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.     

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。     

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。     

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.     

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.     

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.     

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.     

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.     

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.     

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)     

蒜氨酸微丸-蒜酶肠溶双层片制备工艺优选及体外抗肿瘤作用评价

目的 优选蒜氨酸微丸-蒜酶肠溶双层片的制备工艺,并评价其体外抗肿瘤作用.方法 以蒜氨酸的累积释放率为评价指标,以崩解剂种类和用量、肠溶衣用量及微晶纤维素(MCC)与蒜氨酸的质量比为考察因素,采用单因素试验优选蒜氨酸微丸的制备工艺;以大蒜辣素产率为评价指标,以蒜酶层中MCC的用量、包衣温度为考察因素,采用单因素试验优选蒜...     

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.     

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。     

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.     

Induction and inhibition of indole production of intestinal bacteria

The fecal tryptophanase activities were 0.267±0.10 for rats and 0.185±0.01 μmole/min/g wet feces for humans. The activities of indole pyruvate degradation to indole, indole pyruvate lyase, of these feces were 0.051±0.02 and 0.046±0.01 μmole/min/g wet feces, respectively. The optimal pH values of tryptophanase and indole pyruvate lyase were 5.5–7.5 and 5.5–6.5, respectively. When the intestinal flora orE. coli HGU-3 was cultured in GAM broth having six different pH values (5 to 10), the activities of tryptophanase and indole pyruvate lyase in the medium adjusted at pH 6 were dramatically induced by elevating the pH to 9. However, when intestinal microflora were inoculated in the medium containing lactulose, the productions of these enzymes were dramatically inhibited and the pH of the medium was lower than that of the control.     

HPLC法研究还原型谷胱甘肽降解产物

目的研究还原型谷胱甘肽(GSH)在不同破坏条件下的降解产物;在此研究基础上,建立了氧化型谷胱甘肽(GSSG)杂质测定方法。方法采用CenturySIL AQ C18色谱柱(4.6 mm×200 mm,5μm),50 mmol.L-1磷酸二氢钾溶液(含10 mmol.L-1庚烷磺酸钠,用磷酸调节pH值至3.0)-甲醇(体积比97∶3)为流动相;检测波长为210 nm。结果通过对比HPLC保留时间,确定破坏样品中主要降解产物为GSSG、二肽半胱氨酰甘氨酸、谷氨酸、半胱氨酸、甘氨酸和胱氨酸。按外标法计算GSH原料中GSSG的含量为0.32%。结论建立的GSSG测定方法结果准确,重现性好,可用于GSH原料中GSSG的控制。     

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.     

HPLC-MS/MS法研究精制蒜氨酸中有关物质

研究精制蒜氨酸样品中的有关物质。采用HPLC-MS/MS一级质谱扫描总离子流谱测定有关物质的准分子离子，并利用他们的色谱保留时间、二级质谱及/或对照品对照进行结构鉴定。全扫描总离子流谱结果表明，精制蒜氨酸中主要含7个有关物质成分，［M+H］⁺离子m/z分别为116，133，147，152，175及两个m/z同为178的蒜氨酸同分异构体。其中m/z为116，133，147，175的［M+H］⁺离子相应的物质分别鉴定为脯氨酸、门冬酰胺、谷氨酰胺、精氨酸，m/z为152的［M+H］⁺离子鉴定为甲基-L-半胱氨酸亚砜，m/z 178的两个蒜氨酸同分异构体分别经二级质谱鉴定为异蒜氨酸和环蒜氨酸。精制蒜氨酸中主要有关物质分别为氨基酸及蒜氨酸的同系物及同分异构体。     

Reactive sulfur species: kinetics and mechanism of the hydrolysis of cysteine thiosulfinate ester

The kinetics and mechanisms of the hydrolysis of cysteine thiosulfinate ester (CyS(O)SCy ( x- ), x = 0-2) have been investigated by stopped-flow spectrophotometry between pH 6 and pH 14. The rate-limiting reaction of hydroxide is observed for pH < 13. More complicated kinetics are observed above pH 13, where the hydrolysis of CyS(O)SCy (2-) can be fast relative to subsequent reactions. The eventual products of hydrolysis are a 1:1 molar ratio of cystine (CySSCy) and cysteine sulfinic acid (CySO 2H) under all reaction conditions. The rate of hydrolysis is dependent upon the proton state of CyS(O)SCy ( x- ). Furthermore, cysteine thiosulfonate ester (CyS(O) 2SCy) was observed as an intermediate during the hydrolysis of CyS(O)SCy ( x- ) at lower pH. CyS(O) 2SCy eventually hydrolyzes to give stoichiometric amounts of CySSCy and CySO 2H. However, CySO 2H is observed under some conditions for which hydrolysis of CyS(O) 2SCy is relatively slow, thus suggesting multiple hydrolysis pathways for CyS(O)SCy ( x- ). The mechanism up to the rate-limiting step is proposed to be as follows: CyS(O)SCy (0) = H (+) + CyS(O)SCy (-), p K a3 = 7.32; CyS(O)SCy (-) = H (+) + CyS(O)SCy (2-), p K a4 = 7.92; CyS(O)SCy (0) + OH (-) --> products, P 0 k 0 = (5.0 +/- 0.01) x 10 (3) M (-1) s (-1); CyS(O)SCy (-) + OH (-) --> products, P 1 k 1 = 60 +/- 18 M (-1) s (-1); and CyS(O)SCy (2-) + OH (-) --> products, P 2 k 2 = 0.36 +/- 0.01 M (-1) s (-1), where P x is a constant (1 相似文献