Shilpi Singh1, Dhananjay Kumar Singh1, Abha Meena1, Vijaya Dubey1, Nusrat Masood1, Suaib Luqman2. 1. Molecular Bioprospection Department of Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, Uttar Pradesh, India. 2. Molecular Bioprospection Department of Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, Uttar Pradesh, India. Electronic address: s.luqman@cimap.res.in.
Abstract
BACKGROUND: Rutin (quercetin-3-O-rutinoside), a flavonoid, is predominantly found in the buckwheat, cranberries, mulberry and citrus fruits. It is used as a restorative in the preparation of herbal medicine, multivitamin and known to reduce the fate of heart attack. HYPOTHESIS: We aimed to elucidate whether rutin attenuates oxidative stress and its possible mechanism of action in ameliorating the deleterious effect of t-BHP. We also provide evidence that rutin protects the antioxidant status of erythrocytes and liver via Nrf2 and iNOS pathway from oxidative stress. STUDY DESIGN/ METHOD: Human erythrocytes and mice liver were used for the evaluation of rutin's effect against t-BHP induced oxidative stress. The non-enzymatic (GSH, MDA, -CO, -SH) and enzymatic stress markers (SOD, CAT, GPx, GR and GST) were estimated by the colorimetric method. The level of Nrf2, iNOS, liver marker enzymes, triglycerides, cholesterol, HDL-cholesterol, albumin, BUN was measured using ELISA kits. Reactive oxygen species (ROS) was quantified using flow cytometry and fluorometry. RT-PCR was used for the quantification of Nrf2 and iNOS expression levels in the liver tissue of mice. In silico studies were done through receptor-ligand binding interaction. RESULTS: Pre-treatment with the rutin ameliorated the toxic effect of t-BHP by modulating the basal level of GSH, -SH, MDA and -CO significantly (p < 0.01) with respect to untreated control. Rutin also protected the erythrocytes against the t-BHP-induced oxidative stress as evidenced by augmented activity of antioxidant enzymes (CAT, SOD, GPX, GR and GST). Furthermore, at the highest tested concentration (16.3 µM), it protected the morphology of the erythrocytes by decreasing the ROS level (p < 0.01). In addition, the lower MEF values of rutin (0.520 ± 0.005) alone or along with t-BHP (0.630 ± 0.021) indicated its non-toxic and protective behavior. The qPCR analyses revealed that t-BHP potently up-regulates the iNOS and down regulate the Nrf2 expression which was ameliorated with rutin treatment in a dose-dependent manner like silymarin. CONCLUSION: Our findings demonstrate that rutin potentiates its beneficial aspect by displaying a profound role in iNOS-Nrf2 signaling pathway. Accordingly, it may be concluded that the dietary factors wherein rutin is an ingredient could be helpful in the maintenance of the intracellular redox-homeostasis and thus may be effective against oxidative stress related secondary complications.
BACKGROUND:Rutin (quercetin-3-O-rutinoside), a flavonoid, is predominantly found in the buckwheat, cranberries, mulberry and citrus fruits. It is used as a restorative in the preparation of herbal medicine, multivitamin and known to reduce the fate of heart attack. HYPOTHESIS: We aimed to elucidate whether rutin attenuates oxidative stress and its possible mechanism of action in ameliorating the deleterious effect of t-BHP. We also provide evidence that rutin protects the antioxidant status of erythrocytes and liver via Nrf2 and iNOS pathway from oxidative stress. STUDY DESIGN/ METHOD:Human erythrocytes and mice liver were used for the evaluation of rutin's effect against t-BHP induced oxidative stress. The non-enzymatic (GSH, MDA, -CO, -SH) and enzymatic stress markers (SOD, CAT, GPx, GR and GST) were estimated by the colorimetric method. The level of Nrf2, iNOS, liver marker enzymes, triglycerides, cholesterol, HDL-cholesterol, albumin, BUN was measured using ELISA kits. Reactive oxygen species (ROS) was quantified using flow cytometry and fluorometry. RT-PCR was used for the quantification of Nrf2 and iNOS expression levels in the liver tissue of mice. In silico studies were done through receptor-ligand binding interaction. RESULTS: Pre-treatment with the rutin ameliorated the toxic effect of t-BHP by modulating the basal level of GSH, -SH, MDA and -CO significantly (p < 0.01) with respect to untreated control. Rutin also protected the erythrocytes against the t-BHP-induced oxidative stress as evidenced by augmented activity of antioxidant enzymes (CAT, SOD, GPX, GR and GST). Furthermore, at the highest tested concentration (16.3 µM), it protected the morphology of the erythrocytes by decreasing the ROS level (p < 0.01). In addition, the lower MEF values of rutin (0.520 ± 0.005) alone or along with t-BHP (0.630 ± 0.021) indicated its non-toxic and protective behavior. The qPCR analyses revealed that t-BHP potently up-regulates the iNOS and down regulate the Nrf2 expression which was ameliorated with rutin treatment in a dose-dependent manner like silymarin. CONCLUSION: Our findings demonstrate that rutin potentiates its beneficial aspect by displaying a profound role in iNOS-Nrf2 signaling pathway. Accordingly, it may be concluded that the dietary factors wherein rutin is an ingredient could be helpful in the maintenance of the intracellular redox-homeostasis and thus may be effective against oxidative stress related secondary complications.