Dalspinin isolated from Spermacoce hispida (Linn.) protects H9c2 cardiomyocytes from hypoxic injury by modulating oxidative stress and apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Spermacoce hispida (S.hispida), a potential medicinal plant has been traditionally used as an antibacterial, antieczemic, antihypertensive, antidiabetic and antihyperlipidemic agent. Although, this plant has been claimed to protect against oxidative injury and inflammatory conditions in recent studies, its cardioprotective effect and the active constituents responsible for its bioactivity is sparsely studied. Hence this work is undertaken to study the active biomolecule responsible for modulating the cardiomyocytes on hypoxic injury relevant to its ethanopharmacology. AIM OF THE STUDY: The current study is to isolate and characterize a bioactive molecule from S.hispida, which exhibits protection against hypoxic injury in an in vitro hypoxic model of cultured H9c2 cardiomyocytes. MATERIALS AND METHODS: The methanolic extract of S.hispida plant was fractionated with various solvents sequentially. The ethyl acetate fraction that was concentrated and chromatographed over silica gel column eluted 18 fractions, which yielded 5 compounds, which were characterized using spectral data. The isolated new compound was further tested for its protective effect against hypoxic injury, wherein cobalt chloride (CoCl2) was used to induce hypoxia in H9c2 cardiomyoblasts. To evaluate the protective effect of the isolated compound, the markers of oxidative stress, apoptosis, and cell death were checked by endogenous levels of antioxidants, [malondialdehyde (MDA), superoxide dismutase (SOD), reduced glutathione (GSH)], lactate dehydrogenase (LDH) activity and immunoblot (HIF-α, Bcl2, Bax, procaspase and cleaved caspase-3). RESULTS: Among the five compounds isolated and characterized from S. hispida methanolic extract, ß-sitosterol, ursolic acid, quercetin and rutin were known phytochemicals, while the new isoflavone was identified as dalspinin-7-0-ß-D-galactopyranoside (DBG). Among the isolated compounds, the antioxidant potential of DBG confirmed by DPPH free radical scavenging and ORAC assays was superior. CoCl2-induced hypoxic condition significantly decreased cell viability, SOD activity, GSH concentration and increased the level of MDA and LDH activity. Western blot studies revealed an upregulation of HIF-1α, Bax and caspase and down regulation of Bcl-2 expression. The oxidative abnormalities were ameliorated by DBG pretreatment, as deduced by the reduced CoCl2-induced cytotoxicity, MDA concentration, LDH activity and the expression of HIF-1α, Bax and caspase and the enhanced levels of SOD, GSH and Bcl2 expression in a dose-dependent manner. CONCLUSION: DBG protects H9c2 cells from CoCl2-induced hypoxic damage by mitigating oxidative stress and preserving cell viability. The overall findings highlight the protective action of DBG, a potential source of antioxidant of natural origin against hypoxic injury and may help in mitigating the progress of oxidative stress in cardiac cell death.

A natural anticancer pigment,Pheophytin a,from a seagrass acts as a high affinity human mitochondrial translocator protein (TSPO) ligand, in silico, to reduce mitochondrial membrane Potential (∆ψmit) in adenocarcinomic A549 cells.

BACKGROUND: The present investigation looks at the most likely possibilities of usage of a naturally occurring photosynthetic pigment, Pheophytin a, from the seagrass, Syringodium isoetifolium, for plausible use as human TSPO ligand. METHODS: Pheophytin a isolated in our laboratory previously was administered to A549 cell lines in vitro to examine its effects on cell migrations, DNA, cell cycle, Mitochondrial Membrane Potential and gene expressions. In silico tools were used to predict the nature of the compound and target binding. RESULTS: Pheophytin a hadIC50 values of 22.9 ±â€¯5.8 µM for cancerous A549 cell lines, whilst not targeting non-cancerous vero cells [IC50: 183.6 ±â€¯1.92 µM]. Pheophytin a hindered cellular migration, fragmented DNA, arrested cell cycle precisely at S phase, reduced ∆ψmit and directed mRNA expressions toward apoptosis. In silico tools indicate that the compound binds to TSPO with high effectiveness to collapse ∆ψmit(which is proved using wet lab experiments) to promote mitophagy. CONCLUSION: Hence Pheophytin a could be seen as a possible TSPO ligand for targeting metastatic alveolar cancers like A549 via intrinsic apoptotic pathway. GENERAL SIGNIFICANCE: Given the inherent non-toxic nature of the compound and easy extractability from almost all autotrophic eukaryotes, one could be confident to testing in animal models.

Bioassay guided fractionation and identification of active anti-inflammatory constituent from Delonix elata flowers using RAW 264.7 cells.

CONTEXT: Delonix elata (L.) Gamble (Fabaceae) has been used in the Indian traditional medicine system to treat rheumatism and inflammation. AIM: To assess the anti-inflammatory effect of Delonix elata flowers and to isolate the active principle. MATERIALS AND METHODS: The prompt anti-inflammatory constituent was isolated from Delonix elata flower extracts using bioassay guided fractionation in liposaccharide (LPS) stimulated RAW 264.7 macrophage cell line. The anti-inflammatory activity of extracts/fractions/sub-fractions/compounds (10, 25, and 50 µg/ml) was evaluated by estimating the levels of nitric oxide (NO), TNF-α, and IL-1ß after 24 h of LPS induction (1 µg/ml). The isolated active compound was subjected to NMR, IR, and UV analyses for structure determination. RESULTS: In an attempt to search for anti-inflammatory constituents, the active pure principle was isolated and crystallized as a white compound from Delonix elata flowers methanol extract. This active compound (50 µg/ml) decreased the release of inflammatory mediators levels such as NO (0.263 ± 0.03 µM), TNFα (160.20 ± 17.57 pg/ml), and IL-1ß (285.79 ± 15.16 pg/ml) significantly (p < 0.05); when compared to the levels of NO (0.774 ± 0.08 µM), TNFα (501.71 ± 25.14 pg/ml), and IL-1ß (712.68 ± 52.25 pg/ml) from LPS-stimulated macrophage cells. The active compound was confirmed as hesperidin with NMR, IR, and UV spectroscopy data. This is the first report of this compound from Delonix elata flowers. CONCLUSION: The findings of the study support the traditional use of Delonix elata flowers to treat inflammation.

Spectroscopic investigations, antimicrobial, and cytotoxic activity of green synthesized gold nanoparticles.

The gold nanoparticles (AuNPs) were synthesized by using naturally available Punica Granatum fruit extract as reducing and stabilizing agent. The biosynthesized AuNPs was characterized by using UV-Vis, fluorescence, high resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) and thermogravimetric (TGA) analysis. The surface plasmon resonance (SPR) band at 585nm confirmed the reduction of auric chloride to AuNPs. The crystalline nature of the biosynthesized AuNPs was confirmed from the HRTEM images, XRD and selected area electron diffraction (SAED) pattern. The HRTEM images showed the mixture of triangular and spherical-like AuNPs having size between 5 and 20nm. The weight loss of the AuNPs was measured by TGA as a function of temperature under a controlled atmosphere. The biomolecules are responsible for the reduction of AuCl4(-) ions and the formation of stable AuNPs which was confirmed by FTIR measurement. The synthesized AuNPs showed an excellent antibacterial activity against Candida albicans (ATCC 90028), Aspergillus flavus (ATCC 10124), Staphylococcus aureus (ATCC 25175), Salmonella typhi (ATCC 14028) and Vibrio cholerae (ATCC 14033). The minimum inhibitory concentration (MIC) of AuNPs was recorded against various microorganisms. Further, the synthesized AuNPs shows an excellent cytotoxic result against HeLa cancer cell lines at different concentrations.

In vitro studies on α-glucosidase inhibition, antioxidant and free radical scavenging activities of Hedyotis biflora L.

Aim of this study was to evaluate the in vitro α-glucosidase inhibition and antioxidant activity of hexane, ethyl acetate and methanol extracts of Hedyotis biflora L. (Rubiaceae). In in vitro α-glucosidase inhibition and antioxidant activity, the methanol extract showed potent effect compared to hexane and ethyl acetate extracts. The methanol extract of H. biflora (HBMe) showed 50% α-glucosidase inhibition at the concentration of 480.20 ± 2.37 µg/ml. The total phenolic content of HBMe was 206.81 ± 1.11 mg of catechol equivalents/g extract. HBMe showed great scavenging activity on 2,2-diphenyl-picrylhydrazyl (DPPH) (IC(50) 520.21 ± 1.02 µg/ml), hydroxyl (IC(50) 510.21 ± 1.51 µg/ml), nitric oxide (IC(50) 690.20 ± 2.13 µg/ml) and superoxide (IC(50) 510.31 ± 1.45 µg/ml) radicals, as well as high reducing power. HBMe also showed a strong suppressive effect on lipid peroxidation. Using the ß-carotene method, the scavenging values of HBMe was significantly lower than BHT, and metal chelating ability of HBMe also showed a strong inhibition effect when compared to the reference standard. The active compound ursolic acid from HBMe was identified using various spectroscopical studies. The results obtained in this study clearly indicate that HBMe has a significant potential to use as a natural α-glucosidase inhibition, antioxidant agent.