蒜氨酸+蒜酶抗肿瘤及其机制的研究

大蒜制剂、大蒜提取物和大蒜提取成分的抗肿瘤作用已有广泛的研究[1 ] 。蒜氨酸 (Alliin ,2 烯丙基半胱氨酸亚砜 )为大蒜活性成分的主要前体物质 ,蒜氨酸在蒜氨酸酶 (Alli inase ,EC 4 .4.1 .4)的催化作用下产生大蒜辣素 (Allicin ,二烯丙基硫代亚磺酸酯 )。本课题组发现蒜氨酸 +蒜氨酸酶生成的大蒜辣素 1 3h内基本稳定。据此 ,本文采用MGC 80 3和HeLa肿瘤细胞株 ,利用蒜氨酸 +蒜氨酸酶生成的大蒜辣素研究其体外的抗肿瘤作用及其作用机制。1　材料和方法1 .1　细胞培养及药物　细胞培养的传代接种见文献[2 ] 。蒜氨酸及蒜氨酸酶由新疆…     

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。     

可使大蒜无气味吗?

大蒜辣素——气味很强的物质大蒜中蒜氨酸(Alliin)稳定,且无气味,实际上也不起作用。但是,它一旦与大蒜中的蒜氨酸酶接触,就极其迅速地转化为气味很强、但极不稳定的大蒜辣素(Allicin)。因为植物中的蒜氨酸酶通常与蒜氨酸分开,只有组织受到破坏时,它才使底物转化。几种蒜氨酸的同系物性质完全相同。尤其在高温时,大蒜辣素及其同系物自动转化为一系列第二产物,通过水蒸汽蒸馏     

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。     

含蒜酶的大蒜提取物可改善高血压等症的治疗活性

一种新制剂内含有活性的蒜酶和蒜氨酸的干大蒜提取物。据蒜酶量计,干大蒜提取残渣中比大蒜粉含有更多的蒜氨酸,也含至少0.1％的粉状水溶性酸,至少2％的蒜氨酸(千重)。不含蒜酶,仅含蒜氨酸的大蒜提取物     

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。     

HPLC定量测定大蒜的蒜辣素和蒜氨酸

蒜辣素(allicin)及其前体蒜氨酸(alliin)已被认为是大蒜及其制品评价的标志成分。我们根据文献(1)和(3)改进了蒜辣素的合成和纯化方法,并用反相HPLC硅胶吸附物作为内标进行蒜辣素的含量测定。另外我们还建立了两种洗脱系统以缩短分析时间而获得相同的分析数据。蒜辣素的保留时间原为6.7min,而现在的保留时间分别减少到3.5和3.9min。为了建立蒜氨酸的内标,我们改进了氨基酸的合成方法,异构体反复重结晶进行分离,用HPLC进行蒜氨酸定量测定。     

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...     

大蒜甙注射液的制备方法

百合科植物大蒜具有抗菌等性能。其有效成分大蒜辣素由于性质很不稳定,所以迄今无实用价值,而大蒜粗制剂早已广泛应用于临床。对于数种细菌性、真菌性与原虫性感染已充分证明其治疗与预防的价值。前人报导大蒜有效成分蒜辣素很不稳定,蒜辣素的pH 约6.5的水溶液放置过程中     

高效液相色谱法考察蒜酶活力测定中的影响因素

目的考察底物蒜氨酸纯度对蒜酶活力测定数值的影响,比较催化温度为25,35,37℃时蒜酶活力测定数值的差异。建立可行的蒜酶活力检测方法。方法采用高效液相色谱法,以蒜氨酸的减少量计算蒜酶活力。结果进行蒜酶活力测定时,底物蒜氨酸的纯度应不小于90%,确定蒜酶催化反应温度为35℃。结论建立了以95%蒜氨酸为底物,催化温度为35℃的蒜酶活力测定条件。     

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.     

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.     

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.     

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)     

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.     

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.     

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.     

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。     

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.     

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.