Diallylthiosulfinate (Allicin), a Volatile Antimicrobial from Garlic (Allium
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molecules Article Diallylthiosulfinate (Allicin), a Volatile Antimicrobial from Garlic (Allium sativum), Kills Human Lung Pathogenic Bacteria, Including MDR Strains, as a Vapor Jana Reiter 1, Natalja Levina 2, Mark van der Linden 2, Martin Gruhlke 1, Christian Martin 3 and Alan J. Slusarenko 1,* 1 Department of Plant Physiology, RWTH Aachen University, 52056 Aachen, Germany; [email protected] (J.R.); [email protected] (M.G.) 2 German National Reference Centre of Streptococci (GNRCS), University Hospital RWTH Aachen, 52074 Aachen, Germany; [email protected] (N.L.); [email protected] (M.v.d.L.) 3 Institute of Pharmacology and Toxicology, Medical Faculty of RWTH Aachen University, 52074 Aachen, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-(0)241-802-6650 Received: 13 September 2017; Accepted: 9 October 2017; Published: 12 October 2017 Abstract: 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. Keywords: allicin; garlic; Allium sativum; volatile antimicrobial agent; lung pathogenic bacteria; MDR strains; antimicrobial; Streptococcus pneumoniae; Pseudomonas aeruginosa 1. Introduction The antibacterial activity associated with garlic (Allium sativum L.) was identified by Cavallito in 1944 as being due to diallylthiosulfinate which was given the trivial name allicin [1,2]. In vivo allicin is formed by the catalytic action of alliin-lyase (E.C.4.4.1.4) on alliin (S-allyl-L-cysteine sulfoxide) (Scheme1). Enzyme and substrate are compartmentalized separately in cells and become mixed after mechanical damage. Allicin is the first major sulfur-containing volatile to be produced and gives freshly crushed garlic its typical odor. Allicin has a broad spectrum of cellular targets and it is effective against bacteria, fungi, oomycetes, and protozoa [3–8]. Allicin’s mode of action is still not fully understood [9]. Molecules 2017, 22, 1711; doi:10.3390/molecules22101711 www.mdpi.com/journal/molecules Molecules 2017, 22, 1711 2 of 14 Molecules 2017, 22, 1711 2 of 14 Scheme 1. Enzyme-catalyzed biosynthesisbiosynthesis of allicin. However, allicinallicin is is a reactivea reactive sulfur sulfur species species and undergoesand undergoes a thiol-disulfide a thiol-disulfide type exchange type exchange reaction (Schemereaction 2(Schemea) with available 2a) withthiol available groups, thiol or moregroups, specifically, or more withspecifically, thiolate with ions [thiolate10–12]. ions However, [10–12]. in contrastHowever, to in a contrast standard to thiol-disulfide a standard thiol-disulfide exchange reaction, exchange a moleculereaction, a of molecule water is of generated water is generated from the thiosulfinatefrom the thiosulfinate oxygen atom. oxygen The atom. mixed The disulfide mixed formeddisulfide can formed enter can into enter further into standard further exchangestandard reactionsexchange withreactions fresh with thiols fresh and thiols a redox and cycling a redox cascade cycling can cascade ensue, can which ensue, in cellswhich is alsoin cells driven is also by variousdriven by catalytic various enzymes, catalytic e.g.,enzymes, thioredoxins e.g., thiore anddoxins glutaredoxins and glutaredoxins (Scheme2b). (Scheme Thus, 2b). the effectsThus, the of allicineffects onof cellularallicin thiolon cellular homeostasis thiol ofhomeostasis proteins and of cellular proteins redox and buffers, cellular such redox as glutathione, buffers, such can beas profound.glutathione, For can example, be profound. allicin For reacts example, with accessible allicin reacts cysteines with accessible in proteins cysteines and can in inactivate proteins essential and can enzymesinactivate [13 essential]. Allicin enzymes reacts with [13]. glutathione, Allicin reacts shifts with the glutathione, cell redox potential shifts the to a cell more redox oxidized potential state andto a causesmore oxidized disulfide state stress and [9, 14causes,15]. Indisulfide this regard, stress allicin [9,14, has15]. beenIn this described regard, allicin as a cellular has been redox described toxin [15 as]. Allicina cellular is taken redox up readilytoxin [15]. by cells Allicin and hasis ataken calculated up logPreadily = 1.29by ±cells0.13 [and16]. Thehas abilitya calculated to pass easilylogP = through 1.29 ± 0.13 cell membranes[16]. The ability contributes to pass to easily allicin’s through effectiveness cell membranes as an antibiotic contributes [17]. Furthermore, to allicin’s independentlyeffectiveness as of an its antibiotic chemical reactivity,[17]. Furthermore, allicin’s physicalindependently properties of its allow chemical it to cause reactivity, transient allicin’s pore formationphysical properties in biological allow membranes it to cause and transient artificial pore lipid formation bilayers [ 18in]. biological This effect membranes of allicin on and membranes artificial mightlipid bilayers explain [18]. the synergisticThis effect effectof allicin of allicin on membranes with membrane-active might explain antibiotics the synergistic like amphotericin-B effect of allicin andwith polymixin-B membrane-active [19,20 ].antibiotics like amphotericin-B and polymixin-B [19,20]. Importantly, allicinallicin is is a a volatile volatile substance substance and and can can be be effective effective against against microorganisms microorganisms via thevia gasthe phase.gas phase. Since Since there there are no are volatile no volatile antibiotics antibiotics currently currently available available for clinical for clinical use, this use, makes this makes allicin allicin a very interestinga very interesting candidate candidate molecule. molecule. It therefore It makestherefore sense makes to develop sense newto develop strategies new using strategies allicin-based using drugs,allicin-based perhaps drugs, combined perhaps with combined other conventional with other antibiotics, conventional for antibiotics, direct treatment for direct of lung treatment infections of vialung the infections pulmonary via ratherthe pulmonary than the oralrather route. than In the this oral regard, route. the In this successful regard, treatment the successful of tuberculosis treatment patientsof tuberculosis by inhalation patients of by garlic inhalation vapor hasof garlic been vapor reported hasin been the re ‘pre-antibiotic’ported in the ‘pre-antibiotic’ era [21]. era [21]. The antimicrobialantimicrobial effect effect of garlicof garlic or allicin or allicin was already was already documented documented for several for human several pathogenic human bacteriapathogenicin vitrobacteriaand inin animalvitro and trials in [9 ,animal22–24]. trials Generally, [9,22–24]. these Generally, reports have these been reports restricted have to abeen few particularrestricted bacterialto a few species,particular rather bact thanerial a species, systematic rather treatment than a of syst a pathogenicematic treatment group, forof examplea pathogenic lung pathogenicgroup, for example bacteria, lung as we pathogenic report here. bacteria, as we report here. Currently, thethe emergenceemergence ofof bacteriabacteria pan-resistantpan-resistant to current clinical antibiotics is a threat to effective treatmenttreatment ofof infectiousinfectious disease.disease. Resistance to penicillin, the firstfirst commerciallycommercially availableavailable antibiotic, was was already already documented documented in in 1940 1940 before before its itsrelease release into into clinical clinical practice practice in 1943. in 1943.The Theappearance appearance of resistance, of resistance, rapidly rapidly following following discovery discovery or introduction or introduction into into clinical clinical practice, practice, is also is also the thetrend trend for later for later antibiotics antibiotics [25,26]. [25 ,The26]. majority The majority of antibiotic of antibiotic classes classes and drugs and drugswere discovered were discovered before beforethe 1970s the and 1970s there and have there been have few been new few discov new discoverieseries reported reported since (Figure since (Figure 1) [27,28].1)[27