Identification of Antibacterial Compounds in Black Garlic against Salmonella typhimurium using HPLC-Fingerprinting and Chemometrics

ObjectivesGentamicin is a potent aminoglycoside antibiotic. Ototoxicity and nephrotoxicity are the main side effects which restrict the use of gentamicin. Garlic with its intrinsic antioxidant activity may prove beneficial in prevention from ototoxicity. S-allylmercaptocysteine (SAMC), diallyl disulfide (DD), and S-allylcysteine (SAC) are three active compounds found in garlic. In this study, we investigated the effect of SAMC, DD, and SAC on the ototoxicity induced by gentamicin in rats, by using brainstem evoked response audiometry (BERA).MethodsThirty male Wistar rats with intact Preyer’s reflex initially weighing 220–260 g were randomly assigned to either the gentamicin injection with SAMC treatment group (Genta-w SAMC), DD treatment group (Genta-w DD), SAC treatment group (Genta-w SAC), gentamicin injection without any active compounds (AC) treatment groups (Genta-w/o AC), or control group (n=6 rats each group). Gentamicin was given 120-mg/kg body weight, intraperitoneally once daily for 25 days to subjects in all groups except the control group. SAMC 100-mg/kg, and DD 50-mg/kg body weight were given intragastrically, and SAC 250-mg/kg body weight was given intraperitoneally once daily to subjects in Genta-w SAMC, and Genta-w DD, and Genta-w SAC groups, respectively during the study. After 25 days hearing thresholds were evaluated by using BERA test.ResultsThe mean amplitude of auditory thresholds (sensation level [SL]) measured by using BERA for the Genta-w SAMC, Genta-w DD, Genta-w SAC, Genta-w/o AC, and control groups were 22±8, 25±5, 30±9, 54±11, and 10±7 dB SL, respectively (mean±SD). The differences between every active compound group (Genta-w SAMC, Genta-w DD, and Genta-w SAC) and Genta-w/o AC were statistically significant (P<0.016).ConclusionSAMC, DD, and SAC are derivative of garlic seems to attenuate aminoglycoside-induced hearing loss. The effect of SAMC and DD seems to be more prominent than that of SAC.

Aged garlic extract (AGE) contains various biologically active sulfur-containing amino acids, such as S-allylcysteine (SAC), S-1-propenylcysteine (S1PC) and S-allylmercaptocysteine (SAMC). These amino acids have been demonstrated to lower hypertension, improve atherosclerosis and enhance immunity through their anti-inflammatory and antioxidant activities. It was recently reported that the administration of AGE alleviated gingivitis in a clinical trial. In this study, to gain insight into this effect of AGE, the authors examined whether AGE and the three above-mentioned sulfur compounds influence the effects of tumor necrosis factor-α (TNF-α) in inducing intercellular adhesion molecule-1 (ICAM-1) expression and interleukin-6 (IL-6) secretion in Ca9-22 human gingival epithelial cells. It was found that S1PC reduced the level of ICAM-1 protein induced by TNF-α possibly through post-translational levels without affecting the TNF-α-induced mRNA expression. However, SAC and SAMC had no effect. It was also confirmed the inhibitory effect of an antimicrobial peptide [human-β defensin-3 (hβD3)] and found that the inhibitory effects of hbD3 and S1PC were synergistic. On the other hand, the TNF-α-induced IL-6 secretion was attenuated by SAC and SAMC in a dose-dependent manner, whereas S1PC was ineffective. In addition, SAC and SAMC, but not S1PC inhibited the phosphorylation of the transcription factor nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), which is involved in the expression of inflammatory molecules, suggesting that the anti-inflammatory effects of SAC and SAMC are mediated, at least partly, by NF-κB. On the whole, the findings of this study suggest that the three sulfur amino acids in AGE function synergistically in alleviating inflammation in human gingival epithelial cells.

Metastatic cancer is one of the main causes of cancer-related death since they rarely respond to available treatments. Recently, certain compounds isolated from the dietary supplement, garlic, have shown anti-proliferation effect on cancer cells. The aim of this study was to investigate whether certain garlic derivatives had any effect on the potentially invasive androgen-independent prostate cancer (PCa) cells. Using colony-forming, wound-closure as well as matrigel-invasion assays, we found that two main water-soluble constituents of the garlic, S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC), were able to suppress PCa cell proliferation and invasive abilities. This inhibitory effect was associated with induction of mesenchymal to epithelial transition. Most importantly, the SAC and SAMC treatment led to restoration of E-cadherin expression at transcription and protein levels. In contrast, the expression of E-cadherin repressor, Snail, was reduced in the SAC- and SAMC-treated cells. Furthermore, examination of cell lines from other types of cancer (ovarian, nasopharyngeal and esophageal carcinomas) also confirmed that the effect of SAC and SAMC on activation of E-cadherin might be a general effect on human cancer cells. Our results demonstrate a novel anticancer effect of garlic and suggest that certain garlic-derived compounds may be potential agents for suppression of invasive growth through restoration of E-cadherin expression in cancer cells.

The medicinal uses of garlic (Allium sativum) and its constituents have been known for centuries, though its mode of action is still undetermined. Several epidemiological and laboratory studies indicate a potential anti-carcinogenic effect of garlic and some of its constituents. In this study we investigated the effect of aged garlic extract (AGE) and two of its components, S-allylcysteine (SAC), and S-allyl-mercaptocysteine (SAMC) on human breast cancer cells, MCF-7 and MCF-7(ras). Transfection of v-H-ras in MCF-7 cells gives rise to a highly aggressive subset of cells that is estrogen independent and has a five fold greater colony forming efficiency in soft agar than the parent cell line. The modulatory effect of AGE, SAC and SAMC on growth and glutathione cycle was examined in the two cell lines. We noted an anti-proliferative response to SAC and SAMC on both anchorage dependent and independent conditions and an alteration in glutathione level without significant concurrent changes in the glutathione metabolizing enzymes.

Introduction: Incomplete, slow or limited wound healing is one of the major disabling events accompanying diabetic patients. At the present time, there is no effective treatments which enable efficient wound healing in diabetic patients. Aim: To optimise the method of loading of Human Mesenchymal Stem Cells (MSC) with S-Allyl Cysteine (SAC) from aged garlic extract and invitro optimisation of the delivery process, attachment and optimisation of stem cells and loaded stem cells to the fibroin matrix in induction of wound recovery completion in a model of diabetic wound healing. Materials and Methods: It was a preclinical invitro model which employed the anti-inflammatory and anti-bacterial properties of fibroin matrix after populating them with drug-loaded human MSCs. This combination allowed slow and sustained release of the active substance into the wound and with the stem cells, promoted tissue remodelling thereby, inducing enhanced wound recovery in diabetic model. Results: Mixture of SAC and N-Acetylcysteine (NAC) was most effective. S-Allyl Mercaptocysteine (SAMC) protected angiogenesis and showed tube formation in the presence of advanced glycation end-products. Protection against oxidative stress and apoptosis-Propidium Iodide (PI) staining shows protective effects of the cysteine compounds particularly SAC and SAMC. Conclusion: Present study results provide possibilities of utilising tissue-engineered fibroin matrix with drug loaded human MSC as a potential therapeutic technique, due to its beneficial effects in terms of cell attachment and bioactive factor loading, for diabetic wound healing.

Identification of biologically active natural compounds that promote health and longevity, and understanding how they act, will provide insights into aging and metabolism, and strategies for developing agents that prevent chronic disease. The garlic-derived thioallyl compounds S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) have been shown to have multiple biological activities. Here we show that SAC and SAMC increase lifespan and stress resistance in Caenorhabditis elegans and reduce accumulation of reactive oxygen species (ROS). These compounds do not appear to activate DAF-16 (FOXO orthologue) or mimic dietary restriction (DR) effects, but selectively induce SKN-1 (Nrf1/2/3 orthologue) targets involved in oxidative stress defense. Interestingly, their treatments do not facilitate SKN-1 nuclear accumulation, but slightly increased intracellular SKN-1 levels. Our data also indicate that thioallyl structure and the number of sulfur atoms are important for SKN-1 target induction. Our results indicate that SAC and SAMC may serve as potential agents that slow aging.

Thermal processing effects on the chemical constituent and antioxidant activity of s-alk(en)ylcysteine s-oxides (alliin) extract

Aged garlic extract (AGE) is produced by aging raw garlic (Allium sativum L.) in an alcoholic solution for >10 months. AGE is rich in sulfur-containing amino acids, such as S-allylcysteine (SAC), S-1-propenylcysteine (S1PC), S-methylcysteine (SMC) and S-allylmercaptocysteine (SAMC). These sulfur-containing amino acids exert various beneficial pharmacological effects and have different pharmacokinetic properties. For instance, SAC, S1PC and SMC are well absorbed in rats with high bioavailability (88.0-95.8%), whereas SAMC is not detected in the plasma after oral administration. Orally administered SAC and S1PC are excreted in urine in their N-acetylated forms and ~50% of SMC is excreted as inorganic sulfur compounds, whereas SAMC immediately reacts with blood and is metabolized into volatile sulfur compounds. The present review summarizes and discusses the pharmacokinetic profiles (absorption, distribution, metabolism and excretion) of sulfur-containing compounds present in AGE and other garlic-derived substances, such as allicin.

Aged garlic extract (AGE) has been shown to improve peripheral circulatory disturbances in both clinical trials and experimental animal models. To investigate the effect of S-1-propenylcysteine (S1PC), a characteristic sulfur compound in AGE, on cold-induced reduction in tail blood flow of rat, Wistar rats were individually placed in a restraint cage and given the treatment with cold water (15˚C) after the oral administration of AGE or its constituents S1PC, S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC). After the cold-treatment the tail blood flow of rats was measured at the indicated times. The pretreatment with AGE (2 g/kg BW) and S1PC (6.5 mg/kg BW) significantly alleviated the reduction of rat tail blood flow induced by cold treatment. The effect of S1PC was dose-dependent and maximal at the dose of 6.5 mg/kg BW, whereas SAC and SAMC were ineffective. To gain insight into the mechanism of S1PC action, the concentration of nitrogen oxide metabolites (NOx) in the plasma and the levels of phosphorylated endothelial nitric oxide synthase (eNOS) and 5'-AMP-activated protein kinase (AMPK) in the aorta were measured. The pretreatment with S1PC significantly increased the plasma concentration of NOx as well as the level of phosphorylated form of AMPK and eNOS in the aorta after cold-treatment. The present findings suggest that S1PC is a major constituent responsible for the effect of AGE to alleviate the cold-induced reduction of peripheral blood flow in rat by acting on the AMPK/eNOS/NO pathway in the aorta.

The study was undertaken to test the inhibitory potential on cholesterogenesis of organosulfur compounds derived from garlic. The primary rat hepatocytes maintained in Dulbecco's modified Eagle's medium were treated with [2-14C]acetate as substrate for cholesterol synthesis in the presence or absence of test compounds at 0.05 to 4.0 mmol/L. Eleven water-soluble and six lipid-soluble compounds of garlic were tested. Among water-soluble compounds, S-allyl cysteine (SAC), S-ethyl cysteine (SEC), and S-propyl cysteine (SPC) inhibited [2-14C]acetate incorporation into cholesterol in a concentration-dependent manner, achieving 42 to 55% maximal inhibition. Gamma-glutamyl-S-allyl cysteine, gamma-glutamyl-S-methyl cysteine, and gamma-glutamyl-S-propyl cysteine were less potent, exerting only 16 to 29% maximal inhibitions. Alliin, S-allyl-N-acetyl cysteine, S-allylsulfonyl alanine, and S-methyl cysteine had no effect on cholesterol synthesis. Of the lipid-soluble compounds, diallyl disulfide (DADS), diallyl trisulfide (DATS), and dipropyl disulfide (DPDS) depressed cholesterol synthesis by 10 to 25% at low concentrations (< or =0.5 mmol/L), and abolished the synthesis at high concentrations (> or =1.0 mmol/L). Diallyl sulfide, dipropyl sulfide, and methyl allyl sulfide slightly inhibited [2-14C]acetate incorporation into cholesterol only at high concentrations. The complete depression of cholesterol synthesis by DADS, DATS, and DPDS was associated with cytotoxicity as indicated by marked increase in cellular LDH release. There was no apparent increase in LDH secretion by water-soluble compounds except S-allyl mercaptocysteine, which also abolished cholesterol synthesis. Judging from maximal inhibition and IC50 (concentration required for 50% of maximal inhibition), SAC, SEC, and SPC are equally potent in inhibiting cholesterol synthesis.

Today, antibiotic resistance is increasing and evolving into an important health problem. Therefore, it is important to research on alternative therapies to antibiotics. This study aimed to investigate the inhibitory effect of four garlic derivatives on microorganisms commonly isolated in ear infections. The antimicrobial activities of allicin, s-allyl cysteine (SAC), diallyl disulfide (DADS), and s-allyl mercaptocysteine (SAMC) were investigated on standard strains of commonly isolated microorganisms using the broth microdilution method. The test strains were selected among the microorganisms responsible for chronic suppurative otitis media and otitis externa. These microorganisms were Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Candida albicans, and Candida tropicalis. Minimum inhibitory concentration (MIC) values of allicin and SAC ranged from 0.125 to 20 μg/mL for fermentative bacteria (E. coli and K. pneumoniae), 20 to 80 μg/mL for non-fermentative bacteria (P. aeruginosa and A. baumannii), 5 to 10 μg/mL for gram-positive cocci (S. aureus and E. faecium), and 40 to 80 μg/mL for yeasts (C. albicans and C. tropicalis). MIC values of DADS ranged from 40 to 80 μg/mL for fermentative bacteria, 40 to 160 μg/mL for non-fermentative bacteria, 40 to 80 μg/mL for gram-positive cocci, and 20 to 40 μg/mL for yeasts. The MICs of SAMC were >640 μg/mL for the tested bacteria and yeasts. Both allicin and SAC showed antimicrobial activity against the tested microorganisms, even at low concentrations. These two derivatives may be used to treat infections in the future.

To evaluate the effect of S-allylcysteine (SAC) on CWR22R, a human androgen-independent (AI) prostate cancer xenograft, in nude mice. Despite extensive research worldwide there is no effective way to control the growth of prostate cancer, and we previously reported that SAC and S-allylmercaptocysteine (SAMC), two water-soluble derivatives of garlic, inhibit cancer cell invasion through restoration of E-cadherin expression in vitro. The effects of SAC on tumour cell proliferation markers such as Ki-67 and proliferating cell nuclear antigen, and apoptotic regulators including Bcl-2 and cleaved caspase-3, were assessed by immunohistochemical staining. The inhibitory effects of SAC on prostate cancer invasion was examined by immunoreactivity of E-cadherin and its binding proteins alpha, beta and gamma-catenins. The serum prostate-specific antigen (PSA) level at three different times (initiation, middle and end of treatment) and toxicity of SAC on several organs after treatment were assessed. Treatment with SAC resulted in inhibition of the growth of CWR22R, with no detectable toxic effect on nude mice. The SAC-induced growth reduction was correlated with a concurrent reduction in serum PSA level and proliferation rate of xenografts, together with an inhibition of invasion through the restoration of E-cadherin and gamma-catenin expression. Furthermore, the apoptotic rate of SAC-treated tumours increased together with a decrease in Bcl-2 and increase in cleaved caspase-3. These results suggest that this garlic-derived compound might be a potential therapeutic agent for suppressing AI prostate cancer.

Aged garlic extract (AGE) is an odorless derivative of garlic prepared by extracting garlic cloves in an aqueous solution for twenty months. During the process of aging, reactive organosulfur compounds such as allicin present in garlic are converted to their stable isoforms such as S- Allyl cysteine. The unstable organo sulfurs in garlic (Allium sativum L.) have been reported to cause problems in the gastrointestinal (GI) tract with an extremely pungent odor to attain its therapeutic potential. But these pharmacologically safer sulfur compounds of AGE have been studied and reported to have exceptional therapeutic potential in human health and various diseases. SAllyl cysteine (SAC), Diallyl disulfide (DADS), Diallyl trisulfide (DATS), S-allyl-mercaptocysteine (SAMC), are the most studied organosulfur compounds in in-vitro as well as in-vivo research. Biomedical research suggests that these phytoconstituents exhibit antioxidant, cardioprotective, cancer preventive, neuroprotective, immunomodulatory, antilipidemic, antidiabetic, hepatoprotective, and antiobesity effects. The therapeutic potential of aged garlic extract has been found to be extensively beneficial in these conditions, and provide a vast future in biomedical chemistry, herbdrug synergy and drug designing. The purpose of this review is to provide a mechanistic understanding of various organosulfur compounds of AGE in human health and disease based on data provided in the literature.

This study was conducted at the Department of Horticulture, college of Agriculture, University of Baghdad, Abu-Ghraib, during the growing season, 2002-2003 to study the effect of NPK fertilizer and foliar spray with Zn, Mg, and Fe on the Vegetative, yield and chemical characters of five garlic cultivars (Local, chinese, French, Al-sharq and Lebanese). NPK fertilizer at the rates of (0 : 0 : 0), (90 : 90 : 90)and (120 : 120 : 120) Kg / ha were used after one month from planting, followed by foliar spray with Zn, Mg and Fe at the rates of (0 : 0 : 0) (0.1% : 0.2% : 1%) and (0.1% : 0.4% : 0.2%). Split-split plot Design were adapted with 3 replicates for each treatment. Plant hight, yied and garlic extracl were analysied using HPLC. The results of this study can be summarized as follows 1- The french cultivar signifcantly was superior in the average plant height (82. 129 cm). Asignificant increases in plant hight due to the increases in the concentration of NPK or foliar sprays of the studied nutient elrments Leading to the highest value of 92.246cm in the A3F2S2 treatment. 2- Al-Sharq cultivar gave the highest yield of 28.673 ton / ha swhilc the lowest yield of 13.285 tonlha was in lebanese cultivar. NPK treatments and foliar sprays significantly increased the yield and the highest value of 30.360 tonlh in the A4F2S2.treatment. 3- The local garlic cultivar were significantly superior in the organossulpher compounds, Allicin, S-Allyl cysteine, S-allyl Mercapto cysteine and N-Fruetosyl Arginine, which reached the highest value (2.761, 2.318, 1.315, 0.896 mglgm) respectively. These compounds was increased by increasing the level of NPK and foliar sprays and their interactions.

For centuries, garlic (Allium sativum) has been used both as a traditional remedy for most health-related ailments and for culinary purposes. Current preclinical investigations have suggested that dietary garlic intake has beneficial health effects, such as antioxidant, anti-inflammatory, antitumor, antiobesity, antidiabetic, antiallergic, cardioprotective, and hepatoprotective effects. Its therapeutic potential is influenced by the methods of use, preparation, and extraction. Of particular importance is the Aged Garlic Extract (AGE). During the aging process, the odorous, sour, and irritating compounds in fresh raw garlic, such as allicin, are naturally converted into stable and safe compounds that have significantly greater therapeutic effects than fresh garlic. In AGE, S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) are the major water-soluble organosulfurized compounds (OSCs). SAC has been extensively studied, demonstrating remarkable antioxidant, anti-inflammatory, and immunomodulatory capacities. Recently, AGE has been suggested as a promising candidate for the maintenance of immune system homeostasis through modulation of cytokine secretion, promotion of phagocytosis, and activation of macrophages. Since immune dysfunction plays an important role in the development and progress of various diseases, given the therapeutic effects of AGE, it can be thought of exploiting its immunoregulatory capacity to contribute to the treatment and prevention of chronic inflammatory bowel diseases (IBD).