Allicin, the Odor of Freshly Crushed Garlic: A Review of Recent Progress in Understanding Allicin's Effects on Cells

Abstract

The volatile organic sulfur compound allicin (diallyl thiosulfinate) is produced as a defense substance when garlic (Allium sativum) tissues are damaged, for example by the activities of pathogens or pests. Allicin gives crushed garlic its characteristic odor, is membrane permeable and readily taken up by exposed cells. It is a reactive thiol-trapping sulfur compound that S-thioallylates accessible cysteine residues in proteins and low molecular weight thiols including the cellular redox buffer glutathione (GSH) in eukaryotes and Gram-negative bacteria, as well as bacillithiol (BSH) in Gram-positive firmicutes. Allicin shows dose-dependent antimicrobial activity. At higher doses in eukaryotes allicin can induce apoptosis or necrosis, whereas lower, biocompatible amounts can modulate the activity of redox-sensitive proteins and affect cellular signaling. This review summarizes our current knowledge of how bacterial and eukaryotic cells are specifically affected by, and respond to, allicin.

Keywords: Arabidopsis; DNA gyrase; Haemophilus influenzae; Keap1-Nrf2; TRPA1; Yap1; apoptosis; cancer; glutathione; glutathione reductase; lung pathogens; ornithine decarboxylase; yeast.

Scheme 1

Enzyme-catalyzed biosynthesis of allicin. The internal dipole within the molecule is shown in red.

Scheme 2

Reaction of allicin with thiols. Two mol of reduced glutathione, other low molecular weight thiol, or accessible cysteine thiols in proteins (R—SH), can react with one mol of allicin to yield two mol of S-thioallylated product.

Figure 1

Sensing oxidative stress by specific thiol oxidation in yeast and mammalian cells. The sensing of low M_r electrophilic oxidants that cause oxidative stress is predominantly based on the oxidation of sensitive cysteine thiols, e.g., in Yap1 in yeast and in Keap1-Nrf2 in mammals. Allicin stimulates the expression of OSR genes in both yeast and mammals by a similar mechanism.

Figure 2

The influence of allicin on the organization of tubulin. Three-day-old Arabidopsis seedlings expressing GFP-tagged tubulin were incubated for 10 min in 18.5 mM allicin and subsequently viewed with a confocal laser scanning microscope (Leica, Wetzlar, Germany). While the tubulin filament structure is clearly visible in the water control (A) it takes on a more amorphous appearance after treatment with allicin (B). The scale bar = 20 µm.

Figure 3

Summary of mutants in GSH synthesis and metabolism shown to affect allicin resistance. The diagram shows (A) the interplay between the NADPH-generating steps of the Pentose Phosphate Pathway and (B) GSH synthesis and metabolism. The steps at which the respective deletion mutants (Δzwf1, Δgnd1, Δyap1, Δgsh1, Δgsh2, Δglr1) act in yeast cells are shown in red. Allicin reacts directly with reduced glutathione (GSH) to form S-allylmercaptoglutathione (GSSA). The Pentose Phosphate Pathway provides NADPH as an electron donor for the enzyme glutathione reductase (Glr), that reduces GSSA to GSH and allylmercaptan (ASH, prop-2-ene-1-thiol). Yap1 is the central regulator for the oxidative stress response in yeast and activates several genes important for the antioxidant response, including gsh1 and gsh2 whose protein products catalyze GSH biosynthesis, and glr1 which is necessary for the reduction of GSSG and GSSA to GSH by Glr1. Cysteine (Cys) and glutamine (Glu) are converted to γ-l-glutamyl-l-cysteine by Gsh1, and the addition of glycine (Gly) is catalyzed by Gsh2 to form GSH.

Figure 4

Summary of some of the effects of allicin on bacterial cells. The bacteria are: Ec = E. coli K12 [31,78], Sa = S. aureus USA300 [59,80,87], Bs = Bacillus subtilis strain 168 [60,78], Pa = Pseudomonas aeruginosa PA01 [78], 01 = P. fluorescens 01 [88], AR1 = P. fluorescens AR1 [77]. Abbreviations are: Trx = thioredoxin; AhpD = alkylhydroperoxidase D; DsbA = disulfide bond protein A; GSH = glutathione; GSSG = glutathione disulfide; GSSA = S-allylmercaptoglutathione; BSH = bacillithiol; BSSB = bacillithiol disulfide; Gor = glutathione disulfide reductase; GyrA = DNA gyrase subunit A. The heat and oxidative stress regulons induced under allicin stress in different bacteria are indicated, that are either regulated by S-thioallylation of the redox-sensitive regulators (YodB, OhrR, HypR, CatR) or by other mechanisms, such as increased oxidatively damaged proteins (e.g., RpoH, HrcA, CtsR). Background information about the functions of the transcription factors and their regulons is given in Table S1.

Figure 5

Summary of some of the effects of allicin on eukaryotic cells. Selected proteins are directly S-thioallylated and their biochemical function is thus altered (in most cases inhibition). This manifests itself in a general oxidative stress response, changes in metabolism and the cytoskeleton, an altered immune response and an influence on polyamine metabolism. A direct influence on cell membrane integrity can also be observed. At the same time, cells also respond to allicin exposure, for example through redox-sensitive transcription factors whose activity can be stimulated by allicin. Typical factors are the Nrf2-Keap1 system in mammals or the Yap1 transcription factor in yeast. These lead to the activation of protective mechanisms against oxidative stress. Various defense mechanisms are subsequently activated, with GSH synthesis being of particular importance. Associated with this is the Pentose Phosphate Pathway, in heterotrophic organisms the most important source of NADPH, the electron donor for glutathione reductase (Glr, GR). However, the expression of anti-apoptotic genes is also significant for protection against allicin, as is the phytohormone jasmonic acid in plants. Thioredoxins, especially Trx2, also seem to play a subordinate role, and their contribution is not as marked as that of the glutathione system. At = Arabidopsis thaliana, Cc = Chara corallina, Hs = Homo sapiens, Rd = Rhoeo discolor, Sc = Saccharomyces cerevisiae [24,49,53,63,75,88,90,92].

Figure 6

Inhibition of bacteria by allicin vapor and solutions. (A) Bacteria-seeded agar in a Petri plate inverted over a drop of allicin solution in the lid prevents the growth of the bacteria and leads to a clear zone after incubation. (B) In this variation of the test the inoculum was spread over the surface of agar medium with a Drigalski spatula and allowed to grow overnight at 37 °C to make a lawn on the surface before inverting the plate over a droplet of allicin solution. A replica plate was then prepared from the lawn using a sterile velvet pad and this showed that the growth of bacteria was not only prevented by allicin vapor, but that bacteria in a pre-existing lawn can also be killed outright. (C) Agar diffusion test showing the inhibition of the growth of three strains of lung pathogenic Haemophilus influenzae (strains 2019, R2866, and 86-028 NP) spread over the surface of agar medium with a Drigalski spatula. Allicin solution (20 µL) at 5, 20 or 80 mM was pipetted into wells in the bacteria-seeded agar. (D) Agar plates of the 2019 and R2866 H. influenzae strains were inverted over a 20 µL droplet of allicin solution at 10, 20 or 80 mM (absolute amounts = 32.5, 65, 130, and 260 µg allicin, respectively). Plates were incubated for 17 h at 37 °C.