Martin C.H. Gruhlke

Allicin: chemistry and biological properties.

Allicin (diallylthiosulfinate) is a defence molecule from garlic (Allium sativum L.) with a broad range of biological activities. Allicin is produced upon tissue damage from the non-proteinogenic amino acid alliin (S-allylcysteine sulfoxide) in a reaction that is catalyzed by the enzyme alliinase. Current understanding of the allicin biosynthetic pathway will be presented in this review. Being a thiosulfinate, allicin is a reactive sulfur species (RSS) and undergoes a redox-reaction with thiol groups in glutathione and proteins that is thought to be essential for its biological activity. Allicin is physiologically active in microbial, plant and mammalian cells. In a dose-dependent manner allicin can inhibit the proliferation of both bacteria and fungi or kill cells outright, including antibiotic-resistant strains like methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, in mammalian cell lines, including cancer cells, allicin induces cell-death and inhibits cell proliferation. In plants allicin inhibits seed germination and attenuates root-development. The majority of allicin’s effects are believed to be mediated via redox-dependent mechanisms. In sub-lethal concentrations, allicin has a variety of health-promoting properties, for example cholesterol- and blood pressure-lowering effects that are advantageous for the cardio-vascular system. Clearly, allicin has wide-ranging and interesting applications in medicine and (green) agriculture, hence the detailed discussion of its enormous potential in this review. Taken together, allicin is a fascinating biologically active compound whose properties are a direct consequence of the molecule’s chemistry.

The biology of reactive sulfur species (RSS).

Sulfur is an essential and quantitatively important element for living organisms. Plants contain on average approximately 1 g S kg⁻¹ dry weight (for comparison plants contain approximately 15 g N kg⁻¹ dry weight). Sulfur is a constituent of many organic molecules, for example amino acids such as cysteine and methionine and the small tripeptide glutathione, but sulfur is also essential in the form of Fe-S clusters for the activity of many enzymes, particularly those involved in redox reactions. Sulfur chemistry is therefore important. In particular, sulfur in the form of thiol groups is central to manifold aspects of metabolism. Because thiol groups are oxidized and reduced easily and reversibly, the redox control of cellular metabolism has become an increasing focus of research. In the same way that oxygen and nitrogen have reactive species (ROS and RNS), sulfur too can form reactive molecular species (RSS), for example when a -SH group is oxidized. Indeed, several redox reactions occur via RSS intermediates. Several naturally occurring S-containing molecules are themselves RSS and because they are physiologically active they make up part of the intrinsic plant defence repertoire against herbivore and pathogen attack. Furthermore, RSS can also be used as redox-active pharmacological tools to study cell metabolism. The aim of this review is to familiarize the general reader with some of the chemical concepts, terminology and biology of selected RSS.

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.

Intracellular Diagnostics: Hunting for the Mode of Action of Redox-Modulating Selenium Compounds in Selected Model Systems

Redox-modulating compounds derived from natural sources, such as redox active secondary metabolites, are currently of considerable interest in the field of chemoprevention, drug and phytoprotectant development. Unfortunately, the exact and occasionally even selective activity of such products, and the underlying (bio-)chemical causes thereof, are often only poorly understood. A combination of the nematode- and yeast-based assays provides a powerful platform to investigate a possible biological activity of a new compound and also to explore the “redox link” which may exist between its activity on the one side and its chemistry on the other. Here, we will demonstrate the usefulness of this platform for screening several selenium and tellurium compounds for their activity and action. We will also show how the nematode-based assay can be used to obtain information on compound uptake and distribution inside a multicellular organism, whilst the yeast-based system can be employed to explore possible intracellular mechanisms via chemogenetic screening and intracellular diagnostics. Whilst none of these simple and easy-to-use assays can ultimately substitute for in-depth studies in human cells and animals, these methods nonetheless provide a first glimpse on the possible biological activities of new compounds and offer direction for more complicated future investigations. They may also uncover some rather unpleasant biochemical actions of certain compounds, such as the ability of the trace element supplement selenite to induce DNA strand breaks.

The Effects of Allicin, a Reactive Sulfur Species from Garlic, on a Selection of Mammalian Cell Lines

Garlic (Allium sativum L.) has been used as a spice and medicinal plant since ancient times. Garlic produces the thiol-reactive defence substance, allicin, upon wounding. The effects of allicin on human lung epithelium carcinoma (A549), mouse fibroblast (3T3), human umbilical vein endothelial cell (HUVEC), human colon carcinoma (HT29) and human breast cancer (MCF7) cell lines were tested. To estimate toxic effects of allicin, we used a standard MTT-test (methylthiazoltetrazolium) for cell viability and 3H-thymidine incorporation for cell proliferation. The glutathione pool was measured using monobromobimane and the formation of reactive species was identified using 2′,7′-dichlorofluoresceine-diacetate. The YO-PRO-1 iodide staining procedure was used to estimate apoptosis. Allicin reduced cell viability and cell proliferation in a concentration dependent manner. In the bimane test, it was observed that cells treated with allicin showed reduced fluorescence, suggesting glutathione oxidation. The cell lines tested differed in sensitivity to allicin in regard to viability, cell proliferation and glutathione oxidation. The 3T3 and MCF-7 cells showed a higher proportion of apoptosis compared to the other cell types. These data show that mammalian cell lines differ in their sensitivity and responses to allicin.

The defense substance allicin from garlic permeabilizes membranes of Beta vulgaris, Rhoeo discolor, Chara corallina and artificial lipid bilayers

BACKGROUND Allicin (diallylthiosulfinate) is the major volatile- and antimicrobial substance produced by garlic cells upon wounding. We tested the hypothesis that allicin affects membrane function and investigated 1) betanine pigment leakage from beetroot (Beta vulgaris) tissue, 2) the semipermeability of the vacuolar membrane of Rhoeo discolor cells, 3) the electrophysiology of plasmalemma and tonoplast of Chara corallina and 4) electrical conductivity of artificial lipid bilayers. METHODS Garlic juice and chemically synthesized allicin were used and betanine loss into the medium was monitored spectrophotometrically. Rhoeo cells were studied microscopically and Chara- and artificial membranes were patch clamped. RESULTS Beet cell membranes were approximately 200-fold more sensitive to allicin on a mol-for-mol basis than to dimethyl sulfoxide (DMSO) and approximately 400-fold more sensitive to allicin than to ethanol. Allicin-treated Rhoeo discolor cells lost the ability to plasmolyse in an osmoticum, confirming that their membranes had lost semipermeability after allicin treatment. Furthermore, allicin and garlic juice diluted in artificial pond water caused an immediate strong depolarization, and a decrease in membrane resistance at the plasmalemma of Chara, and caused pore formation in the tonoplast and artificial lipid bilayers. CONCLUSIONS Allicin increases the permeability of membranes. GENERAL SIGNIFICANCE Since garlic is a common foodstuff the physiological effects of its constituents are important. Allicins ability to permeabilize cell membranes may contribute to its antimicrobial activity independently of its activity as a thiol reagent.

Diallylthiosulfinate (Allicin), a Volatile Antimicrobial from Garlic (Allium sativum), Kills Human Lung Pathogenic Bacteria, Including MDR Strains, as a Vapor

Garlic (Allium sativum) has potent antimicrobial activity due to allicin (diallylthiosulfinate) synthesized by enzyme catalysis in damaged garlic tissues. Allicin gives crushed garlic its characteristic odor and its volatility makes it potentially useful for combating lung infections. Allicin was synthesized (>98% pure) by oxidation of diallyl disulfide by H2O2 using formic acid as a catalyst and the growth inhibitory effect of allicin vapor and allicin in solution to clinical isolates of lung pathogenic bacteria from the genera Pseudomonas, Streptococcus, and Staphylococcus, including multi-drug resistant (MDR) strains, was demonstrated. Minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were determined and compared to clinical antibiotics using standard European Committee on Antimicrobial Susceptibility Testing (EUCAST) procedures. The cytotoxicity of allicin to human lung and colon epithelial and murine fibroblast cells was tested in vitro and shown to be ameliorated by glutathione (GSH). Similarly, the sensitivity of rat precision-cut lung slices (PCLS) to allicin was decreased by raising the [GSH] to the approximate blood plasma level of 1 mM. Because allicin inhibited bacterial growth as a vapor, it could be used to combat bacterial lung infections via direct inhalation. Since there are no volatile antibiotics available to treat pulmonary infections, allicin, particularly at sublethal doses in combination with oral antibiotics, could make a valuable addition to currently available treatments.

Yap1p, the central regulator of the S. cerevisiae oxidative stress response, is activated by allicin, a natural oxidant and defence substance of garlic

Abstract Allicin is a thiol‐reactive sulfur‐containing natural product from garlic with a broad range of antimicrobial effects against prokaryotes and eukaryotes. Previous work showed that the S. cerevisiae OSI1 gene is highly induced by allicin and other thiol‐reactive compounds, and in silico analysis revealed multiple Yap1p binding motifs in the OSI1 promoter sequence. An OSI1‐promoter::luciferase reporter construct expressed in Wt and &Dgr;yap1 cells showed absolute Yap1p‐dependence for allicin‐induced OSI1‐expression. A GFP::Yap1p fusion protein accumulated in the nucleus within 10 min of allicin treatment and a &Dgr;yap1 mutant was highly sensitive to allicin. Yap1p regulates glutathione (GSH) metabolism genes, and &Dgr;gsh1, &Dgr;gsh2 and &Dgr;glr1 mutants showed increased sensitivity to allicin. Allicin activated the OSI1‐promoter::luciferase reporter construct in &Dgr;gpx3 and &Dgr;ybp1 cells, indicating that allicin activates Yap1p directly rather than via H2O2 production. A systematic series of C‐to‐A Yap1p exchange mutants showed that the C‐term C598 and C620 residues were necessary for allicin activation. These data suggest that Yap1p is an important transcriptional regulator for the resistance of yeast cells to allicin, and that activation occurs by direct modification of C‐term cysteines as shown for other electrophiles. Graphical abstract Figure. No Caption available. HighlightsYap1 mutants are hypersensitive to allicin.Allicin causes Yap1p to accumulate in the nucleus within 10 min of treatment.Allicin activates Yap1p directly at cys598 and cys620, independently of Gpx3p and Ybp1p.First demonstration of direct Yap1p activation by a natural product from a common foodstuff.OSI1 gene (YKL071W) is induced by allicin in a Yap1p‐dependent manner.

Effect of silver nanoparticles on the standard soil arthropod Folsomia candida (Collembola) and the eukaryote model organism Saccharomyces cerevisiae

BackgroundBecause of their antimicrobial properties, silver nanoparticles (AgNPs) have been widely used and have come into contact with the environment. In the present work, an effect of AgNPs on a standard soil organism, Folsomia candida, was studied (in comparison to silver nitrate) focusing on molecular and cellular alterations as ecotoxicological endpoints.ResultsAt the molecular level, an up-regulation of metallothionein-containing protein (MTC) mRNA in AgNP-treated groups indicated toxic heavy metal stress effects caused by the release of silver ions from AgNPs, which is similar to animal groups treated with silver nitrate. Alteration of the steady-state level of glutathione S-transferase (GST) mRNA was detected in animal treated with AgNPs and AgNO3. At the cellular level, the relation between GST activity and the size of the glutathione (GSH) was examined. Change of GST activity from different animal groups was not significant, whereas the GSH pool (reduced and oxidized forms) decreased with increasing concentration of AgNPs. In order to obtain direct evidence whether AgNPs cause oxidative stress, treated animals were incubated with the non-fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). A fluorescence signal was observed in both AgNPs- and AgNO3-treated groups pointing to the production of reactive species (RS). Since RS formation in F.candida is difficult to quantify, yeast strain BY4742 (wild-type) and mutants lacking of oxidative stress-related protective enzymes were exploited as a further eukaryote model organism. AgNPs and AgNO3 were found to also affect growth of yeast and induced oxidative stress.ConclusionsAn effect of AgNPs on Collembola and yeast strains is similar to the one from AgNO3. However, AgNPs is less toxic due to the slow release of silver ions. In summary, the toxic effect of AgNPs on F. candida is caused by the combination of the release of silver ions from AgNPs and the formation of reactive species.

The Cellular ‘Thiolstat’ as an Emerging Potential Target of Some Plant Secondary Metabolites

Glutathione (GSH) is quantitatively the major thiol compound in animal and plant cells and in many prokaryotes. In unstressed cells, glutathione is almost exclusively in the reduced thiol form GSH and serves to buffer the cellular redox balance against detrimental changes. Thus, it can absorb oxidative insults to the cell by being oxidized to glutathione disulfide (GSSG) and by reversibly glutathiolating –SH groups in proteins to protect them from further oxidation. Under varying oxidizing- and reducing conditions, not only GSH but many other thiol-containing molecules in cells can undergo reversible conversion between the R-SH and R-S-S-R’ forms. Targeted oxidation of –SH groups in regulatory and signalling proteins helps the cell recognize oxidative stress and sets in motion the coordinated expression of genes involved in anti-oxidative stress responses. In addition to this targeted infrastructure however, the concentrations of reduced and oxidized GSH and other thiol compounds determine the electrochemical cell potential (redox potential) and this has an indiscriminate global effect on the redox status of many cellular proteins. It is becoming increasingly recognized that the dynamic equilibrium between reduced and oxidized thiol-containing compounds in cells is an important regulator of the cell’s physiology. The proportion of oxidized to reduced thiol groups in the cell is intimately linked with the cell’s redox status and changes in this ‘thiolstat’ are monitored and linked to signal perception and transduction. This marks the thiolstat as an important target for redox active compounds, including many physiologically active secondary metabolites from plants, bacteria, fungi, and other organisms. Here, we provide an overview of how the redox environment of the cell is controlled and how selected secondary metabolites are able to affect the cellular thiolstat and we illustrate some of the known biochemical consequences for the cell of tweaking the thiolstat.