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.

The reduction of methane emissions by ruminants is a highly desirable goal to mitigate greenhouse gas emissions. Various feed additives have already been tested for their ability to decrease methane production; however, practical use is often limited due to negative effects on rumen fermentation or high costs. Organosulphur compounds from garlic (Allium sativum) and flavonoids have been identified as promising plant-derived compounds which are able to reduce methane production. Here, we evaluated the effects of a combination of garlic powder and bitter orange (Citrus aurantium) extracts, Mootral, on ruminal methane production, ruminal fermentation and the community of methanogenic Archaea by using the rumen simulation technique as ex vivo model. The experiment consisted of an equilibration period of 7 days, an experimental period of 8 days and a withdrawal period of 4 days. During the experimental period three fermenters each were either treated as controls (CON), received a low dose of Mootral (LD), a high dose of Mootral (HD), or monensin (MON) as positive control. Application of Mootral strongly reduced the proportion of methane in the fermentation gas and the production rate of methane. Moreover, the experimental mixture induced a dose-dependent increase in the production rate of short chain fatty acids and in the molar proportion of butyrate. Some effects persisted during the withdrawal period. Both, single strand conformation polymorphism and Illumina MiSeq 16S rRNA amplicon sequencing indicated an archaeal community distinct from CON and MON samples in the LD and HD samples. Among archaeal families the percentage of Methanobacteriaceae was reduced during application of both doses of Mootral. Moreover, several significant differences were observed on OTU level among treatment groups and after withdrawal of the additives for LD and HD group. At day 14, 4 OTUs were positively correlated with methane production. In conclusion this mixture of garlic and citrus compounds appears to effectively reduce methane production by alteration of the archaeal community without exhibiting negative side effects on rumen fermentation.

Two metal oxide (i.e., Al2O3 and TiO2) nanoparticles and ajoene, a garlic-derived organosulfur compound, were identified to be effective antimicrobials against Campylobacter jejuni, a leading cause of human gastrointestinal diseases worldwide. A significant synergistic antimicrobial effect was observed using ajoene and Al2O3/TiO2 nanoparticles in a combined manner to cause at least 8 log10 CFU/mL reduction of C. jejuni cells. Whole transcriptome sequencing (RNA-seq) and confocal micro-Raman spectroscopic analyses revealed the antimicrobial mechanism and identified the roles of ajoene and metal oxide nanoparticles in the synergistic treatment. Ajoene and metal oxide nanoparticles mediated a two-phase antimicrobial mechanism. Ajoene served as the inducing factor at the first phase that caused injury of cell membranes and increased the susceptibility of C. jejuni to stress. Metal oxide nanoparticles served as the active factor at the second phase that targeted sensitive cells and physically disrupted cell structure. This synergistic antimicrobial treatment demonstrates a potential to reduce the prevalence of C. jejuni and other pathogens on food contact surfaces and in the food chain.

Epilepsy is a brain disorder characterized by sudden recurrent seizures. Considering the fact that epileptogenesis is a process that affects the quality of life, our goal is to delay the process of epileptogenesis and to increase the latency of epileptic attacks, offering better quality of life to patients. Traditional system of medicines has a promise in some of the medicines, which have been used for the treatment of epilepsy. One such medicinal plant is Eclipta alba (EA). According to Ayurvedic philosophy, the juice of leaves of EA is pounded with garlic and pepper for the treatment of epilepsy. Taking clue from the Ayurvedic system of medicines, we formulated coumarin fraction of EA, namely, coumarin nasal formulation (CNF) for its nasal delivery. CNF was analyzed by using high performance liquid chromatography (HPLC) and ultraviolet absorption spectroscopy for its drug content determination. In vitro drug release studies were performed in simulated nasal electrolyte solution (SNES) maintaining constant pH of 5.5 at 37°C. Irritation by CNF was evaluated using hen's egg test chorioallantoic membrane (HET-CAM) assay. Formulation was found to be non-irritant in HET-CAM assay. CNF was further assessed in vivo by measuring the progress and attainment of pentylenetetrazole (PTZ) kindling in mice. Neuronal changes were assessed by hematoxylin and eosin (H&E) and Nissl staining technique. Glial fibrillary acidic protein (GFAP) a neuroinflammatory marker and tumor necrosis factor alpha (TNF-α) an inflammatory marker were also measured. CNF (10 mg/kg, nasal route) when given as a pretreatment lowered seizure score and delayed the progression of seizure similar to diazepam. CNF decreased the PTZ induced oxidative damage, TNF-α as well as GFAP levels in the midbrain tissue particularly in hippocampus region. The results suggest that CNF may be a promising therapeutic approach to offer protection from sudden recurrent seizures alone or in combination with current drugs in management of epilepsy.

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 1 month to 22 months. The formation of these compounds occurred mostly during the early stage of the aging period: SAMC content reached a maximum at approximately 4 months, whereas GSAMC content reached a maximum at 1 month and then decreased during the subsequent aging period. In order 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 aging process.

Several plants of agricultural and medicinal importance utilize defense chemistry that involves deployment of highly labile, reactive and lachrymatory organosulfur molecules. However, this chemistry is difficult to investigate because the compounds are often short-lived and prone to degradation under the conditions required for analysis by common analytical techniques. This issue has complicated efforts to study the defense chemistry of plants that exploit the use of sulfur in their defense arsenals. This work illustrated how direct analysis in real time-high resolution mass spectrometry (DART-HRMS) can be used to track organosulfur defense compound chemistry under mild conditions. Petiveria alliacea was used a model plant that exploits the enzyme alliinase to generate induced organosulfur compounds in response to herbivory. Tracking of the organosulfur compounds it produces and quantifying them by DART-HRMS using isotopically-labeled analogues, revealed a feedback inhibition loop through which the activities of the alliinase are stymied shortly after their activation. The results show that the downstream thiosulfinate product petivericin (100 µM) and pyruvate (8.4 mM) inhibit alliinase activity by 60% and 29%, respectively after 1 h, and a mixture of the two inhibited alliinase activity by 65%. By 2 h, alliinase activity in the presence of these alliinase-derived products had ceased completely. Since thiosulfinate, pyruvate and lachrymatory sulfine compounds are produced via the same alliinase-derived sulfenic acid intermediate, the inhibition of alliinase activity by increasing concentrations of downstream products shows how production of these defense compounds is modulated in real-time in response to a tissue breach. These findings provide a framework within which heretofore unexplained phenomena observed in the defense chemistry of P. alliacea, onion, garlic, and other plants can be explained, and an approach by which to track labile compounds as well as enzymatic activity by DART-HRMS.

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.

Garlic virus X (GarVX) encodes a 15 kDa cysteine-rich protein (CRP). To investigate the function(s) of p15, its subcellular localization, role as a symptom determinant and capacity to act as a viral suppressor of RNA silencing (VSR) were analysed. Results showed that GFP-tagged p15 was distributed in the cytoplasm, nucleus and nucleolus. Expression of p15 from PVX caused additional systemic foliar malformation and led to increased accumulation of PVX, showing that p15 is a virulence factor for reconstructed PVX-p15. Moreover, using a transient agro-infiltration patch assay and a Turnip crinkle virus (TCV) movement complementation assay, it was demonstrated that p15 possesses weak RNA silencing suppressor activity. Removal of an amino acid motif resembling a nuclear localization signal (NLS) prevented p15 from accumulating in the nucleus but did not abolish its silencing suppression activity. This study provides the first insights into the multiple functions of the GarVX p15 protein.