Exposure of Helicobacter pylori to clarithromycin in vitro resulting in the development of resistance and triggers metabolic reprogramming associated with virulence and pathogenicity.

In H. pylori infection, antibiotic-resistance is one of the most common causes of treatment failure. Bacterial metabolic activities, such as energy production, bacterial growth, cell wall construction, and cell-cell communication, all play important roles in antimicrobial resistance mechanisms. Identification of microbial metabolites may result in the discovery of novel antimicrobial therapeutic targets and treatments. The purpose of this work is to assess H. pylori metabolomic reprogramming in order to reveal the underlying mechanisms associated with the development of clarithromycin resistance. Previously, four H. pylori isolates were induced to become resistant to clarithromycin in vitro by incrementally increasing the concentrations of clarithromycin. Bacterial metabolites were extracted using the Bligh and Dyer technique and analyzed using metabolomic fingerprinting based on Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-Q-ToF-MS). The data was processed and analyzed using the MassHunter Qualitative Analysis and Mass Profiler Professional software. In parental sensitivity (S), breakpoint isolates (B), and induced resistance isolates (R) H. pylori isolates, 982 metabolites were found. Furthermore, based on accurate mass, isotope ratios, abundances, and spacing, 292 metabolites matched the metabolites in the Agilent METLIN precise Mass-Personal Metabolite Database and Library (AM-PCDL). Several metabolites associated with bacterial virulence, pathogenicity, survival, and proliferation (L-leucine, Pyridoxone [Vitamine B6], D-Mannitol, Sphingolipids, Indoleacrylic acid, Dulcitol, and D-Proline) were found to be elevated in generated resistant H. pylori isolates when compared to parental sensitive isolates. The elevated metabolites could be part of antibiotics resistance mechanisms. Understanding the fundamental metabolome changes in the course of progressing from clarithromycin-sensitive to breakpoint to resistant in H. pylori clinical isolates may be a promising strategy for discovering novel alternatives therapeutic targets.

Understanding the effects of Moringa oleifera in chronic unpredictable stressed zebrafish using metabolomics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Moringa leaves have been used for thousands of years to maintain skin health and mental fitness. People also use it to relieves pain and stress. AIM OF THE STUDY: To determine the effects of Moringa oleifera leaves ethanol-aqueous (ratio 7:3) extract (MOLE) on the chronically stressed zebrafish. METHOD: The changes in the stress-related behaviour and the metabolic pathways in response to MOLE treatment in zebrafish were studied. A chronic unpredictable stress model was adopted in which zebrafish were induced with different stressors for 14 days. Stress-related behaviour was assessed using a depth-preference test and a light and dark test. Three doses of MOLE (500, 1000, and 2000 mg/L) were administered to the zebrafish. Upon sacrifice, the brains were harvested and processed for LC-MS QTOF based, global metabolomics analysis. RESULTS: We observed significant changes in the behavioural parameters, where the swimming time at the light phase and upper phase of the tank were increased in the chronically stressed zebrafish treated with MOLE compared to those zebrafish which were not treated. Further, distinctive metabolite profiles were observed in zebrafish with different treatments. Several pathways that shed light on effects of MOLE were identified. MOLE is believed to relieve stress by regulating pathways that are involved in the metabolism of purine, glutathione, arginine and proline, D-glutamine, and D-glutamate. CONCLUSION: MOLE is potentially an effective stress reliever. However, its effects in human needs to be confirmed with a systematic randomised control trial.

Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Vardenafil and Its Application of Bioequivalence.

A simple, rapid, and sensitive method of liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of vardenafil in rabbit plasma. A simple protein precipitation method with ice-cold acetonitrile was used for plasma extraction. The mass transitions m/z 489⟶151 and m/z 390⟶169 were used to measure vardenafil and tadalafil (internal standard), respectively, with a total assay run time of 6 min. The limit of detection was 0.2 ng/mL. The assay was reproducible with intra-assay and interassay precision ranging 1.17%-9.17% and 1.31%-5.86%, respectively. There was also good intra-assay and interassay accuracy between 89.3%-105.3% and 94%-102% of the expected value, respectively. The linearity range was 0.5-60 ng/mL in rabbit plasma (r 2 ≥ 0.99). The measured AUC from 0 to 24 h (AUC0 - 24t ) for the test and reference formulations were 174.38 ± 95.91 and 176.45 ± 76.88, respectively. For the test, C max and T max were 75.36 ± 59.53 ng/mL and 1.42 ± 0.19 h, whereas, for the reference, these were 58.22 ± 36.11 ng/mL and 2.04 ± 0.33 h, respectively. The test formulation achieved a slightly lower AUC0 - 24t value (p > 0.05), higher C max values (p > 0.05), faster T max (p < 0.05), and almost equal bioavailability compared with the reference formulation.

Combined extract of Moringa oleifera and Centella asiatica modulates oxidative stress and senescence in hydrogen peroxide-induced human dermal fibroblasts.

Moringa oleifera Lam. and Centella asiatica (L.) Urb. leaves have been previously reported to exhibit antioxidant activity. The objective of the present study is to determine the in vitro antioxidant activity of the combined extracts of M. oleifera and C. asiatica (TGT-PRIMAAGE) and its effect on hydrogen peroxide (H 2O2)-induced oxidative stress in human dermal fibroblasts. TGTPRIMAAGE acted on the mechanism of hydrogen transfer as it showed scavenging activity in the DPPH assay. This is due to the presence of phenolics and flavonoids in TGT-PRIMAAGE. TGT-PRIMAAGE effectively reduced cellular generation of reactive oxygen species induced by H O2. The activities of superoxide dismutase and catalase were also increased in cells treated with TGT-PRIMAAGE. 2 Treatment with TGT-PRIMAAGE showed significant reduction (P < 0.05) in the number of senescent cells. Significant reduction (P < 0.05) of malondialdehyde was also seen in cells treated with TGT-PRIMAAGE. The p53 protein level was reduced in TGT-PRIMAAGEtreated cells, which indicates its potential in protecting the cells from oxidative stress induced by H2O2.

Antiulcer activity of the chloroform extract of Bauhinia purpurea leaf.

CONTEXT: Bauhinia purpurea L. (Fabaceae) is a native plant species of many Asian countries, including Malaysia and India. In India, the root, stem, bark, and leaf of B. purpurea are used to treat various ailments, including ulcers and stomach cancer. OBJECTIVE: In an attempt to establish its pharmacological potential, we studied the antiulcer activity of lipid-soluble extract of B. purpurea obtained via extraction of air-dried leaves using chloroform. MATERIALS AND METHODS: The rats were administered the chloroform extract (dose range of 100-1000 mg/kg) orally after 24 h fasting. They were subjected to the absolute ethanol- and indomethacin-induced gastric ulcer, and pyloric ligation assays after 30 min. The acute toxicity study was conducted using a single oral dose of 5000 mg/kg extract and the rats were observed for the period of 14 days. omeprazole (30 mg/kg) was used as the standard control. RESULTS: At 5000 mg/kg, the extract produced no sign of toxicity in rats. The extract exhibited significant (p < 0.05) dose-dependent antiulcer activity for the ethanol-induced model. The extract also significantly (p < 0.05) increased the gastric wall mucus production and pH of gastric content, while significantly (p < 0.05) reducing the total volume and total acidity of the gastric content in the pylorus ligation assay. DISCUSSION AND CONCLUSION: The extract possesses antiulcer, antisecretory and cytoprotective activities, which could be attributed to its flavonoid and tannin content. These findings provide new information regarding the potential of lipid-soluble compounds of B. purpurea for the prevention and treatment of gastric ulcers.