Allyl Sulfur Research Articles

Astaxanthin‐s‐allyl cysteine diester attenuated high glucose‐induced oxidative stress toxicity in neuronal cells through protection of mitochondria in vitro

AbstractBackgroundCognitive dysfunction associate with diabetes is related to high glucose‐induced oxidative stress toxicity in neuronal cells. Hence, identifying compounds that protect the neuronal cells against high glucose toxicity is important to overcome cognitive deterioration under diabetic condition. Astaxanthin‐s‐allyl cysteine diester (AST‐SAC) was synthesized by esterification of AST from the source H. pluvialis with SAC, an allyl sulfur compound found in aged garlic extract. AST‐SAC has shown antidiabetic effect in in vivo rat model. Hence, in the present study the effect of AST‐SAC against high glucose‐induced oxidative stress toxicity in neuronal cell line in in vitro was studied.MethodAST‐SAC was pre‐treated to neuronal cell line and high glucose toxicity was induced. The cell viability, reactive oxidant species generation, antioxidant status and apoptosis induction was studied. Further, mitochondrial membrane potential, mitochondrial oxidative phosphorylation activity and antioxidant status of mitochondria was measured.ResultHigh glucose‐induced toxicity increased oxidative stress and decreased over all antioxidant status of the neuronal cells. Due to which functions (membrane potential and oxidative phosphorylation) and antioxidant status of mitochondria were reduced. All these dysfunction cumulatively lead to the induction of apoptosis in neuronal cells. However, AST‐SAC pretreatment has protected the neuronal cells against high glucose‐induced apoptosis through reduction of oxidative stress and protection of mitochondrial function.ConclusionOverall AST‐SAC attenuated high‐glucose toxicity induced neuronal injury in in vitro through preservation of mitochondrial function.

Biological interaction of newly synthesized astaxanthin-s-allyl cysteine biconjugate with Saccharomyces cerevisiae and mammalian α-glucosidase: In vitro kinetics and in silico docking analysis

In humans, alpha-glucosidase activity is present in sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM). α-glucosidase is involved in the hydrolyses of disaccharide into monosaccharides and results in hyperglycemia. Subsequently chronic hyperglycemia induces oxidative stress and ultimately leads to the secondary complications of diabetes. Hence, identifying compounds with dual beneficial activity such as efficient antioxidant and α-glucosidase inhibition property has attracted the attention in recent years. Keeping these views, in the present study astaxanthin (AST; a natural antioxidant present in marine microalgae) was biconjugated with allyl sulfur amino acid such as s-allyl cysteine (SAC). The synthesized AST-SAC (with molecular weight of 883.28) was characterized using UV–visible spectrophotometer, ESI-MS, and NMR analysis. AST-SAC showed potent antioxidant property in vitro. AST-SAC inhibited Saccharomyces cerevisiae α-glucosidase (IC50 = 3.98 μM; Ki = 1 μM) and mammalian α-glucosidase [rat intestinal maltase (IC50 = 6.4 μM; Ki = 1.3 μM) and sucrase (IC50 = 1.6 μM; Ki = 0.18 μM)] enzyme activity in a dose-dependent manner. Kinetic analysis revealed that AST-SAC inhibited all the α-glucosidases in a competitive mode. In silico analysis determined the interaction of AST-SAC with the amino acids present in the active site of S. cerevisiae and human (MGAM and SI) α-glucosidases.

Preparation of Pb(II) ion-imprinted polymers and their application in selective removal from wastewater

A novel hydrophilic Pb(II) ion-imprinted polymer (Pb(II)-IIP) was synthesized using lead ion as a template ion, 2-(allyl sulfur) nicotinic acid as functional monomer by precipitation polymerization method. The adsorption capacity of Pb(II)-IIP to Pb(II) was saturated at 16 min, and the adsorption process is consistent with the quasi-second-order kinetic adsorption model. The optimum adsorption capacity of Pb(II)-IIP was 29.67 mg/g, about triple than Pb(II)-NIP, and the adsorption is in accordance with the Langmuir isothermal model, which indicates that it is dominated by single layer chemical adsorption. X-ray Photoelectron Spectroscopy (XPS) and Energy Dispersive Spectrometer (EDS) data confirm that the chelate ratio of Pb(II) to 2-(allylthio) nicotinic acid is 1:2. In the presence of competing ions, the adsorption capacity of Pb(II)-IIP to Pb(II) is much larger than that of other ions, which indicates that Pb(II)-IIP is less disturbed by competitive ions. The experiment of application of practical wastewater shows that Pb(II)-IIP has a Pb(II) removal rate of 97.2% or more in practical industrial wastewater, which is in accordance with national emission standards. It is proved that Pb(II)-IIP is an effective material to remove Pb(II) in industrial wastewater.

Vacuolar H+-ATPase subunit Vma1p functions as the molecular ligand in the vacuole-targeting fungicidal activity of polymyxin B.

Polymyxin B (PMB) is a cationic cyclic peptide that can selectively inhibit the growth of Gram-negative bacteria by disrupting the outer membrane permeability barrier through binding to lipopolysaccharide (LPS). Here, a fluorescent PMB derivative (PMB-Ds) was applied to visually confirm the vacuole as a direct lethal target of PMB against fungal cells, which lack LPS. PMB-Ds could be visualized in the normal rounded vacuolar membrane of Saccharomyces cerevisiae cells, suggesting the presence of a molecular ligand assisting the vacuole-targeting mobilization of the peptide in the organism. Vma1p, a cytoplasmic subunit constituent of the yeast vacuolar-type ATPase, was identified as one of the PMB-binding proteins by means of mass spectrometry. Mutant cells carrying a deletion of Vma1p but not those with deletions in two separate PMB-binding proteins were shown to be resistant to the vacuolar membrane disruptive action of PMB. Furthermore, the mutant cells were resistant to PMB lethality even when treated with PMB in combination with allicin, an allyl sulfur compound, which can selectively enhance the vacuole-targeting fungicidal activity of the peptide. In contrast, the parent cells were not made resistant to the vacuolar membrane disruptive action of PMB even if cells were pre-treated with bafilomycin A1, a specific inhibitor of the yeast vacuolar-type H+-ATPase. However, the parent cells were rendered more resistant to PMB consequent to Vma1p-GFP localization in the cytoplasm. These findings suggested a role for Vma1p in the vacuole-targeting fungicidal activity of PMB comparable to that of LPS in the outer membrane of Gram-negative bacteria.

H2S-releasing nanoemulsions: a new formulation to inhibit tumor cells proliferation and improve tissue repair.

The improvement of solubility and/or dissolution rate of poorly soluble natural compounds is an ideal strategy to make them optimal candidates as new potential drugs. Accordingly, the allyl sulfur compounds and omega-3 fatty acids are natural hydrophobic compounds that exhibit two important combined properties: cardiovascular protection and antitumor activity. Here, we have synthesized and characterized a novel formulation of diallyl disulfide (DADS) and α-linolenic acid (ALA) as protein-nanoemulsions (BAD-NEs), using ultrasounds. BAD-NEs are stable over time at room temperature and show antioxidant and radical scavenging property. These NEs are also optimal H2S slow-release donors and show a significant anti-proliferative effect on different human cancer cell lines: MCF-7 breast cancer and HuT 78 T-cell lymphoma cells. BAD-NEs are able to regulate the ERK1/2 pathway, inducing apoptosis and cell cycle arrest at the G0/G1 phase. We have also investigated their effect on cell proliferation of human adult stem/progenitor cells. Interestingly, BAD-NEs are able to improve the Lin– Sca1+ human cardiac progenitor cells (hCPC) proliferation. This stem cell growth stimulation is combined with the expression and activation of proteins involved in tissue-repair, such as P-AKT, α-sma and connexin 43. Altogether, our results suggest that these antioxidant nanoemulsions might have potential application in selective cancer therapy and for promoting the muscle tissue repair.

Glutathione-garlic sulfur conjugates: slow hydrogen sulfide releasing agents for therapeutic applications.

Natural organosulfur compounds (OSCs) from Allium sativum L. display antioxidant and chemo-sensitization properties, including the in vitro inhibition of tumor cell proliferation through the induction of apoptosis. Garlic water- and oil-soluble allyl sulfur compounds show distinct properties and the capability to inhibit the proliferation of tumor cells. In the present study, we optimized a new protocol for the extraction of water-soluble compounds from garlic at low temperatures and the production of glutathionyl-OSC conjugates during the extraction. Spontaneously, Cys/GSH-mixed-disulfide conjugates are produced by in vivo metabolism of OSCs and represent active molecules able to affect cellular metabolism. Water-soluble extracts, with (GSGaWS) or without (GaWS) glutathione conjugates, were here produced and tested for their ability to release hydrogen sulfide (H2S), also in the presence of reductants and of thiosulfate:cyanide sulfurtransferase (TST) enzyme. Thus, the TST catalysis of the H2S-release from garlic OSCs and their conjugates has been investigated by molecular in vitro experiments. The antiproliferative properties of these extracts on the human T-cell lymphoma cell line, HuT 78, were observed and related to histone hyperacetylation and downregulation of GAPDH expression. Altogether, the results presented here pave the way for the production of a GSGaWS as new, slowly-releasing hydrogen sulfide extract for potential therapeutic applications.

John Austin Milner, PhD (1947–2013)1,2

John Austin Milner was born on 11 June 1947 in Pine Bluff, AR, and died unexpectedly of a heart attack on 31 December 2013. He was a dear friend to so many scientists that his passing is an irreplaceable loss to the nutrition community and to all those who were fortunate enough to know him. John had a formidable capacity for work and for multitasking on many different projects while never turning away requests for help from colleagues, never failing to ask provocative questions, and always answering every single e-mail. He had a great sense of humor and a unique ability to integrate science with humanistic considerations. Although most of his own research focused on the anticancer properties of garlic and associated allyl sulfur compounds, his interests gradually expanded to bioactives of all sorts. He was an internationally respected expert in all areas of nutrition and cancer prevention, including nutrigenomics, selenium nutriture, antioxidants and health, and functional foods. For 16 y, he chaired the popular sessions on Saturday mornings sponsored by the International Life Sciences Institute (ILSI) at the Experimental Biology meetings, and he edited several of them for publication. John A. Milner (1947–2013)

Oxidative species and S‐glutathionyl conjugates in the apoptosis induction by allyl thiosulfate

Natural allyl sulfur compounds show antiproliferative effects on tumor cells, but the biochemical mechanisms underlying the antitumorigenic properties of the organ sulfur compounds are not yet fully understood. Sodium 2-propenyl-thiosulfate is a garlic water-soluble organo-sulfane sulfur compound able to promote apoptosis in cancer cells, affecting the 'managing' of the redox state in the cell. Our studies show that sodium 2-propenyl-thiosulfate reacts spontaneously with reduced glutathione at physiological pH, leading to the formation of S-allyl-mercapto-glutathione, radicals and peroxyl species, which are able to induce inhibition of enzymes with cysteine in the catalytic site, such as sulfurtransferases. S-Allyl-mercapto-glutathione was purified and characterized by NMR and MS, and its cytotoxic effect at 500 μm on HuT 78 cells was analyzed, showing activation of the p38-MAPK pathway. Many allyl sulfur compounds are also able to promote chemoprevention by induction of xenobiotic-metabolizing enzymes, inducing down-activation or detoxification of the carcinogens. Thus, the effects of the S-allyl-mercapto-glutathione on proteins involved in the cellular detoxification system, such as glutathione S-transferase, have been evaluated both in vitro and in HuT 78 cells. Although the antitumor properties of water-soluble sulfur compounds may arise from several mechanisms and it is likely that more cellular events occur simultaneously, a relevant role is played by the formation of both reduced glutathione conjugates and radical species that affect the activity of the thiol-proteins involved in fundamental cellular processes.

Dietary factors and epigenetic regulation for prostate cancer prevention.

The role of epigenetic alterations in various human chronic diseases has gained increasing attention and has resulted in a paradigm shift in our understanding of disease susceptibility. In the field of cancer research, e.g., genetic abnormalities/mutations historically were viewed as primary underlying causes; however, epigenetic mechanisms that alter gene expression without affecting DNA sequence are now recognized as being of equal or greater importance for oncogenesis. Methylation of DNA, modification of histones, and interfering microRNA (miRNA) collectively represent a cadre of epigenetic elements dysregulated in cancer. Targeting the epigenome with compounds that modulate DNA methylation, histone marks, and miRNA profiles represents an evolving strategy for cancer chemoprevention, and these approaches are starting to show promise in human clinical trials. Essential micronutrients such as folate, vitamin B-12, selenium, and zinc as well as the dietary phytochemicals sulforaphane, tea polyphenols, curcumin, and allyl sulfur compounds are among a growing list of agents that affect epigenetic events as novel mechanisms of chemoprevention. To illustrate these concepts, the current review highlights the interactions among nutrients, epigenetics, and prostate cancer susceptibility. In particular, we focus on epigenetic dysregulation and the impact of specific nutrients and food components on DNA methylation and histone modifications that can alter gene expression and influence prostate cancer progression.

Garlic in health and disease.

The present article reviews the historical and popular uses of garlic, its antioxidant, haematological, antimicrobial, hepatoprotective and antineoplastic properties and its potential toxicity (from sulfoxide). Garlic has been suggested to affect several cardiovascular risk factors. It has also been shown that garlic and its organic allyl sulfur components are effective inhibitors of the cancer process. Since garlic and its constituents can suppress carcinogen formation, bioactivation and tumour proliferation, it is imperative that biomarkers be established to identify which individuals might benefit most. Garlic powder, aged garlic and garlic oil have demonstrated antiplatelet and anticoagulant effects by interfering with cyclo-oxygenase-mediated thromboxane synthesis. Garlic has also been found to have synergistic effects against Helicobacter pylori with a proton pump inhibitor. The active compound allicin may affect atherosclerosis not only by acting as an antioxidant, but also by other mechanisms, such as lipoprotein modification and inhibition of LDL uptake and degradation by macrophages. Freshly prepared garlic homogenate protects against isoniazid+rifampicin-induced liver injury in experimental animal models. Several mechanisms are likely to account for this protection.

The Cyclic Organosulfur Compound Zwiebelane A from Onion (Allium cepa) Functions as an Enhancer of Polymyxin B in Fungal Vacuole Disruption

Zwiebelane A (CIS-2,3-dimethyl-5,6-dithiabicyclo[2.1.1]hexane 5-oxide), a natural product of onion bulbs (Allium cepa L.), is found to enhance the potential fungicidal activity of polymyxin B (PMB). As is the case with allicin, an allyl sulfur compound from garlic, zwiebelane A amplifies the disruptive effect of PMB on the vacuole of Saccharomyces cerevisiae, which has been found to represent a target for antifungal agents.

Kimchi lactic acid bacteria and health benefits

Kimchi contains a high number of lactic acid bacteria (108~9/ml), organic acids (lactic, acetic, succinic, propionic acids, etc.), vitamin B complex, and modified functional phytochemicals formed during the fermentation process. Vitamin C, carotenoids, chlorophylls, tocopherol, isothiocyanates, indole‐3‐carbinol, flavonoids, β‐sitosterol, dietary fiber, capsaicinoids, unsaturated fatty acids, allyl sulfur compounds, gingerel, organic acid, lactic acid bacteria, se, etc are the nutraceuticals found in kimchi. The representative kimchi lactic acid bacteria (LAB) reported so far are Leu. mesenteroides, Leu. citreum, Leu. gasicomitatum, Leu. kimchii, Leu. inhae, Weissella Koreensis, Weissella cibaria, Lac. plantarum, Lac. sakei, Lac. delbrueckii, Lac. buchneri, Lac. brevis, Lac. fermentum, Ped. acidilactici and Ped. pentosaceus. LAB from kimchi revealed high preventive capability of the mutation induced by 4‐NQO, MeIQ and Trp‐p2 as much as (or higher than) Lac. acidophilus from dairy products, regardless of their viability. The cell wall fraction, rather than in the cytosol fraction was responsible for the antimutagenic activity of the lactic acid bacteria. Kimchi LAB exhibited antitumor, antimutagenic effect, increase immune function, decrease avian influenza virus, decrease atopy, antiallergy, production of GABA, etc. During the administration of kimchi in humans, the cell counts of Lactobacillus and Leuconostoc increased (p<0.05). The enzymes of β‐glucosidase, β‐glucuronidase and nitroreductase in the colon decreased during the kimchi intake (p<0.05). Kimchi LAB fermentation increased anticancer effect in HT‐29 human colon carcinoma cells. Optimally ripened (OpR) kimchi induced apoptosis as determined by DAPI staining and increased Bax, and decreased in the expression of Bcl‐2.

Allyl sulfur compounds and cellular detoxification system: effects and perspectives in cancer therapy

Natural organosulfur compounds (OSCs) have been shown to have chemopreventive effects and to suppress the proliferation of tumor cells in vitro through the induction of apoptosis. The biochemical mechanisms underlying the antitumorigenic and anti-proliferative effects of garlic-derived OSCs are not fully understood. Several modes of action of these compounds have been proposed, and it seems likely that the rate of clearance of allyl sulfur groups from cells is a determinant of the overall response. The aim of this review is to focus attention on the effects of natural allyl sulfur compounds on the cell detoxification system in normal and tumor cells. It has been already reported that several natural allyl sulfur compounds induce chemopreventive effects by affecting xenobiotic metabolizing enzymes and inducing their down-activation. Moreover, different effects of water- and oil-soluble allyl sulfur compounds on enzymes involved in the detoxification system of rat tissues have been observed. A direct interaction of the garlic allyl sulfur compounds with proteins involved in the detoxification system was studied in order to support the hypothesis that proteins possessing reactive thiol groups and that are involved in the detoxification system and in the cellular redox homeostasis, are likely the preferential targets of these compounds. The biochemical transformation of the OSCs in the cell and their adducts with thiol functional groups of these proteins, could be considered relevant events to uncover the anticancer properties of the allyl sulfur compounds. Although additional studies, using proteomic approaches and transgenic models, are needed to identify the molecular targets and modes of action of these natural compounds, the allyl sulfur compounds can represent potential ideal agents in anticancer therapy, either alone or in association with other antitumor drugs.

In Vitro Investigation of Antifungal Activity of Allicin Alone and in Combination with Azoles Against Candida Species

Candidiasis is a term describing infections by yeasts from the genus Candida, and the type of infection encompassed by candidiasis ranges from superficial to systemic. Treatment of such infections often requires antifungals such as the azoles, but increased use of these drugs has led to selection of yeasts with increased resistance to these drugs. In this study, we used allicin, an allyl sulfur derivative of garlic, to demonstrate both its intrinsic antifungal activity and its synergy with the azoles, in the treatment of these yeasts in vitro. In this study, the MIC(50) and MIC(90) of allicin alone against six Candida spp. ranged from 0.05 to 25 microg/ml. However, when allicin was used in combination with fluconazole or ketoconazole, the MICs were decreased in some isolates. Our results demonstrated the existing synergistic effect between allicin and azoles in some of the Candida spp. such as C. albicans, C. glabrata and C. tropicalis, but synergy was not demonstrated in the majority of Candida spp. tested. Nonetheless, In vivo testing needs to be performed to support these findings.

A gas chromatography–mass spectrometry method for the quantitation of N-nitrosoproline and N-acetyl- S-allylcysteine in human urine: Application to a study of the effects of garlic consumption on nitrosation

Biomarkers in urine can provide useful information about the bioactivation of chemical carcinogens and can be used to investigate the chemoprotective properties of dietary nutrients. N-Nitrosoproline (NPRO) excretion has been used as an index for endogenous nitrosation. In vitro and animal studies have reported that compounds in garlic may suppress nitrosation and inhibit carcinogenesis. We present a new method for extraction and sensitive detection of both NPRO and N-acetyl- S-allylcysteine from urine. The latter is a metabolite of S-allylcysteine, which is found in garlic. Urine was acidified and the organic acids were extracted by reversed-phase extraction (RP–SPE) and use of a polymeric weak anion exchange (WAX–SPE) resin. NPRO was quantified by isotope dilution gas chromatography–mass spectrometry (GC–MS) using [ 13C 5]NPRO and N-nitrosopipecolinic acid (NPIC) as internal standards. This method was used to analyze urine samples from a study that was designed to test whether garlic supplementation inhibits NPRO synthesis. Using this method, 2.4 to 46.0 ng NPRO/ml urine was detected. The method is straightforward and reliable, and it can be performed with readily available GC–MS instruments. N-Acetyl- S-allylcysteine was quantified in the same fraction and detectable at levels of 4.1 to 176.4 ng/ml urine. The results suggest that 3 to 5 g of garlic supplements inhibited NPRO synthesis to an extent similar to a 0.5-g dose of ascorbic acid or a commercial supplement of aged garlic extract. Urinary NPRO concentration was inversely associated with the N-acetyl- S-allylcysteine concentration. It is possible that allyl sulfur compounds found in garlic may inhibit nitrosation in humans.

Garlic: its anticarcinogenic and antitumorigenic properties.

Overall, several investigations indicate that garlic and its organic allyl sulfur components inhibit the cancer process. Furthermore, these studies reveal that the benefits of garlic are not limited to a specific species, a particular tissue, or a specific carcinogen. Finally, odor is not a prerequisite for the protection provided by garlic against the initiation of chemical carcinogenesis. Although the water-soluble compound S-allyl cysteine is effective in reducing the risk of chemically induced tumors in experimental animals, it has no effect on established tumors. However, oil-soluble compounds such as diallyl disulfide are effective in reducing the proliferation of neoplasms. Although the evidence supports the benefits of garlic, additional evidence is needed to determine the quantity needed by humans to minimize cancer risk.

Diallyl disulfide increases histone acetylation in colon cells in vitro and in vivo

Since epidemiological studies have suggested there is an inverse association between garlic consumption and risks of stomach and colorectal cancers,1,2 a large number of experimental studies have been carried out to determine whether allyl sulfur compounds could exhibit anticarcinogenic properties. In particular, diallyl disulfide (DADS) has been shown to inhibit rodent chemically induced carcinogenesis in various organs including colon. Available data reveal that DADS has the ability to modulate numerous biological mechanisms that may influence carcinogenesis (e.g., inhibition of cell proliferation, induction of apoptosis).3,4 It has also been shown that DADS upregulates (H)-ferritin, a gene whose transcription is partly regulated by histone acetylation.5 In our laboratory we, at first, investigated the in vitro effect of DADS on histone acetylation in human tumor colon cell lines. We observed that DADS induced a rapid histone hyperacetylation that was transient with a single treatment or prolonged with a …

Rhodanese–thioredoxin system and allyl sulfur compounds

Sodium 2-propenyl thiosulfate, a water-soluble organo-sulfane sulfur compound isolated from garlic, induces apoptosis in a number of cancer cells. The molecular mechanism of action of sodium 2-propenyl thiosulfate has not been completely clarified. In this work we investigated, by in vivo and in vitro experiments, the effects of this compound on the expression and activity of rhodanese. Rhodanese is a protein belonging to a family of enzymes widely present in all phyla and reputed to play a number of distinct biological roles, such as cyanide detoxification, regeneration of iron-sulfur clusters and metabolism of sulfur sulfane compounds. The cytotoxic effects of sodium 2-propenyl thiosulfate on HuT 78 cells were evaluated by flow cytometry and DNA fragmentation and by monitoring the progressive formation of mobile lipids by NMR spectroscopy. Sodium 2-propenyl thiosulfate was also found to induce inhibition of the sulfurtransferase activity in tumor cells. Interestingly, in vitro experiments using fluorescence spectroscopy, kinetic studies and MS analysis showed that sodium 2-propenyl thiosulfate was able to bind the sulfur-free form of the rhodanese, inhibiting its thiosulfate:cyanide-sulfurtransferase activity by thiolation of the catalytic cysteine. The activity of the enzyme was restored by thioredoxin in a concentration-dependent and time-dependent manner. Our results suggest an important involvement of the essential thioredoxin-thioredoxin reductase system in cancer cell cytotoxicity by organo-sulfane sulfur compounds and highlight the correlation between apoptosis induced by these compounds and the damage to the mitochondrial enzymes involved in the repair of the Fe-S cluster and in the detoxification system.

Amplification of Vacuole-targeting Fungicidal Activity of Antibacterial Antibiotic Polymyxin B by Allicin, an Allyl Sulfur Compound from Garlic

A cationic antibacterial peptide, polymyxin B (PMB), was evaluated as an antifungal antibiotic against various yeasts and filamentous fungi when used in combination with allicin, an allyl sulfur compound from garlic. Allicin was not lethal but could markedly amplify the fungicidal activity of PMB, which was weakly detected with the increase in the plasma membrane permeability in Saccharomyces cerevisiae. Their combined actions caused a dynamic structural damage to the yeast vacuole as judged by the disappearance of its swollen spherical architecture. The vacuole-targeting activity of PMB was similarly amplified in medium with t-butyl hydroperoxide as a substitute for the action of allicin. These findings suggest that the allicin-mediated lipoperoxide production in fungal plasma membrane is the cause of the enhancement in the cellular uptake of PMB as well as its action against the vacuole.

Dependence of Synergistic Fungicidal Activity of Cu2+ and Allicin, an Allyl Sulfur Compound from Garlic, on Selective Accumulation of the Ion in the Plasma Membrane Fraction via Allicin-Mediated Phospholipid Peroxidation

Allicin was effective in decreasing the lethal concentration of Cu (2+) against various fungal strains including a plant pathogen, Fusarium oxysporum, so that the minimum fungicidal concentration (MFC) of the ion for the fungus could be reduced to 2 % of that detected without allicin. In Saccharomyces cerevisiae, Cu (2+) was not apparently taken up by cells when added alone at a non-lethal concentration, whereas the ion was efficiently incorporated into cells in the presence of allicin, as in the case of cells treated with the ion at a lethal concentration. Although allicin likely increased cellular permeability to Cu (2+) due to its promotive effect on plasma membrane phospholipid peroxidation, these cell-surface events did not result in endogenous reactive oxygen species (ROS) production, a typical toxic effect of the ion. Cu (2+) was detected in the cytoplasmic fraction of cells that had been treated with the ion at a lethal concentration, whereas the ion was entrapped in the plasma membrane fraction upon their treatment with the ion at a low concentration in combination with allicin. Cu (2+) could be solubilized from the plasma membrane fraction by a procedure for the extraction of hydrophobic proteins rather than the extraction of phospholipids, suggesting its complexation with a plasma membrane protein as a result of allicin treatment. Such a subcellular localization of Cu (2+) resulted in the selective leakage of intracellular K (+), but not in the disruptive damage on the plasma membrane, and was considered to underlie the synergistic fungicidal activity of Cu (2+) and allicin.