S-Allylmercaptoglutathione: the reaction product of allicin with glutathione possesses SH-modifying and antioxidant properties.

The reaction between allicin (diallylthiosulfinate), the active component of garlic and reduced glutathione was investigated. The product of this reaction, mixed disulfide S-allylmercaptoglutathione (GSSA) was separated by high performance liquid chromatography and identified by 1H and (13)C nuclear magnetic resonance and mass spectroscopy. The reaction is fast (with an apparent bimolecular reaction rate constant of 3.0 M(-1) s(-1)). It is pH-dependent, which reveals a direct correlation to the actual concentration of mercaptide ion (GS(-)). Both GSSA and S-allylmercaptocysteine (prepared from allicin and cysteine) reacted with SH-containing enzymes, papain and alcohol dehydrogenase from Thermoanaerobium brockii yielding the corresponding S-allylmercapto proteins, and caused inactivation of the enzymes. The activity was restored with dithiothreitol or 2-mercaptoethanol. In addition, GSSA also exhibited high antioxidant properties. It showed significant inhibition of the reaction between OH radicals and the spin trap 5,5'-dimethyl-1-pyroline N-oxide in the Fenton system as well as in the UV photolysis of H2O2. In ex vivo experiments done with fetal brain slices under iron-induced oxidative stress, GSSA significantly lowered the production levels of lipid peroxides. The similar activity of GSSA and allicin as SH-modifiers and antioxidants suggests that the thioallyl moiety has a key role in the biological activity of allicin and its derivatives.

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.

Effect of purified allicin, the major ingredient of freshly crushed garlic, on cancer cell proliferation.

The diverse health benefit effects of garlic include its anticancer activity. However, very little is known about such activity of isolated garlic compounds, among which allicin (the major ingredient of crushed garlic) has been the least studied. The aim of this work was to determine whether pure allicin exhibits the antiproliferative effect reported for garlic in in vitro models. Allicin, but not its precursor alliin, inhibited proliferation of human mammary (MCF-7), endometrial (Ishikawa), and colon (HT-29) cancer cells (50% inhibitory concentration = 10-25 microM). Two of three tested primary lines of human fibroblasts displayed a similar response to allicin (50% inhibitory concentration = 16-40 microM), whereas the third line was almost unaffected by this compound. The pure allicin and water extract of garlic powder with equivalent allicin concentrations displayed a similar potency, suggesting that allicin is responsible for the antiproliferative effect of the extract. The growth inhibition was accompanied by accumulation of cells in the G0/G1 and G2/M phases of the cell cycle (MCF-7 cells) and not by a significant increase in cell death. Allicin caused a transient drop in the intracellular glutathione (GSH) level, the magnitude and kinetics of which significantly varied depending on cell type. The extent of the decrease in GSH levels correlated well (r = 0.75) with the growth inhibitory activity of allicin. On the basis of these findings, we suggest that allicin plays a major role in the antiproliferative effect of water-soluble garlic preparations and that this effect may be attributed to the ability of allicin to transiently deplete the intracellular GSH level.

Antimicrobial properties of allicin from garlic.

Allicin, one of the active principles of freshly crushed garlic homogenates, has a variety of antimicrobial activities. Allicin in its pure form was found to exhibit i) antibacterial activity against a wide range of Gram-negative and Gram-positive bacteria, including multidrug-resistant enterotoxicogenic strains of Escherichia coli; ii) antifungal activity, particularly against Candida albicans; iii) antiparasitic activity, including some major human intestinal protozoan parasites such as Entamoeba histolytica and Giardia lamblia; and iv) antiviral activity. The main antimicrobial effect of allicin is due to its chemical reaction with thiol groups of various enzymes, e.g. alcohol dehydrogenase, thioredoxin reductase, and RNA polymerase, which can affect essential metabolism of cysteine proteinase activity involved in the virulence of E. histolytica.

The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins.

Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) is the main biologically active component of garlic clove extracts. Its biological activity was attributed to either antioxidant activity or thiol disulfide exchange. Antioxidant properties of both allicin and its precursor, alliin (+S-allyl-L-cysteine sulfoxide), were investigated in the Fenton oxygen-radical generating system [H2O2-Fe(II)]. Using the spin trapping technique and ESR, it was found that both compounds possessed significant antioxidant activity. The reaction between allicin and L-cysteine was studied by 1H and 13C-NMR, and a S-thiolation product, S-allylmercaptocysteine, was identified. Allicin irreversibly inhibited SH-protease papain, NADP(+)-dependent alcohol dehydrogenase from Thermoanaerobium brockii (TBAD), and the NAD(+)-dependent alcohol dehydrogenase from horse liver (HLAD). All the three enzymes could be reactivated with thiol containing compounds. Papain could be reactivated with glutathione, TBAD with dithiothreitol or 2-mercaptoethanol (2-ME) but not by glutathione, while HLAD could be reactivated only with 2-ME. This study demonstrates that in addition to its antioxidant activity, the major biological effect of allicin should be attributed to its rapid reaction with thiol containing proteins.

Allicin from garlic strongly inhibits cysteine proteinases and cytopathic effects of Entamoeba histolytica.

The ability of Entamoeba histolytica trophozoites to destroy monolayers of baby hamster kidney cells is inhibited by allicin, one of the active principles of garlic. Cysteine proteinases, an important contributor to amebic virulence, as well as alcohol dehydrogenase, are strongly inhibited by allicin.

Alteration of lipid profile in hyperlipidemic rabbits by allicin, an active constituent of garlic.

BACKGROUND: The effect of garlic on the serum lipid profile has been the subject of controversy. This study was therefore designed to examine the effects of allicin, an active constituent of garlic, on the lipid profile in a rabbit model. METHODS: Allicin was produced by reacting alliin, synthesized in our laboratory, with purified alliinase. Nineteen New Zealand White rabbits were fed a cholesterol-rich diet (0.25% cholesterol) for 18 weeks. Ten rabbits received freshly produced allicin (3 mg/kg orally) starting at 8 weeks, and nine received placebo. There was no significant difference between the lipid profiles of the two groups at baseline up to 8 weeks. RESULTS: From day 28 of allicin supplementation a significant difference was found between the allicin and placebo groups in the graph regression lines describing the influence of allicin on serum cholesterol: Y = 41.39 + 8.69 multiplied by day (control) versus Y = -877.24 + 17.67 multiplied by day (allicin). The same trend was found for low-density lipoprotein concentrations: Y = 10.3 + 8.4 multiplied by (control) versus Y = -750.4 + 15.7 multiplied by day (allicin). The serum high-density lipoprotein levels also differed significantly between the groups: Y = 20.29 + 0.24 multiplied by day (control) versus Y = -109.9 + 1.65 multiplied by day (allicin). CONCLUSIONS: Our results indicate that allicin has a beneficial effect on the serum lipid profile in hyperlipidemic rabbits, and should be further tested clinically.

Alliinase (alliin lyase) from garlic (Alliium sativum) is glycosylated at ASN146 and forms a complex with a garlic mannose-specific lectin.

Alliinase (EC 4.4.1.4) catalyses the production of allicin (thio-2-propene-1-sulfinic acid S-allyl ester), a biologically active compound which is also responsible for the characteristic smell of garlic. It was demonstrated that alliinase which contains 5.5-6% of neutral sugars, gives clear PAS-staining, binds to Con A and can form a complex with garlic mannose-specific lectin (ASA). Evidence that the formation of such a complex is mediated by the interaction of the carbohydrate of the glycoprotein enzyme with the lectin was obtained from a radioligand assay which demonstrated the binding of alliinase to ASA and competitive inhibition of this binding by methyl alpha-D-mannoside. ASA I was shown as the lectin mainly present in the complex with alliinase. The results of this study also demonstrate that alliinase is glycosylated at Asn146 in the sequence Asn146-Met147-Thr148.

Natural antibodies to dietary proteins: the existence of natural antibodies to alliinase (Alliin lyase) and mannose-specific lectin from garlic (Allium sativum) in human serum.

It is known that human serum contains natural antibodies to self and non-self proteins. We wished to determine whether normal human serum contains antibodies to dietary proteins that were never injected. We found that human serum contains antibodies to the two major proteins from cloves of garlic (Allium sativum) which is used as a flavorigard dietary food additive. The antibodies found were directed against alliinase and mannose-specific Allium sativum agglutinin (ASA). The antibodies were purified by affinity chromatography on their corresponding antigens. The purified immunoglobulins were mainly of the IgG and IgM classes and could be divided into two categories--specific and crossreactive. The anti-alliinase antibodies were highly specific, while anti-ASA antibodies were polyreactive. Some of the possible reasons for this difference in specificity are suggested.

Alliin lyase (Alliinase) from garlic (Allium sativum). Biochemical characterization and cDNA cloning.

The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. Its Km using synthetic S-allylcysteine sulfoxide (+ isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near the N-terminal of the intact polypeptide. The second step involved screening of garlic lambda gt11 and lambda ZAPII cDNA libraries with pAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3' noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from alpha wheat gliadin, and expressed in Xenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.

Effects of covalently bound silica-nitroimidazole drug particles on Entamoeba histolytica.

Most commonly used antiamoebic drugs are effective in invasive amebiasis, but their response against trophozoites of Entamoeba histolytica, present in the lumen of the human colon, is inadequate. We report the development of an antiamoebic drug carrier that may be effective against luminal infections. Our preparation consists of small silica particles (5-10 microns in diameter) covalently linked to a potent antiamoebic drug, 2-(4-aminophenoxymethyl)-5-nitro-1-methyl imidazole. Silica-drug particles were injected into mice, hamsters, and guinea pigs. We found that trophozoites phagocytosed the particles in vivo and in vitro, followed by rapid cell death due to the released drug. Analysis of mouse serum revealed that no drug was absorbed from the intestine after placement of the drug-containing particles in the intestine. The antiamoebic activity of particles recovered from the intestine was almost fully retained. This novel antiamoebic concept may be useful for luminal therapy for asymptomatic amebiasis and may minimize side effects and frequency of administration.

Nonimmunoglobulin fraction of human milk inhibits the adherence of certain enterotoxigenic Escherichia coli strains to guinea pig intestinal tract.

The protecting effect of human milk against intestinal infections has been well documented, but its mechanism not completely understood. We have examined the effect of the nonimmunoglobulin fraction (NIgF) of human milk and colostrum on bacterial adherence to the intestinal tract. The NIgF was prepared by passing the milk through an immunosorbent column containing rabbit antihuman gamma-globulin (IgG and IgA). The effluent fraction did not contain gamma-globulins as shown by immunodiffusion on agarose and by using rabbit antihuman Ig, that was then detected with fluorescently-labeled goat antirabbit Ig. The effect of the NIgF of human milk on the adherence of enterotoxigenic Escherichia coli strains to guinea pig intestinal tract was quantitatively determined using radiolabeled bacteria which were incubated with suspensions of viable intestinal cells. Thirteen to 17 bacteria adhered per intestinal cell. NIgF of human milk and colostrum (300 microliter, 6.7 mg) caused about 50% inhibition of the adherence of enterotoxigenic E. coli strains whose attachment was mediated by colonization factor antigen I and II. No inhibition was noted on the adherence of enterotoxigenic E. coli strains containing type I pili. The inhibitory activity resisted boiling and proteolytic digestion with trypsin, but was completely abolished by periodate treatment, indicating that carbohydrate residues were probably involved. Examination of the effect of NIgF of human milk on bacterial adherence to intact intestinal surfaces revealed comparable results. Observations with scanning electron microscopy confirmed, morphologically, the attachment of the bacteria and the inhibitory effect of human milk.(ABSTRACT TRUNCATED AT 250 WORDS)