HP0986 protein of Helicobacter pylori has been shown to trigger induction of proinflammatory cytokines (IL-8 and TNF-α) through the activation of NF-κB and also to induce Fas mediated apoptosis of human macrophage cells (THP-1). In this study, we unravel mechanistic details of the biological effects of this protein in a murine macrophage environment. Up regulation of MCP-1 and TNF-α in HP0986-induced RAW 264.7 cells occurred subsequent to the activation and translocation of NF-κB to the cell nucleus. Further, HP0986 induced apoptosis of RAW 264.7 cells through Fas activation and this was in agreement with previous observations made with THP-1 cells. Our studies indicated activation of TNFR1 through interaction with HP0986 and this elicited the aforementioned responses independent of TLR2, TLR4 or TNFR2. We found that mouse TNFR1 activation by HP0986 facilitates formation of a complex comprising of TNFR1, TRADD and TRAF2, and this occurs upstream of NF-κB activation. Furthermore, FADD also forms a second complex, at a later stage, together with TNFR1 and TRADD, resulting in caspase-8 activation and thereby the apoptosis of RAW 264.7 cells. In summary, our observations reveal finer details of the functional activity of HP0986 protein in relation to its behavior in a murine macrophage cell environment. These findings reconfirm the proinflammatory and apoptotic role of HP0986 signifying it to be an important trigger of innate responses. These observations form much needed baseline data entailing future in vivo studies of the functions of HP0986 in a murine model.
In this work, silver (Ag) decorated reduced graphene oxide (rGO) coated with ultrafine CuO nanosheets (Ag-rGO@CuO) was prepared by the combination of a microwave-assisted hydrothermal route and a chemical methodology. The prepared Ag-rGO@CuO was characterized for its morphological features by field emission scanning electron microscopy and transmission electron microscopy while the structural characterization was performed by X-ray diffraction and Raman spectroscopy. Energy-dispersive X-ray analysis was undertaken to confirm the elemental composition. The electrochemical performance of prepared samples was studied by cyclic voltammetry and galvanostatic charge-discharge in a 2M KOH electrolyte solution. The CuO nanosheets provided excellent electrical conductivity and the rGO sheets provided a large surface area with good mesoporosity that increases electron and ion mobility during the redox process. Furthermore, the highly conductive Ag nanoparticles upon the rGO@CuO surface further enhanced electrochemical performance by providing extra channels for charge conduction. The ternary Ag-rGO@CuO nanocomposite shows a very high specific capacitance of 612.5 to 210 Fg-1 compared against rGO@CuO which has a specific capacitance of 375 to 87.5 Fg-1 and the CuO nanosheets with a specific capacitance of 113.75 to 87.5 Fg-1 at current densities 0.5 and 7 Ag-1, respectively.
The present work was employing the Quality by Design approach for the development and validation of a LC-MS-MS method to support the clinical advancement in determination of sildenafil in human plasma using lorazepam as an internal standard. Sample preparation involved solid phase extraction and calibration range observed between 3 and 1,700 ng/mL. The method was systematically optimized by employing Box-Behnken design and used mobile phase flow rate, pH and composition of mobile phase as the critical factors, and assessing the design for retention time and peak area as the responses. A substantial decrease in the variability associated with the method variables was shown in optimization studies and confirmed enhanced method robustness. The present studies revealed that developed method achieves all the regulatory requirements for linearity, accuracy, precision, selectivity, sensitivity and stability for the determination of sildenafil in human plasma. There was not any significant change in the stability of the drug shown by stability studies, performed in human plasma through freeze-thaw cycles, bench-top stability, short-term stability, long-term stability and auto sampler stability. In short, this method shows satisfactory results for the analysis of sildenafil in human plasma and possesses high degree of utility in pharmacokinetic and bioequivalence studies.
Traditionally, plants of the genus Calotropis have been used to cure various common diseases. The present research work explores the chemical and biological characterization of one of the most common species of this genus, i.e., Calotropis gigantea (L.) Dryand (syn. Calotropis gigantea (L.) Dryand.), having multiple folklore applications. The ethanolic extract of leaves of Calotropis gigantea (L.) Dryand was analyzed for the phytochemical composition by determining the total bioactive (total phenolic and total flavonoid) contents and UHPLC-MS secondary metabolites analysis. For phytopharmacological evaluation, in vitro antioxidant (including DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum, and metal chelation antioxidant assays) activities, enzyme inhibition potential (against AChE, BChE, α-amylase, and tyrosinase enzymes), and in vivo wound healing potential were determined. The tested extract has been shown to contain considerable flavonoid (46.75 mg RE/g extract) and phenolic (33.71 mg GAE/g extract) contents. The plant extract presented considerable antioxidant potential, being the most active for CUPRAC assays. Secondary metabolite UHPLC-MS characterization, in both the positive and negative ionization modes, indicated the tentative presence of 17 different phytocompounds, mostly derivatives of sesquiterpene, alkaloids, and flavonoids. Similarly, the tested extract exhibited considerable inhibitory effects on tyrosinase (81.72 mg KAE/g extract), whereas it showed weak inhibition ability against other tested enzymes. Moreover, in the case of in vivo wound healing assays, significant improvement in wound healing was observed in both the tested models at the doses of 0.5 percent w/w (p < 0.001) and 2.0 percent w/w (p < 0.01) on the 16th day. The outcomes of the present research work suggested that C. gigantea (L.) Dryand plant extract could be appraised as a potential origin of bioactive molecules having multifunctional medicinal uses.
Trillium govanianum is a high-value medicinal herb, having multifunctional traditional and culinary uses. The present investigation was carried out to evaluate the phytochemical, biological and toxicological parameters of the T. govanianum Wall. ex D. Don (Family: Trilliaceae) roots collected from Azad Kashmir, Pakistan. Phytochemical profiling was achieved by determining total bioactive contents (total phenolic and flavonoid contents) and UHPLC-MS analysis. For biological evaluation, antioxidant activities (DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum, and metal chelation assays) and enzyme inhibition activities (against AChE, BChE, glucosidase, amylase, and tyrosinase) were performed. Moreover, cytotoxicity was assessed against three human carcinoma cell lines (MDA-MB-231, CaSki, and DU-145). The tested extract was found to contain higher total phenolics (7.56 mg GAE/g dry extract) as compared to flavonoid contents (0.45 mg RE/g dry extract). Likewise, for the antioxidant activity, higher CUPRAC activity was noted with 39.84 mg TE/g dry extract values. In the case of enzyme assays, higher activity was pointed out against the cholinesterase, glucosidase and tyrosinase enzymes. The plant extract displayed significant cytotoxicity against the cell lines examined. Moreover, the in-silico studies highlighted the interaction between the important phytochemicals and tested enzymes. To conclude, the assessed biological activity and the existence of bioactive phytochemicals in the studied plant extract may pave the way for the development of novel pharmaceuticals.