Allicin inhibits cell growth and induces apoptosis through the mitochondrial pathway in HL60 and U937 cells

In this article, the effects of allicin, a biological active compound of garlic, on HL60 and U937 cell lines were examined. Allicin induced growth inhibition and elicited apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c release into the cytosol, activation of caspase 9 and caspase 3 and DNA fragmentation. Pretreatment of HL60 cells with cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (mPTP), inhibited allicin-treated cell death. HL60 cell survival after 1 h pretreatment with cyclosporine A, followed by 16 h in presence of allicin (5 microM) was approximately 80% compared to allicin treatment alone (approximately 50%). Also N-acetyl cysteine, a reduced glutathione (GSH) precursor, prevented cell death. The effects of cyclosporine A and N-acetyl cysteine suggest the involvement of mPTP and intracellular GSH level in the cytotoxicity. Indeed, allicin depleted GSH in the cytosol and mitochondria, and buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly augmented allicin-induced apoptosis. In HL60 cells treated with allicin (5 microM, 30 min) the redox state for 2GSH/oxidized glutathione shifted from EGSH -240 to -170 mV. The same shift was observed in U937 cells treated with allicin at a higher concentration for a longer period of incubation (20 microM, 2 h). The apoptotic events induced by various concentrations of allicin correlate to intracellular GSH levels in the two cell types tested (HL60: 3.7 nmol/10(6) cells; U937: 7.7 nmol/10(6) cells). The emerging mechanistic basis for the antiproliferative function of allicin, therefore, involves the activation of the mitochondrial apoptotic pathway by GSH depletion and by changes in the intracellular redox status.     

Protective role of allicin and L-ascorbic acid against the genotoxic damage induced by chlormadinone acetate in cultured human lymphocytes

In our present study, different doses of allicin and L-ascorbic acid were tested against the genotoxic damage induced by chlormadinone acetate (CMA; 40 microM) using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) as the parameters. Treatment with allicin and L-ascorbic acid resulted in reduction of CAs and SCEs. The results suggested a protective role of allicin and L-ascorbic acid against CMA induced genotoxic damage.     

Inhibition of GSH-dependent PGH2 isomerase in mammary adenocarcinoma cells by allicin

We have reported that allicin, a constituent of garlic oil, has no effect on the activities of platelet cyclooxygenase or thromboxane synthase, or vascular PGI2 synthase. The effect of allicin on glutathione (GSH) dependent PGH2 to PGE2 isomerase is unknown. We therefore studied the effect of allicin on PGE2 biosynthesis in a murine mammary adenocarcinoma cell line (No 4526). Intact or sonicated cells were incubated with either 14C-arachidonic acid (AA) or 14C-PGH2, respectively. Following metabolism, products were extracted, separated by TLC and analyzed by radiochromatographic scan. PGE2 was predominantly formed with minimal amounts of PGF2 alpha and PGD2. Formation of 6-keto-PGF1 alpha or TXB2 was not detected indicating the absence of TXA2 and PGI2 synthase activity. Indomethacin and ibuprofen inhibited the PGE2 formation (p less than 0.05). The enzymatic PGE2 formation in sonicates was blocked by depletion of the cellular non-protein thiols by buthionine sulfoximine and was shown to be dependent on GSH. Allicin, over the range of 10-1000 microM, inhibited the formation of PGE2 in cells exposed to 2.0 microM 14C-AA for 20 min. and in sonicated cells incubated with 20.0 microM 14C-PGH2 for 2 min (p less than 0.05). Allicin did not alter cyclooxygenase-mediated oxygen utilization in ram seminal vessicle microsomes, suggesting that allicin selectively inhibits the GSH-dependent PGH2 to PGE2 isomerase in this adenocarcinoma cell line.     

Allicin, a naturally occurring antibiotic from garlic, specifically inhibits acetyl-CoA synthetase

Allicin is shown to be a specific inhibitor of the acetyl-CoA synthetases from plants, yeast and mammals. The bacterial acetyl-CoA-forming system, consisting of acetate kinase and phosphotransacetylase, was inhibited too. Non-specific interaction with sulfhydryl-groups could be excluded in experiments with dithioerythritol and p-hydroxymercuribenzoate. Binding of allicin to the enzyme is non-covalent and reversible. [14C]-Acetate incorporation into fatty acids of isolated plastids was inhibited by allicin with an I50-value lower than 10 microM. Other enzymes of the fatty acid synthesis sequence were not affected, as was shown using precursors other than acetate.     

Allicin-induced suppression of Mycobacterium tuberculosis 85B mRNA in human monocytes

Despite of encountering a robust immune response, Mycobacterium tuberculosis (MTB) successfully survives and persists in the human host. We investigated the early regulation of MTB 85B gene by allicin in MTB-infected human monocytes. During the first 24h of infection, levels of both MTB 85B intracellular mRNA and secreted protein were significantly down-regulated by allicin in a dose-dependent manner, which was mediated by inhibition of glutathione and NF-kappaB pathway. Allicin-induced MTB 85B suppression correlated with suppression of TNF-alpha released from infected monocytes. The allicin-induced up-regulation of glutathione and IFN-gamma with simultaneous decrease in TNF-alpha supports the anti-inflammatory property of allicin by elicitation of protective immune response. Thus, allicin may prove to be valuable in the containment of MTB and therefore be useful as an adjunct in treatment of tuberculosis.     

Allicin disrupts the cell's electrochemical potential and induces apoptosis in yeast

The volatile substance allicin gives crushed garlic (Allium sativum) its characteristic odor and is a pro-oxidant that undergoes thiol-disulfide exchange reactions with -SH groups in proteins and glutathione. The antimicrobial activity of allicin is suspected to be due to the oxidative inactivation of essential thiol-containing enzymes. We investigated the hypothesis that at threshold inhibitory levels allicin can shunt yeast cells into apoptosis by altering their overall redox status. Yeast cells were treated either with chemically synthesized, pure allicin or with allicin in garlic juice. Allicin-dependent cell oxidation was demonstrated with a redox-sensitive GFP construct and the shift in cellular electrochemical potential (E(hc)) from less than -215 to -181mV was calculated using the Nernst equation after the glutathione/glutathione disulfide couple (2GSH/GSSG) in the cell was quantified. Caspase activation occurred after allicin treatment, and yeast expressing a human antiapoptotic Bcl-XL construct was rendered more resistant to allicin. Also, a yeast apoptosis-inducing factor deletion mutant was more resistant to allicin than wild-type cells. We conclude that allicin in garlic juice can activate apoptosis in yeast cells through its oxidizing properties and that this presents an alternative cell-killing mechanism to the previously proposed specific oxidative inactivation of essential enzymes.     

大蒜素诱导人食管癌EC-109细胞凋亡

为寻找毒副作用小并且治疗效果好的抗癌药物,研究大蒜素对人食管癌EC-109细胞凋亡的影响,同时探讨了大蒜素引发细胞凋亡的可能机制。通过激光共聚焦显微镜观察细胞形态变化,琼脂糖凝胶电泳检测DNA片段化情况,流式细胞术检测细胞凋亡率和线粒体膜电位变化,qRT-PCR和Western blotting检测细胞凋亡相关基因Bax、Bcl-2的mRNA和蛋白表达水平。结果显示,大蒜素作用人食管癌EC-109细胞48 h后,线粒体膜电位显著降低,并且早期凋亡细胞和晚期凋亡细胞所占百分比均显著增加。同时,与对照组相比,Bax mRNA和蛋白水平均显著升高(p<0.05),Bcl-2 mRNA和蛋白水平均显著降低(p<0.05)。据此,本研究得出大蒜素可诱导人食管癌EC-109细胞凋亡,并呈剂量依赖性,有潜在的药用价值。     

Suppression of Mycobacterium tuberculosis induced reactive oxygen species (ROS) and TNF-alpha mRNA expression in human monocytes by allicin

Chronic inflammation associated with tumor necrosis factor (TNF)-alpha and reactive oxygen species (ROS) is the hallmark of tuberculosis. Mycobacterium tuberculosis (MTB) directly stimulates human monocytes to secrete TNF-alpha. We show the augmented expression of TNF-alpha mRNA in MTB-infected monocytes by cellular activation and ROS was suppressed by allicin in a dose-dependent manner. Also, allicin enhanced the glutathione peroxidase activity, which correlated inversely with the downregulation of ROS and TNF-alpha in MTB-infected monocytes. Hence, allicin may prove to be a valuable natural antioxidant in combating tuberculosis.     

The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity

Allicin (diallyl thiosulfinate) is the main biologically active component of the freshly crushed garlic extracts. In the present work the ability of allicin to cross through membranes (artificial and biological) was studied. Partition coefficients of allicin in water/octanol, water/hexadecane and water/phospholipids mixtures were determined. Using phospholipid vesicles loaded with hydrophilic thiols (reduced glutathione or 2-nitro-5-thiobenzoate), we observed that allicin freely permeates through phospholipid bilayers and interacts with the SH groups. The reaction rate of allicin with SH containing molecules after crossing the membrane was the same as in solution. Fast diffusion and permeation of allicin across human red blood cell membranes was also demonstrated. Allicin does not induce leakage, fusion or aggregation of membrane. The high permeability of allicin through membranes may greatly enhance the intracellular interaction with thiols.     

Garlic Revisited: Antimicrobial Activity of Allicin-Containing Garlic Extracts against Burkholderia cepacia Complex

The antimicrobial activities of garlic and other plant alliums are primarily based on allicin, a thiosulphinate present in crushed garlic bulbs. We set out to determine if pure allicin and aqueous garlic extracts (AGE) exhibit antimicrobial properties against the Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. We prepared an AGE from commercial garlic bulbs and used HPLC to quantify the amount of allicin therein using an aqueous allicin standard (AAS). Initially we determined the minimum inhibitory concentrations (MICs) of the AGE against 38 Bcc isolates; these MICs ranged from 0.5 to 3% (v/v). The antimicrobial activity of pure allicin (AAS) was confirmed by MIC and minimum bactericidal concentration (MBC) assays against a smaller panel of five Bcc isolates; these included three representative strains of the most clinically important species, B. cenocepacia. Time kill assays, in the presence of ten times MIC, showed that the bactericidal activity of AGE and AAS against B. cenocepacia C6433 correlated with the concentration of allicin. We also used protein mass spectrometry analysis to begin to investigate the possible molecular mechanisms of allicin with a recombinant form of a thiol-dependent peroxiredoxin (BCP, Prx) from B. cenocepacia. This revealed that AAS and AGE modifies an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. To our knowledge, we report the first evidence that allicin and allicin-containing garlic extracts possess inhibitory and bactericidal activities against the Bcc. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit investigation as adjuncts to existing antibiotics.     

Quantitative and qualitative analysis of the antifungal activity of allicin alone and in combination with antifungal drugs

The antifungal activity of allicin and its synergistic effects with the antifungal agents flucytosine and amphotericin B (AmB) were investigated in Candida albicans (C. albicans). C. albicans was treated with different conditions of compounds alone and in combination (allicin, AmB, flucytosine, allicin + AmB, allicin + flucytosine, allicin + AmB + flucytosine). After a 24-hour treatment, cells were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) to measure morphological and biophysical properties associated with cell death. The clearing assay was conducted to confirm the effects of allicin. The viability of C. albicans treated by allicin alone or with one antifungal drug (AmB, flucytosine) in addition was more than 40% after a 24-hr treatment, but the viability of groups treated with combinations of more than two drugs was less than 32%. When the cells were treated with allicin alone or one type of drug, the morphology of the cells did not change noticeably, but when cells were treated with combinations of drugs, there were noticeable morphological changes. In particular, cells treated with allicin + AmB had significant membrane damage (burst or collapsed membranes). Classification of cells according to their cell death phase (CDP) allowed us to determine the relationship between cell viability and treatment conditions in detail. The adhesive force was decreased by the treatment in all groups compare to the control. Cells treated with AmB + allicin had a greater adhesive force than cells treated with AmB alone because of the secretion of molecules due to collapsed membranes. All cells treated with allicin or drugs were softer than the control cells. These results suggest that allicin can reduce MIC of AmB while keeping the same efficacy.     

大蒜素对白色念珠菌感染小鼠神经内分泌免疫系统调节作用的研究(英文)

本研究旨在探讨大蒜素对白色念珠菌感染小鼠神经内分泌免疫系统(NEI)的调节作用。将小鼠随机分为三组:正常对照组、模型组、大蒜素组。除正常对照组外,模型组、大蒜素组分别建立小鼠白色念珠菌感染模型。大蒜素组小鼠给予大蒜素灌胃,其余组给予等量生理盐水。连续用药14 d后,各组小鼠取血测定促肾上腺激素(ACTH)、皮质酮(CORT)、β-内啡肽(β-EP)、5-羟色胺(5-HT)的水平,测定胸腺指数及脾脏淋巴细胞增殖活性。实验结果显示,模型组小鼠NEI功能发生紊乱,与正常对照组相比,ACTH、CORT、β-EP水平均明显升高,5-HT、胸腺指数及脾脏淋巴细胞增殖活性均明显下降,差异有显著性(P<0.05)。而大蒜素组小鼠ACTH、CORT、β-EP水平较之模型组明显下降,5-HT、胸腺指数及脾脏淋巴细胞增殖活性明显升高,差异有显著性(P<0.05)。综上所述,本研究提示大蒜素对白色念珠菌感染小鼠紊乱的NEI具有明显的调节作用。     

大蒜素调控黑腹果蝇产卵偏嗜性和适合度

[目的]研究果蝇对大蒜素的产卵选择,并解析果蝇产卵避性的机制和生物学意义.[方法]应用产卵双向选择装置,检测黑腹果蝇Drosophila melanogaster雌成虫对0.01％,0.015％和0.02％大蒜素的产卵选择性;利用产卵装置,检测黑腹果蝇对大蒜素的位置效应;通过毛细管摄食法检测黑腹果蝇摄食行为;利用黑暗(...     

Thermostability of allicin determined by chemical and biological assays

The garlic-derived antibacterial principle, alk(en)yl sulfinate compounds, has long been considered as very short-lived substance. However, there are some data showing a rather more stable nature of allicin. We determined here the thermostability of allicin by a systematic analyses employing chemical quantification and an antibacterial activity assay. Allicin in an aqueous extract of garlic was degraded stoichiometrically in proportion to the temperature; we estimated the half-life of allicin to be about a year at 4 degrees C (from 1.8 mg/ml to 0.9 mg/ml) and 32 d at 15 degrees C, but only 1 d at 37 degrees C (from 2.0 mg/ml to 1.0 mg/ml). The half-life values for antibacterial activity showed a similar trend in results: 63 d or more at 4 degrees C for both antibacterial activities, 14 d for anti-staphylococcal activity, and 26 d for anti-escherichia activity at 15 degrees C, but only 1.2 d and 1.9 d for the respective activities at 37 degrees C. Such antibacterial activities were attributable to the major allicin, allyl 2-propenylthiosulfinate. Surprisingly, the decline in the quantity of allicin was not accompanied by its degradation; instead, allicin became a larger molecule, ajoene, which was 3-times larger than allicin.     

Sterols and Asparagine in the Rice Plant,Endogenous Factors Related to Resistance against the Brown Planthopper (Nilaparvata lugens)

The garlic-derived antibacterial principle, alk(en)yl sulfinate compounds, has long been considered as very short-lived substance. However, there are some data showing a rather more stable nature of allicin. We determined here the thermostability of allicin by a systematic analyses employing chemical quantification and an antibacterial activity assay. Allicin in an aqueous extract of garlic was degraded stoichiometrically in proportion to the temperature; we estimated the half-life of allicin to be about a year at 4 °C (from 1.8 mg/ml to 0.9 mg/ml) and 32 d at 15 °C, but only 1 d at 37 °C (from 2.0 mg/ml to 1.0 mg/ml). The half-life values for antibacterial activity showed a similar trend in results: 63 d or more at 4 °C for both antibacterial activities, 14 d for anti-staphylococcal activity, and 26 d for anti-escherichia activity at 15 °C, but only 1.2 d and 1.9 d for the respective activities at 37 °C. Such antibacterial activities were attributable to the major allicin, allyl 2-propenylthiosulfinate. Surprisingly, the decline in the quantity of allicin was not accompanied by its degradation; instead, allicin became a larger molecule, ajoene, which was 3-times larger than allicin.     

一株大蒜素降解菌的分离与鉴定

【目的】解决大蒜废水难以生化处理的问题,筛选能够降解大蒜素的细菌,并对其生化特性进行分析。【方法】通过16S r RNA基因测序,对大蒜废水沉积物进行细菌多样性分析,发现其中有多种大蒜素耐受菌;通过富集培养与划线分离大蒜素降解菌,并进一步分析其最适生长和大蒜素降解条件。【结果】得到一株大蒜素降解菌(JX6-2),能够以大蒜素为唯一碳源生长(50-300 mg/L),将大蒜素彻底降解。其最适生长温度和降解大蒜素温度均为35°C,最适pH值在中性左右,添加葡萄糖对大蒜素降解没有明显影响。【结论】分离得到了一株大蒜素降解菌,分析了其生长和降解特性,为生化方法处理大蒜素废水提供了新菌种。     

Effect of allicin on the production of polysaccharide intercellular adhesin in Staphylococcus epidermidis

Aims: Polysaccharide intercellular adhesin (PIA) is the main agglutination agent in the biofilm forming strain Staphylococcus epidermidis. To find an explanation for the observed inhibition of biofilm formation by allicin, we studied the effect of allicin on PIA production in samples treated with sub MIC doses of allicin and compared this with a control culture without allicin. Methods and Results: Bacteria (Staph. epidermidis ATCC 35984) were grown in glass tubes, and PIA was extracted by vortex vibration using microbeads and NN dimethyl acetamide/LiCl as solvent. The extracts were filtered and passed through size exclusion columns. Chromatographic fractions were analysed with an excess of sodium metaperiodate and the excess was determined spectrophotometrically using 2,4,6‐tripyridyl‐s‐triazine. Conclusion: The amount of exopolysaccharides in samples previously treated with allicin is significantly lower than in the control. This finding suggests a specific enzymatic inhibition in PIA synthesis. Significance and Impact of the Study: This study provides an insight into the mechanism of biofilm formation, and is a biochemical model for PIA inhibition by allicin. The analysis proposed may be useful in studies of production of exopolysaccharides responsible for adherence and agglutination of Staph. epidermidis. Prevention of biofilm formation by allicin opens up a new field of in vitro studies and permits us to envisage future clinical applications.     

Evaluation of Allicin for the Treatment of Experimentally Induced Subacute Lead Poisoning in Sheep

Garlic (Allium sativum) is known to reduce lead toxicity in some species of animals. The objective of this study was to evaluate the efficacy of allicin, one of the most active components of garlic, in the treatment of subacute lead intoxication in sheep. Nine female sheep weighing 25-29 kg orally received a daily dose of 80 mg/kg body weight of lead acetate for 5 days. The animals were then assigned into two groups. Group 1 did not receive any further treatment and was used as the control group and group 2 was treated orally by 2.7 mg/kg body weight of allicin twice daily for 7 days. Within one day following allicin treatment, group 2 blood lead levels were significantly lower than that in group 1 (mean of 616.9 microg/l and 290.02 microg/l, respectively; P < 0.05). Also, allicin treatment significantly reduced kidney lead content and considerably reduced bone and ovary lead contents. These results suggest that allicin might have some therapeutic effects on lead poisoning.     

Thermal degradation of allicin in garlic extracts and its implication on the inhibition of the in-vitro growth of Helicobacter pylori

Allicin, the main active principle related to Allium sativum chemistry, is considered to be responsible for the bacteriostatic properties of garlic. The work described here has demonstrated the direct implication of the allicin present in solvent-free garlic extracts obtained with ethanol (ethanolic garlic extract, EGE) and acetone (acetonic garlic extract, AGE) in the inhibition of the in-vitro growth of Helicobacter pylori (Hp), the bacterium responsible for serious gastric diseases such as ulcers and even gastric cancer. The evolution of allicin concentration as a function of time and temperature has been the subject of a kinetic study. The reaction order, activation energy, and preexponential factor (in accordance with Arrhenius theory) have been determined for the decomposition process of allicin in these organic media. First-order decomposition, an activation energy of 97.4 kJ/mol, and an Arrhenius preexponential factor of 8.9 x 10(10) s(-1) have been determined for allicin in EGE. For allicin in AGE the kinetic order determined was 1.5, the activation energy 184.5 kJ/mol, and the preexponential factor 3.1 x 10(24) s(-1) (mg/L)(-0.5). The presence or absence of allicin in these garlic products was found to be crucial for the inhibition of the in-vitro growth of Hp, as demonstrated by microbiological analysis for AGE. A relationship has been identified between the effectiveness and durability of the anti-Hp properties shown by AGE and the allicin content of these products. The bacteriostatic properties were active for up to 10 months if the samples were maintained at 6 degrees C.     

Antibacterial Potential of Garlic-Derived Allicin and Its Cancellation by Sulfhydryl Compounds

Allicin (allyl 2-propenethiosulfinate), an antibacterial principle of garlic, has drawn much attention, since it has potent antimicrobial activity against a range of microorganisms, including methicillin-resistant Staphylococcus aureus. There have been many reports on the antibacterial properties of allicin, but no quantitative comparison of antibacterial activities between freshly prepared garlic extract and clinically useful antibiotics has been performed. To verify the substantial antibacterial effect of aqueous garlic extract, we compared it with those of allicin and several clinically useful antibiotics using two representative bacteria commonly found in the human environment, Gram-positive S. aureus and Gram-negative Escherichia coli. The garlic extract had more potent anti-staphylococcal activity than an equal amount of allicin. In terms of antibiotic potency against Gram-positive and Gram-negative bacteria, authentic allicin had roughly 1–2% of the potency of streptomycin (vs. S. aureus), 8% of that of vancomycin (vs. S. aureus), and only 0.2% of that of colistin (vs. E. coli). The antibacterial activity of allicin was completely abolished by cysteine, glutathione and coenzyme A, but not by non-SH-compounds. The oxygen in the structure (–S(=O)–S–) of allicin therefore functions to liberate the S-allyl moiety, which might be an offensive tool against bacteria.