molecules Review Allicin, the Odor of Freshly Crushed Garlic: A Review of Recent Progress in Understanding Allicin’s Effects on Cells § Jan Borlinghaus 1,*, Jana Foerster (née Reiter) 1, Ulrike Kappler 2, Haike Antelmann 3 , Ulrike Noll 1, Martin C. H. Gruhlke 1 and Alan J. Slusarenko 1,* 1 Department of Plant Physiology, RWTH Aachen University, 52056 Aachen, Germany; [email protected] (J.F.); [email protected] (U.N.); [email protected] (M.C.H.G.) 2 School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, QLD 4072, Australia; [email protected] 3 Institute of Biology-Microbiology, Freie Universität Berlin, 14195 Berlin, Germany; [email protected] * Correspondence: [email protected] (J.B.); [email protected] (A.J.S.) § Dedication: This article is dedicated to Professor emeritus Dr. Eric Block on his 79th birthday in recognition of his lifelong contributions to, and enthusiastic support of, research on the chemistry and biological effects of sulfur-containing natural compounds. Abstract: The volatile organic sulfur compound allicin (diallyl thiosulfinate) is produced as a de- fense 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 Citation: Borlinghaus, J.; Foerster, J.; accessible cysteine residues in proteins and low molecular weight thiols including the cellular redox Kappler, U.; Antelmann, H.; Noll, U.; buffer glutathione (GSH) in eukaryotes and Gram-negative bacteria, as well as bacillithiol (BSH) in Gruhlke, M.C.H.; Slusarenko, A.J. Gram-positive firmicutes. Allicin shows dose-dependent antimicrobial activity. At higher doses in Allicin, the Odor of Freshly Crushed eukaryotes allicin can induce apoptosis or necrosis, whereas lower, biocompatible amounts can mod- Garlic: A Review of Recent Progress ulate the activity of redox-sensitive proteins and affect cellular signaling. This review summarizes in Understanding Allicin’s Effects on our current knowledge of how bacterial and eukaryotic cells are specifically affected by, and respond Cells §. Molecules 2021, 26, 1505. to, allicin. https://doi.org/10.3390/ molecules26061505 Keywords: TRPA1; Yap1; Keap1-Nrf2; DNA gyrase; glutathione; glutathione reductase; lung pathogens; Academic Editor: Michiho Ito Haemophilus influenzae; apoptosis; cancer; ornithine decarboxylase; Arabidopsis; yeast Received: 16 February 2021 Accepted: 6 March 2021 Published: 10 March 2021 1. Introduction Garlic (Allium sativum) is an internationally appreciated culinary ingredient in many Publisher’s Note: MDPI stays neutral dishes and an economically important agricultural crop in several countries. It is well with regard to jurisdictional claims in known that when an intact garlic clove is chopped or crushed it instantly releases a typical published maps and institutional affil- pungent bouquet or odor. The characteristic odor of garlic is due to volatile organic sulfur- iations. containing compounds (VOSCs) that are rapidly produced upon tissue damage and diffuse into the air. The major contributor to the odor of freshly crushed garlic is allicin (diallyl thio- sulfinate, preferred IUPAC name = S-prop-2-en-1-yl prop-2-ene-1-sulfinothioate). Garlic releases VOSCs to defend itself against attacks by pathogens and pests and its antibacterial Copyright: © 2021 by the authors. principle was identified as allicin by Cavallito in 1944 [1,2]. In vivo allicin forms after Licensee MDPI, Basel, Switzerland. cellular damage when the enzyme alliinase, which in intact cells is localized in the vac- This article is an open access article uole, mixes with the substrate alliin which is located in the cytosol (Scheme1)[3–5]. Alliin distributed under the terms and (S-allylcysteine sulfoxide) is an odorless non-protein amino acid. It has been reported that conditions of the Creative Commons alliinase is predominantly located in bundle sheath cells in bulbs, leaves, and inflorescence Attribution (CC BY) license (https:// axes in the garlic plant [6]. Other alkyl-cysteine sulfoxides are also present in the cytoplasm creativecommons.org/licenses/by/ of garlic cells and can serve as alliinase substrates, yielding amongst others, dimethyl, 4.0/). Molecules 2021, 26, 1505. https://doi.org/10.3390/molecules26061505 https://www.mdpi.com/journal/molecules MoleculesMolecules 20212021, 26, 26, ,x 1505 FOR PEER REVIEW 2 of 22 2 of 22 dipropyl and mixed alkyl thiosulfinates. Nevertheless, allicin accounts for 60–80% of the accounts for 60–80% of the total thiosulfinates formed by damaged garlic [3]. Alliinase (S‐ total thiosulfinates formed by damaged garlic [3]. Alliinase (S-alkyl-L-cysteine S-oxide alkyl-sulfenate-lyase,alkyl‐L‐cysteine S‐oxide E.C. 4.4.1.4) alkyl‐sulfenate readily converts‐lyase, itsE.C. substrates 4.4.1.4) toreadily the corresponding converts its substrates sulfenicto the acids, corresponding which condense sulfenic spontaneously acids, to producewhich thiosulfinatescondense spontaneously (Scheme1)[ 7]. The to produce reactionsthiosulfinates are very (Scheme rapid and 1) are [7]. 97% The complete reactions within are 30 very s at 23rapid◦C. Furthermore,and are 97% a complete single within clove30 s at of garlic23 °C. (approximately Furthermore, 10 a g)single can produce clove of up garlic to 5 mg (approximately of allicin, illustrating 10 g) thecan large produce up to evolutionary5 mg of allicin, investment illustrating the plant the places large in this evolutionary defense molecule investment [8]. An easy the and plant efficient places in this indefense vitro synthesis molecule protocol [8]. An for easy >98% and pure efficient allicin in and vitro approximately synthesis protocol 90% yield for has >98% been pure allicin published [9]. and approximately 90% yield has been published [9]. Scheme 1. Enzyme-catalyzed biosynthesis of allicin. The internal dipole within the molecule is shown in red. Scheme 1. Enzyme‐catalyzed biosynthesis of allicin. The internal dipole within the molecule is shown in red. The odor of crushed garlic is usually described as ‘pungent’, which the shorter Oxford EnglishThe dictionary odor of defines crushed as “biting, garlic caustic is usually or piquant, described and affects as the ‘pungent’, skin and the which organs the shorter ofOxford taste and English smell with dictionary a prickling defines sensation” as “biting, [10]. VOSCs caustic are or known piquant, for their and intense affects and the skin and uniquethe organs olfactory of taste properties, and smell in many with cases a prickling being detectable sensation” by some [10]. animal VOSCs species are in known ppb. for their Even the relatively insensitive human nose can detect VOSCs in the low ppm range [7,11]. intense and unique olfactory properties, in many cases being detectable by some animal Garlic-derived preparations were shown to have insect-repellent effects and to dis- couragespecies the in feedingppb. Even of some the birds, relatively for example insensitive European human starlings. nose The can garlic detect preparations VOSCs in the low testedppm againstrange starlings[7,11]. contained mixed diallyl, dimethyl and allyl-methyl polysulfanes but no allicinGarlic [12].‐derived Nevertheless, preparations allicin has repellentwere shown effects onto herbivores have insect and‐ hasrepellent been shown effects and to todiscourage stimulate a subsetthe feeding of TRPA1 of (transientsome birds, receptor for potential) example non-selective European ionstarlings. channels The garlic inpreparations pain-sensing tested (nociceptive) against neurons starlings [13,14 contained]. The TRPA1 mixed ion channel, diallyl, alsodimethyl known and as the allyl‐methyl ‘wasabipolysulfanes receptor’, but is activated no allicin by a[12]. wide Nevertheless, range of electrophiles allicin including has repellent allyl isothiocyanate, effects on herbivores the active ingredient of wasabi [15]. Allyl isothiocyanate activates TRPA1 by covalent mod- ificationand has of been a cysteine shown residue to stimulate within the a cytoplasmicsubset of TRPA1 N terminus (transient in TRPA1 receptor [16]. Due potential) to non‐ itsselective thiol-reactivity ion channels (see later), in it pain seems‐sensing likely that (nociceptive) allicin acts similarly neurons when [13,14]. it stimulates The TRPA1 ion TRPA1-bearingchannel, also known neurons. as Furthermore, the ‘wasabi it receptor’, was shown is to activated be the same by a particular wide range subset of ofelectrophiles TRPA1including neurons allyl that isothiocyanate, were stimulated the by active both allyl ingredient isothiocyanate of wasabi and allicin [15]. [ 13Allyl]. Activa- isothiocyanate tionactivates of TRPA1 TRPA1 results by in covalent local inflammation modification and pain of a and cysteine can evoke residue innate within fear behavior the cytoplasmic in N theterminus sensory in host, TRPA1 and it is[16]. no surpriseDue to thereforeits thiol‐ thatreactivity allicin acts (see as later), an effective it seems deterrent likely of that allicin herbivory [11,17]. In addition, plants can influence one another and other organisms by acts similarly when it stimulates TRPA1‐bearing neurons. Furthermore, it was shown to using volatile signals which act as allelochemicals, i.e., secondary products that affect the growth,be the survivalsame particular or reproduction