Therapeutic effect of aged garlic extract on gingivitis in dogs.

Periodontal disease is one of the most common dental health problems in dogs. Clinical studies in humans have shown that aged garlic extract (AGE), which contains stable and water-soluble sulfur-containing bioactive compounds, improves the symptoms of periodontal diseases. Our previous study demonstrated that oral administration of AGE in healthy Beagle dogs at 90 mg/kg/day for 12 weeks had no adverse effects such as hemolytic anemia, which is well known to occur as a result of ingestion of Allium species, including onions and garlic, in dogs. However, the therapeutic potential of AGE in canine periodontal disease remains unclear. Accordingly, we investigated the therapeutic effects of AGE in Beagle dogs with mild gingivitis. Feeding 18 mg/kg/day of AGE for 8 weeks resulted in the improvement of gingival index score, level of volatile sulfur compounds in exhaled air, and enzyme activity of periodontal pathogens without any adverse effects on clinical signs and hematological and serum biochemical parameters. Moreover, AGE increased the concentration of salivary cathelicidin, an antimicrobial peptide that contributes to the oral innate immune response. These results suggest that AGE could be a potential therapeutic agent for canine gingivitis.

3-Allyltrisulfanyl-alanine Formation during the Preparation of Aged Garlic Extract.

The formation of 3-allyltrisulfanyl-alanine (ATrSA) was investigated during the aging process to prepare aged garlic extract (AGE). In raw garlic, ATrSA and its possible precursor, S-allylmercaptocysteine (SAMC), were barely detectable. However, the ATrSA content in AGE increased steadily during the 22 month of aging, while the SAMC level increased to a maximum at 4 months and then gradually decreased. In a model reaction mimicking the AGE preparation process, ATrSA production was decreased when the formation of SAMC was blocked by a γ-glutamyl-transpeptidase inhibitor but its decrease was reversed by the addition of SAMC. We also found that ATrSA was formed by the incubation of SAMC with allylsulfides such as diallyldisulfide and diallyltrisulfide. These findings suggest that ATrSA is formed via the reaction involving SAMC during the aging process. In addition, we found that ATrSA inhibits the secretion of interleukin-6 induced by lipopolysaccharide in mouse splenic lymphocytes in culture.

Potential benefits of garlic and other dietary supplements for the management of hypertension.

Elevated blood pressure is a major risk factor for cardiovascular diseases. Although some effective drug treatments are available, a relatively large proportion of patients have uncontrolled blood pressure. Dietary supplements are used for the prevention and treatment of hypertension as complementary and alternative medicines. Of the various dietary supplements, antioxidants, fish oil and diverse herbal products are commonly used. Within this context, it is important to determine the actual effectiveness and possible side-effects of these supplements; however, some of the products have been poorly investigated for their effects and safety. In the current review, we focus on garlic and several other dietary supplements, such as coenzyme Q10, fish oil and probiotics, that have exhibited significant beneficial effects on blood pressure in clinical trials. In addition, we discuss the possible mechanisms of action responsible for their anti-hypertensive effects, as well as the safety, active ingredients and their potential use as adjunct therapies for uncontrolled hypertension.

Chemistry of aged garlic: Diversity of constituents in aged garlic extract and their production mechanisms via the combination of chemical and enzymatic reactions.

Raw garlic contains characteristic compounds, such as S-alk(en)ylcysteine sulfoxides, γ-glutamyl-S-alk(en)-ylcysteines and polysaccharides. These compounds undergo various transformation processes during the aging process. Among these compounds, the change of sulfur-containing molecules is diverse and time-dependent. Previously, by means of the liquid chromatography (LC)/LC-mass spectrometry (MS) method, a number of unidentified peaks corresponding to candidates of sulfur-containing molecules were detected in the chromatogram of aged garlic extract (AGE), and identified using MS and nuclear magnetic resonance (NMR). The production mechanisms of these compounds were then examined by model reactions and laboratory experiments mimicking the aging process. Three γ-glutamyl tripeptides [γ-glutamyl-γ-glutamyl-S-methylcysteine, γ-glutamyl-γ-gluta-myl-S-allylcysteine (GGSAC), γ-glutamyl-γ-glutamyl-S-1-propenylcysteine], γ-glutamyl-S-allylmercaptocysteine (GSAMC) and cis-S-1-propenylcysteine (cis-S1PC) were isolated and identified. GGSAC was produced from GSAC through the enzymatic reaction catalyzed by γ-glutamyltranspeptidase (GGT), and two other tripeptides could be produced in similar reactions. GSAMC was produced by the reaction between γ-glutamyl dipeptides and allicin. Furthermore, GSAMC was a precursor compound of S-allyl-mercaptocysteine (SAMC), and thus it was produced from GSAMC by GGT. cis-S1PC was produced from trans-S1PC by the isomerization reaction. A number of other compounds were also identified, including Maillard reaction products; however, their production mechanisms have not been elucidated. In this review, we present the changes in characteristic constituents in raw garlic and garlic extract during the aging process and discuss their production mechanisms involving the various chemical and enzymatic reactions.

Metabolomic study reveals the acute hypotensive effect of S-1-propenylcysteine accompanied by alteration of the plasma histidine level in spontaneously hypertensive rats.

Our previous study has shown that a single dose of S-1-propenylcysteine (S1PC) exerted an antihypertensive effect in spontaneously hypertensive rats (SHR), while its mode of action remained to be further investigated. The aim of this study was to explore the potential mechanism of the antihypertensive effect of S1PC in SHR using a liquid chromatography-mass spectrometry (LC-MS)-based metabolomic approach. Blood samples were serially collected from SHR after a single oral administration of S1PC (6.5 mg/kg body weight). The metabolomics data acquired from the LC-MS analysis of plasma samples were processed using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). In addition, the SHR were treated with S1PC or histidine (10 mg/kg body weight) with and without intravenous preinjection of thioperamide, a histamine H3 receptor antagonist. The blood pressure of SHR was measured by the tail-cuff method at different times after administration. In the PLS-DA score plots, the clusters of the S1PC groups were clearly or partly separated from those of the control groups at 1.5 and 3 h after administration, indicating the metabolic profiles were substantially altered by the S1PC treatment at these time points. Comparative analysis based on variable importance in the projection (VIP) values obtained from PLS-DA led to the identification of 14 and 15 metabolites differing between the two groups at 1.5 and 3 h, respectively, which included various amino acids. Among the metabolites identified, the plasma histidine level in the S1PC group significantly increased at 1.5 and 3 h, and decreased to that in the control group at 6 h. Moreover, pretreatment with thioperamide inhibited the blood pressure lowering effect of S1PC as well as that of histidine. These results suggested that S1PC alters histidine metabolism and consequently exerts the antihypertensive effect via the central histamine H3 receptor.

Changes of S-Allylmercaptocysteine and γ-Glutamyl- S-allylmercaptocysteine Contents and Their Putative Production Mechanisms in Garlic Extract during the Aging Process.

γ-Glutamyl- S-allylmercaptocysteine (GSAMC), a putative precursor compound of S-allylmercaptocysteine (SAMC), was isolated and identified from aged garlic extract (AGE). We analyzed the change of their contents in AGE during the aging process, chronologically from 1 to 22 months. The formation of these compounds occurred mostly during the early stage of the aging period: the SAMC content reached a maximum at approximately 4 months, whereas the GSAMC content reached a maximum at 1 month and then decreased during the subsequent aging period. To assess the possible relationship between the change of the two compounds during the aging process, we set up the model reactions with the hypothesis that GSAMC is produced from γ-glutamyl- S-allylcysteine (GSAC)/γ-gultamyl- S-1-propenylcysteine (GS1PC) and that SAMC is produced from GSAMC by endogenous γ-glutamyl transpeptidase (GGT) in garlic during the early stage of the aging process. We found that, in the model reactions, SAMC was produced from GSAMC by the garlic protein fraction having GGT activity and its production was suppressed by a GGT inhibitor. Furthermore, the production of GSAMC from allicin and GSAC/GS1PC was found in another model reaction. The reaction between allicin and GS1PC was faster than that between allicin and GSAC and, thus, may be involved in the production of GSAMC in the early stage of the aging process.

Anti-inflammatory action of cysteine derivative S-1-propenylcysteine by inducing MyD88 degradation.

The degradation of target proteins by small molecules utilizing the cellular proteolytic system is featured as a treatment strategy of several diseases. We found that S-1-propenylcysteine (S1PC) among several cysteine derivatives in aged garlic extract inhibited TLR-mediated IL-6 production by inducing the degradation of adaptor protein MyD88. We showed that S1PC directly denatured MyD88 and induced the formation of protein aggregates. Consequently, MyD88 was degraded by aggresome-autophagy pathway. On the other hand, S-allylcysteine, a structural analog of S1PC, failed to induce the degradation of MyD88 because of its inability to denature MyD88 although it also activated autophagy. Our findings suggest that S1PC induces MyD88 degradation through the denaturation of MyD88 and the activation of autophagy. Thus, S1PC may serve as the base to develop a therapeutic means for immune diseases associated with aberrant TLR signaling pathways.

Isolation and Identification of Three γ-Glutamyl Tripeptides and Their Putative Production Mechanism in Aged Garlic Extract.

We analyzed aged garlic extract (AGE) to understand its complex sulfur chemistry using post-column high-performance liquid chromatography with an iodoplatinate reagent and liquid chromatography high resolution mass spectrometry (LC-MS). We observed unidentified peaks of putative sulfur compounds. Three compounds were isolated and identified as γ-glutamyl-γ-glutamyl- S-methylcysteine, γ-glutamyl-γ-glutamyl- S-allylcysteine (GGSAC) and γ-glutamyl-γ-glutamyl- S-1-propenyl-cysteine (GGS1PC) by nuclear magnetic resonance and LC-MS analysis based on comparisons with chemically synthesized reference compounds. GGSAC and GGS1PC were novel compounds. Trace amounts of these compounds were detected in raw garlic, but the contents of these compounds increased during the aging process. Production of these compounds was inhibited using a γ-glutamyl transpeptidase (GGT) inhibitor in the model reaction mixtures. These findings suggest that γ-glutamyl tripeptides in AGE are produced by GGT during the aging process.

(2R,3S,2″R,3″R)-Manniflavanone Protects Proliferating Skeletal Muscle Cells against Oxidative Stress and Stimulates Myotube Formation.

We investigated the antioxidative properties of (2R,3S,2″R,3″R)-manniflavanone (MF) using in vitro assays and examined its effects on myogenesis and lactate-induced oxidative stress in C2C12 cells. MF was purified from Garcinia buchananii stem bark. H2O2 and oxygen radical absorbance capacity assays demonstrated that MF is a powerful antioxidant. This finding was supported by diphenylpicrylhydrazine radical scavenging activity of MF. MF was less cytotoxic to C2C12 cells compared to ascorbic acid and myricetin. Moreover, MF accelerated myotube formation in the differentiated C2C12 cells by up-regulating myogenic proteins such as MyoG and myosin heavy chain. Furthermore, MF rescued late differentiation of myoblast suppressed by lactate treatment and up-regulated the expression levels of Nrf2 in lactate-induced oxidative stress, indicating that MF stimulates antioxidative activity inside C2C12 cells. Collectively, MF is a potent antioxidant with a higher safety profile than ascorbic acid and myricetin. It reduces oxidative stress-induced delaying of skeletal muscle differentiation by scavenging reactive oxygen species and regulating myogenic proteins factors.

Chemical and Biological Properties of S-1-Propenyl-l-Cysteine in Aged Garlic Extract.

S-1-Propenyl-l-cysteine (S1PC) is a stereoisomer of S-1-Propenyl-l-cysteine (SAC), an important sulfur-containing amino acid that plays a role for the beneficial pharmacological effects of aged garlic extract (AGE). The existence of S1PC in garlic preparations has been known since the 1960's. However, there was no report regarding the biological and/or pharmacological activity of S1PC until 2016. Recently, we performed a series of studies to examine the chemical, biological, pharmacological and pharmacokinetic properties of S1PC, and obtained some interesting results. S1PC existed only in trace amounts in raw garlic, but its concentration increased almost up to the level similar of SAC through aging process of AGE. S1PC showed immunomodulatory effects in vitro and in vivo, and reduced blood pressure in a hypertensive animal model. A pharmacokinetic study revealed that S1PC was readily absorbed after oral administration in rats and dogs with bioavailability of 88-100%. Additionally, S1PC had little inhibitory influence on human cytochrome P450 activities, even at a concentration of 1 mM. Based on these findings, S1PC was suggested to be another important, pharmacologically active and safe component of AGE similar to SAC. In this review, we highlight some results from recent studies on S1PC and discuss the potential medicinal value of S1PC.

Metabolomic study on the antihypertensive effect of S-1-propenylcysteine in spontaneously hypertensive rats using liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry.

Aged garlic extract (AGE) has been shown to improve hypertension in both clinical trials and experimental animal models. However, the active ingredient of AGE remains unknown. In the present study, we investigated the antihypertensive effects of AGE and its major constituents including S-1-propenylcysteine (S1PC) and S-allylcysteine (SAC) using spontaneously hypertensive rats (SHR) and found that S1PC is an active substance to lower blood pressure in SHR. In addition, the metabolomics approach was used to investigate the potential mechanism of the antihypertensive action of S1PC in SHR. Treatment with AGE (2g/kg body weight) or S1PC (6.5mg/kg body weight; equivalent to AGE 2g/kg body weight) significantly decreased the systolic blood pressure (SBP) of SHR after the repeated administration for 10 weeks, whereas treatment with SAC (7.9mg/kg body weight; equivalent to AGE 2g/kg body weight) did not decrease the SBP. After the treatment for 10 weeks, the plasma samples obtained from Wistar Kyoto (WKY) rats and SHR were analyzed by means of ultra high performance liquid chromatography coupled with high-resolution quadrupole-Orbitrap mass spectrometry. Multivariate statistical analysis of LC-MS data showed a clear difference in the metabolite profiles between WKY rats and SHR. The results indicated that 30 endogenous metabolites significantly contributed to the difference and 7 of 30 metabolites were changed by the S1PC treatment. Furthermore, regression analysis showed correlation between SBP and the plasma levels of betaine, tryptophan and 3 LysoPCs. This metabolomics approach suggested that S1PC could exert its antihypertensive effect by affecting glycine, serine and threonine metabolism, tryptophan metabolism and glycerophospholipid metabolism.

S-1-Propenylcysteine promotes the differentiation of B cells into IgA-producing cells by the induction of Erk1/2-dependent Xbp1 expression in Peyer's patches.

OBJECTIVES: S-Allylcysteine (SAC) and S-1-propenylcysteine (S1PC) are the characteristic sulfur-containing amino acids in aged garlic extract. In this study, we investigated the effect of SAC and S1PC on intestinal immunoglobulin (Ig)A production to gain insight into the immunomodulatory effect of aged garlic extract. METHODS: In vitro study: Mouse splenic lymphocytes were treated with S1PC (0.1 and 0.3 mM) or SAC (0.1 and 0.3 mM) for 3 d, and IgA concentration in the culture medium was examined. In vivo study: Mice were orally administrated S1PC (7.5, 15, and 30 mg/kg) for 5 d and the IgA level in the intestinal lavage fluids as well as the population of IgA-producing cells in Peyer's patches were measured using mouse IgA enzyme-linked immunosorbent assay quantification set and flow cytometer, respectively. RESULTS: S1PC enhanced IgA production in mouse splenic lymphocytes in culture. However, SAC was ineffective. In addition, oral administration of S1PC to mice increased the IgA level and number of IgA-producing cells in Peyer's Patches. Furthermore, S1PC induced the expression of X-box binding protein 1 (Xbp1) mRNA, an inducer of plasma cell differentiation, in Peyer's patches. This induction was accompanied by the degradation of paired box protein 5 and the activation of mitogen activated protein/extracellular signal-regulated kinase signaling pathway. CONCLUSION: These results suggest that S1PC increases IgA-producing cells via the enhancement of Erk1/2-mediated Xbp1 expression in the intestine.

Development of an Analytic Method for Sulfur Compounds in Aged Garlic Extract with the Use of a Postcolumn High Performance Liquid Chromatography Method with Sulfur-Specific Detection.

BACKGROUND: Garlic and its processed preparations contain numerous sulfur compounds that are difficult to analyze in a single run using HPLC. OBJECTIVE: The aim of this study was to develop a rapid and convenient sulfur-specific HPLC method to analyze sulfur compounds in aged garlic extract (AGE). METHODS: We modified a conventional postcolumn HPLC method by employing a hexaiodoplatinate reagent. Identification and structural analysis of sulfur compounds were conducted by LC-mass spectrometry (LC-MS) and nuclear magnetic resonance. The production mechanisms of cis-S-1-propenylcysteine (cis-S1PC) and S-allylmercaptocysteine (SAMC) were examined by model reactions. RESULTS: Our method has the following advantages: less interference from nonsulfur compounds, high sensitivity, good correlation coefficients (r > 0.98), and high resolution that can separate >20 sulfur compounds, including several isomers, in garlic preparations in a single run. This method was adapted for LC-MS analysis. We identified cis-S1PC and γ-glutamyl-S-allyl-mercaptocysteine in AGE. The results of model reactions suggest that cis-S1PC is produced from trans-S1PC through an isomerization reaction and that SAMC is produced by a reaction involving S-allylcysteine/S1PC and diallyldisulfide during the aging period. CONCLUSION: We developed a rapid postcolumn HPLC method for both qualitative and quantitative analyses of sulfur compounds, and this method helped elucidate a potential mechanism of cis-S1PC and SAMC action in AGE.

Dehydrodiconiferyl alcohol suppresses monocyte adhesion to endothelial cells by attenuation of JNK signaling pathway.

Several clinical studies have shown that the intake of aged garlic extract improves endothelial dysfunction. Lignan compounds, (+)-(2S,3R)-dehydrodiconiferyl alcohol (DDC) and (-)-(2R,3S)-dihydrodehydrodiconiferyl alcohol (DDDC), have been isolated as antioxidants in aged garlic extract. There is evidence showing the importance of oxidative stress in endothelial dysfunction. In the present study, we examined whether DDC and DDDC enhance endothelial cell function in vitro. Cell adhesion assay was performed using THP-1 monocyte and human umbilical vein endothelial cells (HUVECs) which were activated by lipopolysaccharide (LPS) or advanced glycation end products (AGEs)-BSA. Cellular ELISA method was used for the evaluation of vascular cell adhesion molecule 1 (VCAM-1) expression on HUVECs. DDC and DDDC suppressed the adhesion of THP-1 to HUVECs which was activated by LPS or AGEs-BSA. DDC and DDDC also inhibited VCAM-1 expression induced by LPS or AGEs-BSA, but DDDC was less effective than DDC. In addition, the inhibitory effect of DDC on VCAM-1 expression involved suppressing JNK/c-Jun pathway rather than NF-κB pathway. DDC has an inhibitory effect on VCAM-1 expression via JNK pathway in endothelial cells and therefore may serve as a novel pharmacological agent to improve endothelial dysfunction.

In vitro activity-guided identification of antioxidants in aged garlic extract.

Activity-guided fractionation was applied on an aged garlic extract (AGE), reported to show strong antioxidant activity, in order to locate the key in vitro antioxidant ingredients by means of the hydrogen peroxide scavenging (HPS) assay as well as the ORAC assay. Besides the previously reported four tetrahydro-ß-carbolines, (1R,3S)- and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid and (1R,3S)- and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-ß-carboline-1,3-dicarboxylic acid, LC-MS/MS, LC-TOF-MS, and 1D/2D-NMR experiments led to the identification of coniferyl alcohol and its dilignols (-)-(2R,3S)-dihydrodehydrodiconiferyl alcohol, (+)-(2S,3R)-dehydrodiconiferyl alcohol, erythro-guaiacylglycerol-ß-O-4'-coniferyl ether, and threo-guaiacylglycerol-ß-O-4'-coniferyl ether as the major antioxidants in AGE. The purified individual compounds showed high antioxidant activity, with EC50 values of 9.7-11.8 µM (HPS assay) and 2.60-3.65 µmol TE/µmol (ORAC assay), respectively.

Isolation and structure elucidation of highly antioxidative 3,8″-linked biflavanones and flavanone-C-glycosides from Garcinia buchananii bark.

The aim of this study was to identify antioxidants from Garcinia buchananii bark extract using hydrogen peroxide scavenging and oxygen radical absorbance capacity (ORAC) assays. LC-MS/MS analysis, 1D- and 2D-NMR, and circular dichroism (CD) spectroscopy led to the unequivocal identification of the major antioxidative molecules as a series of three 3,8″-linked biflavanones and two flavanone-C-glycosides. Besides the previously reported (2R,3R,2″R,3″R)-naringenin-C-3/C-8″ dihydroquercetin linked biflavanone (GB-2; 4) and (2R,3S,2″R,3″R)-manniflavanone (3), whose stereochemistry has been revised, the antioxidants identified for the first time in Garcinia buchananii were (2R,3R)-taxifolin-6-C-ß-D-glucopyranoside (1), (2R,3R)-aromadendrin-6-C-ß-D-glucopyranoside (2), and the new compound (2R,3S,2″S)-buchananiflavanone (5). The H2O2 scavenging and the ORAC assays demonstrated that these natural products have an extraordinarily high antioxidative power, especially (2R,3S,2″R,3″R)-manniflavanone (3) and GB-2 (4), with EC50 values of 2.8 and 2.2 µM, respectively, and 13.73 and 12.10 µmol TE/ µmol. These findings demonstrate that G. buchananii bark extract is a rich natural source of antioxidants.