Mechanism of active acetylcholinesterase inhibition by organic sulfanes in garlic: Non-covalent binding and covalent modifications.

Numerous secondary metabolites in medicinal food homology plants such as Allium inhibit the activity of acetylcholinesterase (AChE), but the current understanding of the inhibition mechanism is limited. In this study, we employed ultrafiltration, spectroscopic, molecular docking, and matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) techniques to investigate the inhibition mechanism of AChE by garlic organic sulfanes, including diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). The results of UV-spectrophotometry and ultrafiltration experiments showed the inhibition of AChE activity by DAS and DADS was reversible (competitive inhibition), but inhibition by DATS was irreversible. Molecular fluorescence and molecular docking indicated DAS and DADS changed the positions of key amino acids inside the catalytic cavity through hydrophobic interactions with AChE. By using MALDI-TOF-MS/MS, we found DATS irreversibly inhibited AChE activity by opening disulfide-bond switching of disulfide bond 1 (Cys-69 and Cys-96) and disulfide bond 2 (Cys-257 and Cys-272) in AChE, as well as by covalently modifying Cys-272 in disulfide bond 2 to generate AChE-SSA derivatives (strengthened switch). This study provides a basis for further exploration of natural AChE inhibitors using organic active substances in garlic and presents a hypothesis of U-shaped spring force arm effect based on the disulfide bond-switching reaction of DATS that can be used to evaluate the stability of disulfide bonds in proteins.

Diallyl Trisulfide, the Antifungal Component of Garlic Essential Oil and the Bioactivity of Its Nanoemulsions Formed by Spontaneous Emulsification.

The aim of this study was to evaluate the chemical compounds of garlic essential oil (EO), and determine the antifungal efficacy of garlic EO and its major components, diallyl trisulfide and its nanoemulsions against wood-rotting fungi, Trametes hirsuta and Laetiporus sulphureus. GC-MS analysis revealed that the major constituents of garlic EO were diallyl trisulfide (39.79%), diallyl disulfide (32.91%), and diallyl sulfide (7.02%). In antifungal activity, the IC50 value of garlic EO against T. hirsuta and L. sulphureus were 137.3 and 44.6 µg/mL, respectively. Results from the antifungal tests demonstrated that the three major constituents were shown to have good antifungal activity, in which, diallyl trisulfide was the most effective against T. hirsuta and L. sulphureus, with the IC50 values of 56.1 and 31.6 µg/mL, respectively. The diallyl trisulfide nanoemulsions showed high antifungal efficacy against the examined wood-rotting fungi, and as the amount of diallyl trisulfide in the lipid phase increases, the antifungal efficacy of the nanoemulsions increases. These results showed that the nanoemulsions and normal emulsion of diallyl trisulfide have potential to develop into a natural wood preservative.

Diallyl disulphide-loaded spherical gold nanoparticles and acorn-like silver nanoparticles synthesised using onion extract: catalytic activity and cytotoxicity.

Onion (Allium cepa) extract was used for the green synthesis of gold and silver nanoparticles. Each colloidal solution exhibited surface plasmon resonance, with a peak at 532 nm for gold nanoparticles and 391 nm for silver nanoparticles. Microscopic results confirmed the presence of spherical shapes. The X-ray diffraction pattern demonstrated a face-centered cubic structure. Both nanoparticles had negative zeta potentials and retained colloidal stability in cell culture medium. Catalytic applications were evaluated for 4-nitrophenol reduction and methyl orange degradation reactions by monitoring with UV-visible spectrophotometry. Furthermore, the nanoparticles demonstrated no significant cytotoxicity against human pancreas ductal adenocarcinoma cells (PANC-1) and human colorectal adenocarcinoma cells (HT-29). PEGylation and diallyl disulphide loading of the gold and silver nanoparticles meaningfully reduced the cell viability of both cell lines. Furthermore, diallyl disulphide loading resulted in more cytotoxicity against PANC-1 cells than against HT-29 cells. Additionally, the gold nanoparticles were more cytotoxic than the silver nanoparticles upon diallyl disulphide loading. Interestingly, after PEGylation and diallyl disulphide loading, the silver nanoparticles exhibited acorn-like shapes, while the gold nanoparticles retained spherical shapes. This result suggested that nanoparticles green-synthesised by onion extract have possibilities as nanocatalysts and drug delivery nanocarriers for catalytic and nanomedicine applications.

H2S-Donating trisulfide linkers confer unexpected biological behaviour to poly(ethylene glycol)-cholesteryl conjugates.

Inspired by the properties of the naturally occurring H2S donor, diallyl trisulfide (DATS, extracted from garlic), the biological behaviour of trisulfide-bearing PEG-conjugates was explored. Specifically, three conjugates comprising an mPEG tail and a cholesteryl head were investigated: conjugates bridged by a trisulfide linker (T), a disulfide linker (D) or a carbamate linker (C), and a fourth comprising two mPEG tails bridged by a trisulfide linker (P). H2S testing using both a fluorescent chemical probe in HEK293 cells and an amperometric sensor to monitor release in suspended cells, demonstrated the ability of the trisulfide conjugates, T and P, to release H2S in the presence of cellular thiols. Cytotoxicity and cyto-protective capacity on HEK293 cells showed that T was the best tolerated of the conjugates studied, and remarkably more so than D or C. Moreover, it was noted that application of T conferred a protective effect to the cells, effectively abolishing the toxicity associated with co-administered C. The interaction of conjugates and combinations thereof with the cell membrane of HEK cells, as well as ROS generation were also investigated. It was found that C caused significant membrane perturbation, correlating with high losses in cell viability and pronounced generation of ROS, especially in the mitochondria. T, however, did not disturb the membrane and was able to mitigate the generation of ROS, especially in the mitochondria. The interplay of the cholesteryl group and H2S donation for conferring cytoprotective effects was clearly demonstrated as P did not display the same beneficial characteristics as T.

Dillapiole in Piper holtonii as an Inhibitor of the Symbiotic Fungus Leucoagaricus gongylophorus of Leaf-Cutting Ants.

Plants of the Piperaceae family are studied for their diverse secondary metabolism with a vast array of compounds that act as chemical defense agents against herbivores. Of all the agricultural pests, the management of insects is a highly significant challenge in the Neotropics, and ants of the Attini tribe pose a major problem. Due to their symbiotic association with the fungus Leucoagaricus gongylophorus (Möller) Singer (Agaricaceae), the species of Atta and Acromyrmex have exhaustive foraging activity which has intensified as deforestation and monoculture farming have increased. The control of leaf-cutting ants is still carried out with synthetic products with negative consequences to the environment and human health. In search for natural and sustainable alternatives to synthetic pesticides, Piper holtonii C. DC. was selected among other plant species after field observations of the foraging activity of Atta cephalotes, which revealed that P. holtonii was never chosen by ants. In vitro evaluation of an ethanol extract of the leaves of P. holtonii resulted in promising inhibitory activity (IC50 102 ppm) against L. gongylophorus. Subsequently, bioassay-guided fractionation led to the isolation of the phenylpropanoid dillapiole, which was also detected in the essential oil. This compound demonstrated inhibition of the fungus with an IC50 of 38 ppm. Considering the symbiotic relationship between the Attini ants and L. gongylophorus, the negative effect on the survival of one of the organisms will affect the survival of the other, so dillapiole or standardized essential oil extracts of P. holtonii containing this active principle could be a unique and useful source as a control agent for leaf cutting-ants.

Diallyl Trisulfide Inhibits Platelet Aggregation through the Modification of Sulfhydryl Groups.

Diallyl trisulfide (DATS) is a secondary metabolite of allicin, a volatile organosulfur flavoring compound generated by the crushing of garlic. These compounds have various medicinal effects such as antiplatelet activity. In this study, we demonstrated for the first time the cellular mechanism involved in the inhibition of platelet aggregation by DATS and dipropyl trisulfide (DPTS), which is a saturated analogue of DATS. Washed murine platelets were incubated with these sulfides, and platelet aggregation was evaluated by light transmission aggregometry. The amount of reaction products produced by DATS, DPTS, and glutathione (GSH) was measured using liquid chromatography-mass spectrometry. Compared with DPTS, DATS potently inhibited platelet aggregation induced by thrombin, U46619, and collagen. N-Ethylmaleimide (NEM), which is commonly used to modify sulfhydryl groups, also suppressed platelet aggregation. The reactivity of DATS with GSH was higher than that of DPTS. These data suggested that DATS inhibited platelet aggregation through the reaction of sulfhydryl groups.

Measurement of diallyl disulfide and allyl methyl sulfide emanating from human skin surface and influence of ingestion of grilled garlic.

Diallyl disulfide (DADS) and allyl methyl sulfide (AMS) have been known as a metabolic product of sulfur-containing foods, typically garlic. The odour of such organosulfur compounds following garlic ingestion is often considered as an unpleasant element. Although previous studies have identified the DADS and AMS associated with garlic breath, no study has been reported on the determination of both compounds emanating from human skin surface. This study aimed to demonstrate the effect of garlic ingestion on the dermal emissions of DADS and AMS using a passive flux sampler coupled with gas chromatography-mass spectrometry. Firstly, baseline levels were investigated for 30 healthy volunteers in their daily life. The results of 1 h-sampling at the forearm showed the emission fluxes of both compounds followed the lognormal distribution with a geometric mean of 0.18 ng cm-2 h-1 for DADS and 0.22 ng cm-2 h-1 for AMS. Subsequently, the garlic ingestion tests were conducted for selected volunteers. The emission flux of DADS increased just after grilled garlic ingestion and decreased gradually thereafter. In contrast, the dermal emission flux of AMS reached a peak at 30 min after ingestion, and then gradually decreased. This peak shift suggests AMS is relatively latent in the skin organs.

A fluorescent probe for carbon monoxide based on allyl ether rather than allyl ester: A practical strategy to avoid the interference of esterase in cell imaging.

Pd0-mediated Tsuji-Trost reaction is a practical strategy to design fluorescent probes for carbon monoxide (CO) sensing, and in such reaction CO can reduce Pd2+ to Pd0 in-situ and remove allyl groups on fluorophores. In most of these probes, esters are commonly used to link allyl on fluorophores. We found that the ester groups could be hydrolyzed by esterase activity of fetal bovine serum (FBS), while FBS is a requisite in cell culture, and the hydrolysis could interfere the Pd0-mediated Tsuji-Trost reaction. In this study, we synthesized a fluorescent probe (Cou-CO) using allyl ether as reaction site rather than allyl ester. Cou-CO is non-fluorescence, and could react with CO under the presence of Pd0 to form Cou with strong fluorescence, and the maximum excitation and emission wavelengths of Cou are 464 nm and 495 nm respectively. Cou-CO shows excellent selectivity to CO and could avoid the effect of FBS with the limit of detection for CO is 78 nm. Finally, Cou-CO was successfully applied for imaging of CO in living cells.

Nanofibers mat as sampling module of direct analysis in real time mass spectrometry for sensitive and high-throughput screening of illegally adulterated sulfonylureas in antidiabetic health-care teas.

In this work, novel ionic liquid (IL) functionalized polyacrylonitrile nanofibers mat (IL/PAN-NFsM) was firstly prepared through thiol-ene "click" reaction and evaluated for the establishment of a sensitive and high-throughput screening method. Because of its excellent pre-separation efficiency, IL/PAN-NFsM can adsorb trace-level targets from complex sample matrix within tens of seconds by performing a solid-phase extraction (SPE) process, and then served as sampling modules of direct analysis real time mass spectrometry (DART-MS) without any additional processing. This means the target analytes concentrated on IL/PAN-NFsM were directly desorbed, ionized, and detected by DART-MS. To verify the feasibility of the proposed method, three illegal added synthetic drugs (gliclazide, glimepiride, and gliquidone) were screened in six types of antidiabetic health-care tea samples. The results indicated that the sensitivity is in the level of ng g-1, while total analysis time does not exceed 1.0 min per sample. Moreover, the stability expressed as relative standard deviation (RSD) varies from 1.7% to 17.3%. We proposed a new screening mode based on the direct combination of functionalized NFsM and DART-MS, which is expected to become a universal method in food safety analysis.

Heating tomato puree in the presence of lipids and onion: The impact of onion on lycopene isomerization.

Z-lycopene isomers are more bioavailable than all-E-lycopene, especially 5-Z-lycopene. Based on our observations, the addition of unblanched onion could favor Z-isomerization of lycopene (by more than 94%) during heating tomato-onion-extra virgin olive oil (EVOO) purees at 90 °C for 2 h. The increase in Z-lycopene was correlated linearly with the addition of unblanched onion, with R2 > 0.92, and increased rates of 5-Z-lycopene were 3-4 times higher than for 9-Z-lycopene and 13-Z-lycopene. Diallyl disulfide (DADS), formed by alliinase-catalyzed breakdown of non-volatile precursors in onion, contributed to these increases and correlated linearly (R2 > 0.79, 0-0.50 mg/g puree) with increased Z-lycopene. Increased rates of 5-Z-lycopene were also 3-4 times higher than for 9-Z-lycopene and 13-Z-lycopene. However, blanching of onion, in tomato-onion-EVOO purees, before heating, significantly decreased the effect of onion on Z-isomerization of lycopene.

Diallyl Trisulfide Enhances Benzo[a]pyrene-induced CYP1A1 Expression and Metabolic Activation in Hepatic HepG2 Cells.

BACKGROUND/AIM: Benzo[a]pyrene (BaP), an environmental pollutant produced by combustion processes, induces expression of cytochrome P450 (CYP) 1A1 via the activation of aryl hydrocarbon receptor (AHR). Induced CYP1A1 is involved in BaP metabolism, resulting in either detoxification or metabolic activation in a context-dependent manner. The effect of diallyl trisulfide (DATS), a garlic-derived organosulfur compound, on BaP metabolism has not been investigated. MATERIALS AND METHODS: The combined effect of DATS and BaP on BaP metabolism in hepatocyte-derived HepG2 cells was examined. RESULTS: DATS enhanced BaP-induced CYP1A1 and CYP1B1 mRNA expression, BaP hydroxylation and BaP-DNA adduct formation. Combined treatment of BaP and DATS also increased reactive oxygen species levels. DATS enhanced BaP-induced AHR recruitment and histone H3 acetylation on the CYP1A1 promoter. CONCLUSION: DATS combined treatment enhances BaP metabolic activation through an AHR-modulating mechanism.

Garlic and its Active Compounds: A Potential Candidate in The Prevention of Cancer by Modulating Various Cell Signalling Pathways.

BACKGROUND: Cancer is a multi-factorial disease including alterations in the cell signalling pathways. Currently, several drugs are in use to treat cancer but such drugs show negative side effects on normal cells and cause severe toxicity. METHODS: The current research is mainly focused on medicinal plants with potential therapeutic efficacy in the treatment of cancer without any adverse effects on normal cells. In this regard, garlic and its active compounds including diallyl sulfide, diallyl trisulfide, ajoene, and allicin have been established to suppress the growth of cancer and killing of cancer cells. RESULT: The review focuses on garlic and its active compounds chemopreventive effect through modulating various cell signalling pathways. Additionally, garlic and its active compound were established to induce cell cycle arrest at the G0/G1 phase and G2/M phases in cancer cells, increase the expression of tumor suppressor genes, inhibit the angiogenesis process, induction of apoptosis and modulation of various other genetic pathways. CONCLUSION: This review sketches the diverse chemopreventive activities of garlic and their active ingredients in the management of cancer mainly focusing on cell signalling pathways.

Increased Sulfiredoxin Expression in Gastric Cancer Cells May Be a Molecular Target of the Anticancer Component Diallyl Trisulfide.

Sulfiredoxin (Srx) is a newly discovered antioxidant enzyme playing a role in the catalytic reduction of oxidative modifications. Srx is overexpressed in a variety of cancers. It may promote carcinogenesis as well as tumor progression. In this study, we report for the first time that Srx expression might be positively associated with the development of gastric cancer and tumor malignancy. Immunohistochemistry showed that, compared to normal tissues (42%, 20/47), Srx expression in gastric tumors (85%, 40/47) was much more common (chi-square test, p<0.01). In addition, the staining of Srx was stronger in poorly differentiated gastric cancer than in well-differentiated gastric cancer. Western blotting showed that, in the gastric tumor cell line BGC823, the Srx protein was upregulated in response to H2O2 treatment, although it was inadequate to counteract the increased oxidative stress, as indicated by the gradually increasing level of malondialdehyde (MDA). In addition, Srx expression, MDA levels, and ROS levels in BGC823 cells were markedly inhibited upon treatment with diallyl trisulfide (DATS), a major constituent of garlic oil with proven anticancer effects. These results suggest that Srx may be an oxidative stress marker. Antioxidation may account for the anticancer potential of garlic.

Purification and identification of 4-allylbenzene-1,2-diol: an antilisterial and biofilm preventing compound from the leaves of Piper betle L. var Pachaikodi.

Antibiotic-resistant food-borne Listeriosis has been rising with up to 30% mortality threat in humans since several decades. Hence, discovering antilisterial from the extracts of ethnomedicinal plants may be of value as a novel antidote. In our preceding study, we reported that ethanolic extract of Piper betle L. var Pachaikodi leaves exhibited antibacterial activity towards Listeria monocytogenes MTCC 657. Consequently in the present study, the bioactive molecule responsible for anti-Listeria activity was purified and identified as 4-allylbenzene-1,2-diol. This identified bioactive compound may have significance while used as antimicrobials and/or food additives in food processing sector as evidenced by dual action: biofilm inhibition and pore formation on cell membrane.

Anti-obesity effect of garlic oil on obese rats via Shenque point administration.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenque is an acupoint located in the umbilicus and connected with the meridians. Thus, acupoint herbs applied at Shenque plays a pivotal role in the Chinese traditional medicine due to its sensitivity, permeability, and absorption. Many studies reported the use of Shenque point as a successful therapeutic approach. However, the effect of garlic oil (GO) applied at Shenque point to combat obesity is unmet. Consequently, we investigated the potential benefit of GO applied at Shenque point against obesity. AIM OF THE STUDY: To investigate GO effects on obese rats applied at Shenque acupoint and orally administered, and to identify the chemical constituents of GO. MATERIALS AND METHODS: Rats were randomly divided into 2 groups: naive and model group. The model group rats were fed with a high fat diet for 7 weeks to induce obesity, and then they were randomly divided into 5 groups: model, GO Shenque point treated groups (25, 50 and 100 mg/kg/day) and oral group (50 mg/kg/day). Biochemical indexes in the serum, weight of adipose tissue and liver histopathology were evaluated after 6 weeks of GO treatment using a Hitachi 7080 analyzer (Hitachi, Japan). Moreover, GO chemical components were detected by gas chromatography-mass spectrometer (GC-MS). RESULTS: Compared with the naive rats, model rats exhibited higher body and liver weight, increased fat deposition, higher triglyceride concentration and alveolar development. In contrast, GO Shenque point treated groups showed a substantial decrease in body weight (P = 0.358, 0.028, 0.031, respectively), fat mass, cholesterol (P = 0.004, 0.041, 0.001, respectively), triglyceride (P = 0.001, 0.001, 0.001, respectively), and low density lipoprotein concentrations (P = 0.001, 0.000, 0.001, respectively). The effect was more remarkable than the GO orally administered. In addition, twelve GO organosulfur compounds were identified by GC-MS and diallyl trisulfide (DATS) was detected as the main compound, with a 32.08% concentration. CONCLUSIONS: These findings demonstrated that GO had a significant anti-obesity effect on obese rats by reducing the body weight and protecting the liver from damage, and the effect of Shenque point treatment was better than oral administration, suggesting that GO was an effective weight-loss drug and Shenque point administration might be considered as a new anti-obesity approach.

Quantification of volatile metabolites derived from garlic in human breast milk.

Maternal garlic intake during pregnancy and the breastfeeding period has been reported to be associated with the potential of modulating later garlic acceptance in infants. However, the metabolism of garlic constituents in humans and their elimination and potential excretion into human milk are not yet fully understood. In previous studies, we identified volatile garlic-derived metabolites in human milk as well as in human urine, namely allyl methyl sulfide, allyl methyl sulfoxide and allyl methyl sulfone. To monitor the excretion of these garlic metabolites in a larger cohort, we quantified these metabolites in a total of 18 human milk sets, whereby each set comprised of one sample collected before and three samples after garlic consumption. The analyses revealed that the concentrations of the metabolites were most abundant 1-3.5 h after garlic consumption, with distinct differences between test persons regarding metabolite concentrations as well as temporal excretion.

Converting organosulfur compounds to inorganic polysulfides against resistant bacterial infections.

The use of natural substance to ward off microbial infections has a long history. However, the large-scale production of natural extracts often reduces antibacterial potency, thus limiting practical applications. Here we present a strategy for converting natural organosulfur compounds into nano-iron sulfides that exhibit enhanced antibacterial activity. We show that compared to garlic-derived organosulfur compounds nano-iron sulfides exhibit an over 500-fold increase in antibacterial efficacy to kill several pathogenic and drug-resistant bacteria. Furthermore, our analysis reveals that hydrogen polysulfanes released from nano-iron sulfides possess potent bactericidal activity and the release of polysulfanes can be accelerated by the enzyme-like activity of nano-iron sulfides. Finally, we demonstrate that topical applications of nano-iron sulfides can effectively disrupt pathogenic biofilms on human teeth and accelerate infected-wound healing. Together, our approach to convert organosulfur compounds into inorganic polysulfides potentially provides an antibacterial alternative to combat bacterial infections.

Salting-in counter-current chromatography separation of tanshinones based on room temperature ionic liquids.

Ionic liquids have been widely used for the extraction and separation of bioactive natural and synthetic mixtures. In this study, we provided an updated example by using an ionic liquid-based salting-in counter-current chromatography (CCC) strategy for the separation of hydrophobic tanshinones without subsequent column chromatography purification. Several ionic liquids such as 1-allyl-3-methylimidazolium chloride ([AMIM]Cl), 1-methallyl-3-methylimidazolium chloride ([MAMIM]Cl) and 1-butyl-3-ethylimidazolium chloride [BMIM]Cl could significantly decrease the partition coefficients (K) of tanshinones in the selected two-phase solvent composed of hexane-ethyl acetate-methanol-ionic liquid aqueous solution (5:5:6:4, v/v). Typically, K values of three target tanshinones including tanshinone I, 1,2-dihydrotanshinquione and tanshinone IIA were reduced from 3.57, 4.57 and 5.50 to 1.62, 2.33 and 3.08, respectively, by the inclusion of 10% [AMIM]Cl in the solvent system. After salting-in CCC separation, the purified tanshinones were obtained only by simple ethyl acetate extraction. In general, the current results demonstrated that the ionic liquid-based salting-in CCC may be as an alternative strategy for the optimization of CCC solvent systems and separation of lipophilic natural products.

Formulation and characterization of garlic (Allium sativum L.) essential oil nanoemulsion and its acaricidal activity on eriophyid olive mites (Acari: Eriophyidae).

Green and nanoacaricides including essential oil (EO) nanoemulsions are important compounds to provide new, active, safe acaricides and lead to improvement of avoiding the risk of synthetic acaricides. This study was carried out for the first time on eriophyid mites to develop nanoemulsion of garlic essential oil by ultrasonic emulsification and evaluate its acaricidal activity against the two eriophyid olive mites Aceria oleae Nalepa and Tegolophus hassani (Keifer). Acute toxicity of nanoemulsion was also studied on male rats. Garlic EO was analyzed by gas chromatography-mass spectrometry (GC-MS), and the major compounds were diallyl sulfide (8.6%), diallyl disulfide (28.36%), dimethyl tetrasulfide (15.26%), trisulfide,di-2-propenyl (10.41%), and tetrasulfide,di-2-propenyl (9.67%). Garlic oil nanoemulsion with droplet size 93.4 nm was formulated by ultrasonic emulsification for 35 min. Emulsification time and oil and surfactant ratio correlated to the emulsion droplet size and stability. The formulated nanoemulsion showed high acaricidal activity against injurious eriophyid mites with LC50 298.225 and 309.634 µg/ml, respectively. No signs of nanoemulsion toxicity were noted in treating rats; thus, it may be considered non-toxic to mammals. Stability of garlic oil nanoemulsion, high acaricidal activity, and the absence of organic toxic solvents make the formulation that may be a possible acaricidal product. Results suggest the possibility of developing suitable natural nanoacaricide from garlic oil.

An Optimized Facile Procedure to Synthesize and Purify Allicin.

Allicin is a reactive sulfur species (RSS) and defence substance from garlic (Allium sativum L.). The compound is a broad-spectrum antibiotic that is also effective against multiple drug resistant (MDR) strains. A detailed protocol for allicin synthesis based on diallyl-disulfide (DADS) oxidation by H2O2 using acetic acid as a catalyst was published in 2001 by Lawson and Wang. Here we report on improvements to this basic method, clarify the mechanism of the reaction and show that it is zero-order with respect to DADS and first-order with respect to the concentration of H2O2. The progress of allicin synthesis and the reaction mechanism were analyzsd by high-performance liquid chromatography (HPLC) and the identity and purity of the products was verified with LC-MS and ¹H-NMR. We were able to obtain allicin of high purity (>98%) and >91% yield, with standard equipment available in any reasonable biological laboratory. This protocol will enable researchers to prepare and work with easily and cheaply prepared allicin of high quality.