration of extracts with a 10,000 molecular sieve did not attenuate relaxation. Inactivation of alliinase and allicin formation, with either boiling of the garlic clove for 30 min or 100% ethanol treatment, completely abolished relaxation. In contrast, similar treatment of crushed garlic with formed allicin retained the relaxation response. 4. Pure allicin produced a similar relaxation as garlic extract, with an EC(50) of approximately 0.8 microg/mL. Disruption of endothelium or N(G)-nitro-L-arginine methyl ester pretreatment attenuated the relaxation, whereas indomethacin had no effect. 5. Prior garlic (500 microg/mL) treatment enhanced acetylcholine relaxation by shifting the response curve to the left, but had no effect on nitric oxide (NO) donor-induced responses. 6. These results demonstrate that garlic and the active metabolite allicin are capable of eliciting a NO-dependent relaxation in RPA and that this response is likely to be mediated via garlic activation of NO formation rather than its stabilization.     

大蒜辣素前体包芯片的制备

目的:制备大蒜辣素前体包芯片,使其口服后在短时间内促发酶促反应,生成大蒜辣素。方法:以蒜氨酸和蒜酶双层片为片芯,控酸颗粒为外层压制得到包芯片。并以人工胃液为介质小杯法考察包芯片大蒜辣素产率。结果:大蒜辣素前体包芯片在人工胃液中10 min内大蒜辣素产率>70%。结论:以蒜氨酸、蒜酶及控酸盐组合制备包芯片能够有效避免蒜酶在胃酸条件下失活,达到在体内获得较高产率大蒜辣素目的。     

大蒜抗氧化活性成分的测定及薄层生物自显影技术分析

目的测定和分析大蒜抗氧化活性成分。方法利用1,1-二苯基苦基苯肼法(DPPH·)测定大蒜灭酶体系和蒜酶激活体系抗氧化活性成分,利用大蒜氨基酸展开系统(正丁醇-冰乙酸-水)、大蒜辣素展开系统(甲苯-乙酸乙酯)和大蒜多糖展开系统(冰乙酸-氯仿-水)对大蒜抗氧化活性成分进行分析。结果大蒜灭酶体系中氨基酸、大蒜多糖和蒜酶激活体系中的大蒜含硫化合物的半数抑制质量浓度(IC50)分别为0.058,0.170和0.068mg·mL-1。大蒜灭酶体系中的蒜氨酸和蒜酶激活体系中的大蒜辣素有抗氧化活性,其他抗氧化活性成分有待进一步确定。结论大蒜中氨基酸和含硫化合物有一定的抗氧化活性,具有研究价值。     

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.     

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.     

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.