R Lakshmi Sundaram1, Hannah R Vasanthi2. 1. Central Research Facility, Sri Ramachandra Medical College and Research Institute, Porur, Chennai, 600 116, Tamil Nadu, India. 2. Deparment of Biotechnology, Pondicherry University, Pondicherry, 605 014, India. Electronic address: hannah.dbt@pondiuni.edu.in.
Abstract
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, β-sitosterol, ursolic acid, quercetin and rutin were known phytochemicals, while the new isoflavone was identified as dalspinin-7-0-β-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.
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. hispidamethanolic extract, β-sitosterol, ursolic acid, quercetin and rutin were known phytochemicals, while the new isoflavone was identified as dalspinin-7-0-β-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.