Influence of Two Garlic-Derived Compounds, Propyl Propane Thiosulfonate (PTS) and Propyl Propane Thiosulfinate (PTSO), on Growth and Mycotoxin Production by Fusarium Species In Vitro and in Stored Cereals.

Two garlic-derived compounds, Propyl Propane Thiosulfonate (PTS) and Propyl Propane Thiosulfinate (PTSO), were examined for their efficacy against mycotoxigenic Fusarium species (F. graminearum, F. langsethiae, F. verticillioides). The objectives were to assess the inhibitory effect of these compounds on growth and mycotoxin production in vitro, and in situ in artificially inoculated wheat, oats and maize with one isolate of each respectively, at different water activity (aw) conditions when stored for up to 20 days at 25 °C. In vitro, 200 ppm of either PTS or PTSO reduced fungal growth by 50-100% and mycotoxin production by >90% depending on species, mycotoxin and aw conditions on milled wheat, oats and maize respectively. PTS was generally more effective than PTSO. Deoxynivalenol (DON) and zearalenone (ZEN) were decreased by 50% with 80 ppm PTSO. One-hundred ppm of PTS reduced DON and ZEN production in wheat stored at 0.93 aw for 20 days, although contamination was still above the legislative limits. Contrasting effects on T-2/HT-2 toxin contamination of oats was found depending on aw, with PTS stimulating production under marginal conditions (0.93 aw), but at 0.95 aw effective control was achieved with 100 ppm. Treatment of stored maize inoculated with F. verticilliodies resulted in a stimulation of total fumonsins in most treatments. The potential use of such compounds for mycotoxin control in stored commodities is discussed.

Potentides: New Cysteine-Rich Peptides with Unusual Disulfide Connectivity from Potentilla anserina.

Cysteine-rich peptides (CRPs), which are disulfide-constrained peptides with 3 to 5 disulfide bonds and molecular weights of 2 to 6 kDa, are generally hyperstable and resistant to thermal, chemical, and enzymatic degradation. Herein, the discovery and characterization of a novel suite of CRPs, collectively named potentides pA1-pA16 from the root of the medicinal herb Potentilla anserina L, are described. Through a combination of proteomic and transcriptomic methods, it is shown that 35-residue potentide pA3, which is the most abundant member of potentides, exhibits high stability against heat, acidic, and proteolytic degradation. Transcriptomic analysis revealed that potentide precursor sequences contained four tandem repeats in the mature domain, which is the first report on tandem repeats being found in the Rosaceae family. Disulfide mapping showed that potentide pA3 displayed a novel disulfide connectivity of C1-C3, C2-C6, and C4-C5; a cystine motif that has not been reported in plant CRPs. Transcriptomic data mining and a neighbor-joining clustering analysis revealed 56 potentide homologues and their distribution in the families of Rosaceae and Ranunculaceae in angiosperm. Altogether, these results reveal a new plant CRP structure with an unusual cystine connectivity. Additionally, this study expands the families and structure diversity of CRPs as potentially active peptide pharmaceuticals.

A salting-out assisted liquid-liquid microextraction procedure for determination of cysteine followed by spectrophotometric detection.

A new analytical method for sensitive determination of cysteine based on its interaction with phenazine methosulfate was developed using salting-out liquid-liquid microextraction followed by spectrophotometric detection. The mechanism of the reaction was studied and confirmed by Fourier transform infrared and mass spectroscopy. Experimental parameters affecting the extraction efficiency were investigated and under the optimal conditions, good linearity was observed in the range 0.2 - 6.0 µg mL-1 with a correlation coefficient of 0.9972. The limit of detection and limit of quantification were found to be 0.07 and 0.21 µg mL -1, respectively. The enrichment factor was 25. The developed methodology was applied for analysis of cysteine in food supplements. The obtained data were in good agreement with LC-MS/MS analysis.

Effects of S-1-propenylcysteine, a sulfur compound in aged garlic extract, on blood pressure and peripheral circulation in spontaneously hypertensive rats.

OBJECTIVES: This study was designed to investigate the antihypertensive effect of S-1-propenylcysteine, a characteristic sulfur compound in aged garlic extract, using a hypertensive rat model. METHODS: The blood pressure and tail blood flow of both spontaneously hypertensive rats and control Wistar Kyoto rats were measured by the tail-cuff method and the noncontact laser Doppler method, respectively, at various times after single oral administration of a test compound for 24 h. KEY FINDINGS: Treatment with S-1-propenylcysteine (6.5 mg/kg BW) significantly decreased the systolic blood pressure of spontaneously hypertensive rat approximately 10% at 3 h after administration, and thereafter, the systolic blood pressure gradually returned to the baseline level in 24 h. The effect of S-1-propenylcysteine was dose-dependent and was maximal at the dose of 6.5 mg/kg BW at 3 h. However, the other compounds such as S-allylcysteine and S-allylmercaptocysteine in aged garlic extract were ineffective. In addition, S-1-propenylcysteine had no effect on systolic blood pressure of control Wistar Kyoto rats. Furthermore, S-1-propenylcysteine significantly increased the blood flow at 3 h after administration at the dose of 6.5 mg/kg BW. CONCLUSIONS: S-1-propenylcysteine is a key constituent of aged garlic extract responsible for its antihypertensive effect, and the effect of S-1-propenylcysteine involves the improvement in peripheral circulation.

High doses of S-methylcysteine cause hypoxia-induced cardiomyocyte apoptosis accompanied by engulfment of mitochondaria by nucleus.

Despite its important role as a medicinal plant, some studies reported a toxic effect for garlic (Allium sativum) when given in higher doses. Herein, we investigated the possible cardiotoxic effects of high doses of S-methylcysteine (SMC), a water soluble organosulfur compound present in garlic. Rats were orally administered SMC at a low dose (50mg), high dose (150mg) and very high dose (300mg)/kg body weight, or saline (control) for 10days. High and very high doses of SMC resulted in a significant increase in serum cardiac injury biomarkers [aspartate transaminase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and cardiac troponin T (cTnT)], as well as oxidative stress marker nitric oxide (NO) concentration in heart and a significant decrease in cardiac superoxide dismutase (SOD) activity. Moreover, ultrastructure findings in myocardium of rats treated by high and very high doses showed inter-bundle vacuolation, loss of myofibrils, and centripetal movement of mitochondria towards nucleus. The mitochondria were partially surrounded by nuclear membrane at high dose SMC, and completely engulfed by nucleus at very high dose. This centripetal movement of mitochondria accompanied by cardiomyocytes hypoxia-induced apoptosis as evident by increasing TUNEL positive cells as well as upregulation of apoptotic genes (caspase3 and Bax), hypoxia inducible factor 1 alpha (HIF1α), dynein light chain 1 (DYNLL1) and downregulation of the anti-apoptotic marker, Bcl2. We conclude that high and very high doses of SMC cause hypoxia induced cardiomyocyte apoptosis accompanied by engulfment of mitochondria by nucleus.

Garlic Organosulfur Compounds Reduce Inflammation and Oxidative Stress during Dengue Virus Infection.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant global human disease and mortality. One approach to develop treatments for DENV infection and the prevention of severe disease is through investigation of natural medicines. Inflammation plays both beneficial and harmful roles during DENV infection. Studies have proposed that the oxidative stress response may be one mechanism responsible for triggering inflammation during DENV infection. Thus, blocking the oxidative stress response could reduce inflammation and the development of severe disease. Garlic has been shown to both reduce inflammation and affect the oxidative stress response. Here, we show that the garlic active compounds diallyl disulfide (DADS), diallyl sulfide (DAS) and alliin reduced inflammation during DENV infection and show that this reduction is due to the effects on the oxidative stress response. These results suggest that garlic could be used as an alternative treatment for DENV infection and for the prevention of severe disease development.

In situ Identification of Labile Precursor Compounds and their Short-lived Intermediates in Plants using in vivo Nanospray High-resolution Mass Spectrometry.

INTRODUCTION: Many secondary metabolites in plants are labile compounds which under environmental stress, are difficult to detect and track due to the lack of rapid in situ identification techniques, making plant metabolomics research difficult. Therefore, developing a reliable analytical method for rapid in situ identification of labile compounds and their short-lived intermediates in plants is of great importance. OBJECTIVE: To develop under atmospheric pressure, a rapid in situ method for effective identification of labile compounds and their short-lived intermediates in fresh plants. METHODOLOGY: An in vivo nanospray high-resolution mass spectrometry (HR-MS) method was used for rapid capture of labile compounds and their short-lived intermediates in plants. A quartz capillary was partially inserted into fresh plant tissues, and the liquid flowed out through the capillary tube owing to the capillary effect. A high direct current (d.c.) voltage was applied to the plant to generate a spray of charged droplets from the tip of the capillary carrying bioactive molecules toward the inlet of mass spectrometer for full-scan and MS/MS analysis. RESULTS: Many labile compounds and short-lived intermediates were identified via this method: including glucosinolates and their short-lived intermediates (existing for only 10 s) in Raphanus sativus roots, alliin and its conversion intermediate (existing for 20 s) in Allium sativum and labile precursor compound chlorogenic acid in Malus pumila Mill. CONCLUSION: The method is an effective approach for in situ identification of internal labile compounds and their short-lived intermediates in fresh plants and it can be used as an auxiliary tool to explore the degradation mechanisms of new labile plant compounds. Copyright © 2016 John Wiley & Sons, Ltd.

The JNK Signaling Pathway Is a Novel Molecular Target for S-Propargyl- L-Cysteine, a Naturally-Occurring Garlic Derivatives: Link to Its Anticancer Activity in Pancreatic Cancer In Vitro and In Vivo.

Pancreatic ductal adenocarcinoma (PDA) remains a devastating disease worldwide. Although significant improvement has been made in understanding its pathophysiology, only small portion of patients with PDA are likely to benefit from curative surgery and current chemotherapy. Thus, there is an urgent need for developing novel effective therapeutic approaches to the treatment of PDA. Today, garlic products have become an important source of effective compounds for the treatment of cancer. We have recently identified a novel garlic active component, S-propargyl-L-cysteine (SPRC), an analogue of S-Allyl Cysteine (SAC). Although its anticancer activity has been shown against several cancers, the mechanism of action is not fully understood. The present study was first designed to determine the anticancer activity of SPRC in PDA and the underlying mechanisms of action in vitro and in vivo. Our results demonstrated that SPRC reduced cell viability and colony formation, inhibited cell proliferation, induced G2/M phase cell cycle arrest and apoptosis in human PDA cells with various p53 statuses (HPAC, p53 wt; Panc-1, p53 mt). Furthermore, SPRC inhibited tumor growth in Panc-1 xenograft models. We also demonstrated that SPRC achieved its anticancer effects by regulation of the JNK protein levels through increasing its phosphorylation and decreasing its polyubiquitination-mediated degradation. In conclusion, SPRC has significant anti-PDA activity and the effects do not depend on p53 status, presumably through activating the JNK signaling pathway, providing a basis for the development of this compound as a novel target anticancer therapeutic agent for PDA.

Combination of aqueous two-phase extraction and cation-exchange chromatography: new strategies for separation and purification of alliin from garlic powder.

In this study, a two-step process combining aqueous two-phase extraction (ATPE) with chromatography was developed for extraction and purification of alliin from garlic powder. The partition coefficient and yield value of alliin in different types of aqueous two-phase system (ATPS) were compared and response surface methodology (RSM) was used for analyzing and optimizing the extraction process. The optimal extraction conditions of 19% (w/w) (NH4)2SO4, 20% (w/w) 1-prpanol, at 30°C, pH 2.35 with 8.54% (w/w) NaCl was chosen based on the higher yield. Compared to the results obtained with the conventional extraction method, this method had an evident advantage on yield (20.4mg/g versus the original yield of 15.0mg/g) and the concentration of alliin in extract solution by ATPE was close to three times of that with conventional extraction. The purification of alliin was carried out with the ammonium form of sulfonic acid cation-exchange resins 001×7. Sample solution with alliin concentration of 1mg/mL was passed through resins and the desorption of alliin was accomplished by water at the flow velocity of 0.5mL/min, 1.5mL/min, respectively. The purity and recovery of alliin after purification were 80% and 76%, respectively.

S-alkenyl cysteine sulfoxide and its antioxidant properties from Allium cepa var. tropeana (red onion) seeds.

A new cysteine sulfoxide, (S(S)R(C))-S-(3-pentenyl)-L-cysteine sulfoxide (1), was identified from the seeds of Allium cepa var. tropeana, together with the known methiin, etiin, alliin, isoalliin, propiin, and butiin. The structure of compound 1 was established by analysis of its physical and spectroscopic data. The antioxidant activity of an extract containing cysteine sulfoxides and compound 1 was evaluated using the FRAP and DPPH tests.

A comparative study of antioxidants S-allyl cysteine sulfoxide and vitamin E on the damages induced by nicotine in rats.

The dietary consumption of antioxidant-rich fruits and vegetables is inversely correlated with the incidence of various diseases like cardiovascular diseases and lung cancer. We have tried to find out how far the S-allyl cysteine sulfoxide (SACS) isolated from garlic (Allium Sativum L.) can combat the nicotine-induced peroxidative damage in rats. The effects have been compared with the standard antioxidant vitamin E. Administration of SACS or vitamin E (100 mg/kg) to nicotine (0.6 mg/kg) treated rats for 21 days showed decreased concentrations of thiobarbituric acid reactive substances, hydroperoxides, and conjugated dienes in liver, lungs, and heart as compared with the values found in rats treated with nicotine alone. The activities of catalase and superoxide dismutase increased. The levels of the antioxidants like vitamins A, C, and E in the liver and glutathione in all tissues increased significantly in SACS-treated or vitamin E fed rats. However, the antioxidant status was higher when vitamin E was administered as compared with SACS administered to nicotine-treated rats.

Isolation of S-n-butylcysteine sulfoxide and six n-butyl-containing thiosulfinates from Allium siculum.

Isolation and identification of (S(S)R(C))-S-n-butylcysteine sulfoxide (1) from the bulbs of Allium siculum is reported. This compound was found in all parts of the plant (bulbs, stem, leaves, and flowers) along with known compounds (S(S)R(C))-S-methyl- and (R(S)R(C))-(E)-S-(1-propenyl)cysteine sulfoxides (2 and 3, respectively). In addition, six n-butyl-containing thiosulfinates (4-9) have been found in a CH2Cl2 extract of the bulbs. Structures were determined by a combination of spectral methods (primarily NMR and MS) and by comparison with authentic compounds obtained by synthesis. Antimicrobial activities of 4-7 and 9 are reported.

Antidiabetic and antioxidant effects of S-methyl cysteine sulfoxide isolated from onions (Allium cepa Linn) as compared to standard drugs in alloxan diabetic rats.

Antidiabetic and antoxidant effects of S-methyl cysteine sulfoxide (SMCS) isolated from A. cepa and two standard drugs, glibenclamide and insulin were studied and compared in alloxan diabetic rats after using each of them for treatment for two months. These drugs ameliorated the diabetic condition significantly, viz. maintenance of body weight and control of blood sugar in rats. Further they lowered the levels of malondialdehyde, hydroperoxide and conjugated dienes in tissues exhibiting antioxidant effect on lipid peroxidation in experimental diabetes. This is achieved by their stimulating effects on glucose utilization and the antioxidant enzymes, viz. superoxide dismutase and catalase. The probable mechanism of action of SMCS and glibenclamide may be partly dependent on the stimulation of insulin secretions and partly due to their individual actions. In the amelioration of diabetes the standard drugs showed a better action, but as an antioxidant SMCS proved to be a better one.

Identification of the two zinc-bound cysteines in the ferric uptake regulation protein from Escherichia coli: chemical modification and mass spectrometry analysis.

Selective chemical modification of thiol groups combined with mass spectrometry analysis was used to characterize cysteine ligands in the zinc-binding site of the Fur protein. Fur is a metalloregulatory protein involved in the regulation of almost all bacterial genes related to iron uptake in Gram-negative bacteria such as Escherichia coli. In addition to the iron site, Fur also possesses a tight-binding zinc site that likely comprises two cysteines. Using a new procedure, we confirm the involvement of two cysteines in zinc binding and identify them within the two pairs of cysteines present in the protein. The protein was treated under nondenaturing conditions with iodoacetamide, and the progressive alkylation of the thiol groups monitored by quenching the reaction at different times and measuring the extent of alkylation by mass spectrometry. Complementary experiments were carried out in the absence or presence of EDTA, a strong zinc chelator, to determine which of the cysteines were protected from alkylation by the zinc atom. Enzymatic digestion of the modified protein and analysis of the peptide mixture by mass spectrometry enabled fast identification of reactive and protected thiol groups. Two cysteines, Cys92 and Cys95, were thus assigned as zinc ligands. Examination of the sequence comprising the zinc site indicates that it may belong to a new type of structural zinc site. Furthermore, Cys132 was shown to be the fastest reacting cysteine, implying it is a surface-exposed residue.

Aged garlic extract and its constituents inhibit Cu(2+)-induced oxidative modification of low density lipoprotein.

Oxygen radical injury and lipid peroxidation have been suggested as major causes of atherosclerosis, cancer, liver disease, and the aging process. More specifically, oxidative modification of low density lipoprotein (LDL) has been recognized as an important process of atherosclerosis. In this study, we determined the effects of aged garlic extract (AGE), four of its constituents, and a metabolite on Cu(2+)-induced oxidative modification of LDL using an in vitro system. All these compounds were shown to inhibit oxidative modification of LDL.

S-allyl cysteine inhibits activation of nuclear factor kappa B in human T cells.

Reactive oxygen species are involved in signal transduction pathways leading to nuclear factor kappa B (NF-kappa B) activation which has been implicated in the regulation of gene transcription. We recently reported that a garlic compound, S-allyl cysteine (SAC), protects bovine pulmonary artery endothelial cells from oxidant injury induced by hydrogen peroxide (H2O2). In this study we determined the effects of SAC on NF-kappa B activation in human T lymphocytes (Jurkat cells) induced by tumor necrosis factor alpha (TNF- alpha) and H2O2. Activated NF-kappa B in nuclear extracts was measured by an electrophoretic mobility shift assay using 32P-labeled probe. SAC consistently exhibited a dose-dependent inhibition of NF-kappa B activation induced by both TNF-alpha and H2O2. Supershift with specific antibodies to NF-kappa B subunits confirmed that the inducible retarded bands observed in the EMSA and p65-p50 heterodimer of the NF-kappa B/Rel protein. Our data suggest that SAC may act via antioxidant mechanisms to block NF-kappa B activation in Jurkat cells.

Antiperoxide effect of S-allyl cysteine sulfoxide, an insulin secretagogue, in diabetic rats.

Treatment of alloxan diabetic rats with the antioxidant S-allyl cysteine sulfoxide (SACS) isolated from garlic (Allium sativum Linn), ameliorated the diabetic condition almost to the same extent as did glibenclamide and insulin. In addition, SACS controlled lipid peroxidation better than the other two drugs. Furthermore, SACS significantly stimulated in vitro insulin secretion from B cells isolated from normal rats. Hence it can be surmised that the beneficial effects of SACS could be due to both its antioxidant and its secretagogue actions. The former effect is more predominant and the latter is only secondary. These effects highlight the therapeutic value of garlic, which is a component of many diets.

Effects of S-allyl cysteine sulfoxide isolated from Allium sativum Linn and gugulipid on some enzymes and fecal excretions of bile acids and sterols in cholesterol fed rats.

S-allyl cysteine sulfoxide, isolated from garlic, A. sativum, is more or less as active as gugulipid in controlling hypercholestermia, obesity and derangement of enzyme activities in cholesterol diet fed rats. The beneficial effects of the drugs are partly due to their inhibitory effects on transaminases, alkaline phosphatase, lipogenic enzymes and HMG CoA reductase and partly due to their stimulatory effects on plasma lecithin-cholesterol acyl transferase lipolytic enzymes and fecal excretion of sterols and bile acids.

Antiperoxide effects of S-allyl cysteine sulphoxide isolated from Allium sativum Linn and gugulipid in cholesterol diet fed rats.

Cholesterol containing diet significantly increased not only the body weight, but also the weight of liver and adipose tissue of rats. This is accompanied by a significant increase in blood lipids, atherogenic index and lipid peroxidation and a significant decrease in reduced glutathione level, superoxide dismutase and catalase activities in tissues. Treatment with S-allyl cysteine sulphoxide reverses the deleterious effects of cholesterol diet significantly and almost as effectively as gugulipid.