In vitro toxicological assessment of an organosulfur compound from Allium extract: Cytotoxicity, mutagenicity and genotoxicity studies.

Garlic (Allium sativum) and onion (Allium cepa) are being used in the food industry as flavoring but also for their antimicrobial activities. These activities are mainly derived from the organosulfur compounds (OSCs). Propyl propane thiosulfinate (PTS) is an OSC with potential use in the active packaging, but its safety should be guaranteed before being commercialized. The aim of this work was to investigate for the first time the cytotoxicity of PTS as well as its in vitro mutagenic/genotoxic potential using the following battery of genotoxicity tests:(1)the bacterial reverse-mutation assay in S. typhimurium (Ames test, OECD 471, 1997); (2) the micronucleus test (MN, OECD 487, 2016); (3) the mouse lymphoma thymidine-kinase assay (MLA, OECD 476, 2015), and (4) the comet assay (standard and modified with restriction enzymes). The results revealed that PTS was not mutagenic neither in the Ames test nor in MLA. However, genotoxic effects were recorded in the MN test on mammalian cells (L5178YTk+/-cells) after PTS exposure at the highest concentration tested (17.25 µM) without S9, and also its metabolites (+S9, from 20 µM). Moreover, in the comet assay, PTS induced DNA breaks damage in Caco-2 cells at the highest concentration tested (280 µM) but it did not induce oxidative DNA damage.

Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging.

Essential oils from onion (Allium cepa L.), garlic (Allium sativum L.), and their main components, such as propyl thiosulfinate oxide (PTSO) are being intended for active packaging with the purpose of maintaining and extending food product quality and shelf life. The present work aims to assess for the first time the potential mutagenicity/genotoxicity of PTSO (0-50 µM) using the following battery of genotoxicity tests: (1) the bacterial reverse-mutation assay in Salmonella typhimurium (Ames test, OECD 471); (2) the micronucleus test (OECD 487) (MN) and (3) the mouse lymphoma thymidine-kinase assay (OECD 476) (MLA) on L5178YTk(+/-), cells; and (4) the comet assay (with and without Endo III and FPG enzymes) on Caco-2 cells. The results revealed that PTSO was not mutagenic in the Ames test, however it was mutagenic in the MLA assay after 24 h of treatment (2.5-20 µM). The parent compound did not induce MN on mammalian cells; however, its metabolites (in the presence S9) produced positive results (from 15 µM). Data from the comet assay indicated that PTSO did not induce DNA breaks or oxidative DNA damage. Further in vivo genotoxicity tests are needed to confirm its safety before it is used as active additive in food packaging.

Acute toxicological studies of the main organosulfur compound derived from Allium sp. intended to be used in active food packaging.

Some plant extracts have been proposed as potential alternative to the use of synthetic preservatives in the food industry. Among those, extracts from Allium species exhibit interesting antimicrobial and antioxidant properties for the food packaging industry. The present work aims to assess the usefulness and potential safety of the major organosulfur compound present in a commercial Allium sp. extract (PROALLIUM AP®), namely propyl thiosulfinate oxide (PTSO). For this purpose, its antimicrobial activity was studied in a wide range of microorganisms. Moreover, cytotoxicity and ultrastructural cellular damages caused by PTSO were studied in two human cell lines, Caco-2 and HepG2, being the colonic cells more sensitive to this compound. Finally, the protective role of PTSO against an induced oxidative situation was evaluated in the human intestinal Caco-2 cells. The results revealed damage at high concentration, although no significant adverse effects were recorded for the concentration to be used in food packaging. Moreover, the in vivo study also revealed the potential safety use at the established concentrations. In addition, the antimicrobial properties and the antioxidant role of PTSO were confirmed. Therefore, this compound could be considered as a good natural alternative to synthetic preservatives used in the food packaging industry.

Allicin from garlic neutralizes the hemolytic activity of intra- and extra-cellular pneumolysin O in vitro.

Pneumolysin (PLY) is a key virulence factor contributes to the pathogenesis of Streptococcus pneumoniae. In this study we investigated the effect of allicin and aqueous garlic extracts on hemolytic activity of PLY both in prelysed and intact cells. Additionally the antimicrobial activity of allicin was tested against the bacteria. All tested materials potently inhibited the PLY hemolytic activity. Allicin neutralizes PLY in a concentration- and time-dependent manner. Twenty five minute incubation of PLY (2 HU/mL) with 0.61 µM/mL concentration of allicin, totally inhibited hemolytic activity of PLY (IC50 = 0.28 µM/mL). The inhibitory activity of old extract of garlic was similar to pure allicin (IC50 = 50.46 µL/mL; 0.31 µM/mL; P < 0.05). In contrast fresh extract of garlic inhibits the PLY hemolytic activity at lower concentrations (IC50 = 13.96 µL/mL; 0.08 µM/mL allicin). Exposure of intact cells to allicin (1.8 µM) completely inhibited hemolytic activity of PLY inside bacterial cells. The inhibitory effect of the allicin was restored by addition of reducing agent DTT at 5 mM, proposing that allicin likely inhibits the PLY by binding to cysteinyl residue in the binding site. The MIC value of allicin was determined to be 512 µg/mL (3.15 µM/mL). These results indicate that PLY is a novel target for allicin and may provide a new line of investigation on pneumococcal diseases in the future.

Dependence of synergistic fungicidal activity of Cu2+ and allicin, an allyl sulfur compound from garlic, on selective accumulation of the ion in the plasma membrane fraction via allicin-mediated phospholipid peroxidation.

Allicin was effective in decreasing the lethal concentration of Cu (2+) against various fungal strains including a plant pathogen, Fusarium oxysporum, so that the minimum fungicidal concentration (MFC) of the ion for the fungus could be reduced to 2 % of that detected without allicin. In Saccharomyces cerevisiae, Cu (2+) was not apparently taken up by cells when added alone at a non-lethal concentration, whereas the ion was efficiently incorporated into cells in the presence of allicin, as in the case of cells treated with the ion at a lethal concentration. Although allicin likely increased cellular permeability to Cu (2+) due to its promotive effect on plasma membrane phospholipid peroxidation, these cell-surface events did not result in endogenous reactive oxygen species (ROS) production, a typical toxic effect of the ion. Cu (2+) was detected in the cytoplasmic fraction of cells that had been treated with the ion at a lethal concentration, whereas the ion was entrapped in the plasma membrane fraction upon their treatment with the ion at a low concentration in combination with allicin. Cu (2+) could be solubilized from the plasma membrane fraction by a procedure for the extraction of hydrophobic proteins rather than the extraction of phospholipids, suggesting its complexation with a plasma membrane protein as a result of allicin treatment. Such a subcellular localization of Cu (2+) resulted in the selective leakage of intracellular K (+), but not in the disruptive damage on the plasma membrane, and was considered to underlie the synergistic fungicidal activity of Cu (2+) and allicin.

The cytotoxic effect of ajoene, a natural product from garlic, investigated with different cell lines.

The sulfur-containing compound ajoene (4,5,9-trithiadodeca-1,6,11-triene-9-oxide) which arises from alliin, a cysteine derivative stored in garlic bulbs, was produced synthetically by decomposition of allicin. Its cytotoxic effect was tested using human primary fibroblasts (FS4), a permanent, non-tumorgenic cell line derived from baby hamster kidney cells (BHK21) and a tumorgenic lymphoid cell line derived from a Burkitt lymphoma (BJA-B). The cytotoxic action was in the range 2-50 micrograms/ml depending on the cell density. ED50 values, estimated on the basis of fmol ajoene/cell, revealed slightly higher doses for the primary cell (FS4) than the permanent line (BHK), whereas the tumorgenic BJA-B cells were most sensitive.

Allergic contact dermatitis to garlic (Allium sativum L.). Identification of the allergens: the role of mono-, di-, and trisulfides present in garlic. A comparative study in man and animal (guinea-pig).

Garlic (Allium sativum L.) water- and ethanol-soluble extracts were prepared and purified by column chromatography. They were tested on garlic-sensitive patients and showed that the allergenic fraction was well located in a few column chromatography fractions. Guinea-pigs were sensitized with garlic water-soluble extracts and tested (open epicutaneous tests) with several fractions. The presence of diallyldisulfide was detected in the sensitizing chromatographic fractions. Guinea-pigs were successfully sensitized to this product and cross-reacted to garlic; animals sensitized to garlic extracts cross-reacted to diallyldisulfide. Both groups reacted to allicin, an oxidized derivative of diallyldisulfide present in garlic. Garlic-sensitive patients showed positive tests to diallyldisulfide, allylpropyldisulfide, allylmercaptan and allicin.