Praveen Kumar Issac1, Rupmanjari Karan2, Ajay Guru1,2, R Pachaiappan3, Mariadhas Valan Arasu4, Naif Abdullah Al-Dhabi4, Ki Choon Choi5, Ramasamy Harikrishnan6, Jesu Arockia Raj7,8. 1. SRM Research Institute, SRM Institute of Science and Technology, Kattankulathur, 603203, Chennai, Tamil Nadu, India. 2. Department of Biotechnology, College of Science and Humanities, SRM Institute of Science and Technology, Kattankulathur, 603203, Chennai, Tamil Nadu, India. 3. Department of Biotechnology, School of Bioengineering, Faculty of Engineering and Technology, SRM Institute of Science and Technology, Kattankulathur, 603203, Chennai, Tamil Nadu, India. 4. Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh, 11451, Saudi Arabia. 5. Grassland and Forage Division, National Institute of Animal Science, RDA, Seonghwan-Eup, Cheonan-Si, Chungnam, 330801, Republic of Korea. 6. Department of Zoology, Pachaiyappa's College for Men, Kanchipuram, Tamil Nadu, 631501, India. 7. SRM Research Institute, SRM Institute of Science and Technology, Kattankulathur, 603203, Chennai, Tamil Nadu, India. jesuaraj@hotmail.com. 8. Department of Biotechnology, College of Science and Humanities, SRM Institute of Science and Technology, Kattankulathur, 603203, Chennai, Tamil Nadu, India. jesuaraj@hotmail.com.
Abstract
BACKGROUND: Plant-derived phytochemicals such as flavonoids have been explored to be powerful antioxidants that protect against oxidative stress-related diseases. In the present study, Morin, a flavonoid compound was studied for its antioxidant and antidiabetic properties in relation to oxidative stress in insulin resistant models conducted in rat skeletal muscle L6 cell line model. METHODS: Evaluation of antioxidant property of morin was assayed using in vitro methods such as cell viability by MTT assay, estimation of SOD and CAT activity and NO scavenging activity. The anti-oxidative nature of morin on L6 cell line was conducted by the DCF-DA fluorescent activity. Glucose uptake in morin treated L6 myotubes are accessed by 2-NBDG assay in the presence or absence of IRTK and PI3K inhibitors. Further glycogen content estimation due to the morin treatment in L6 myotubes was performed. Antioxidant and insulin signaling pathway gene expression was examined over RT-PCR analysis. RESULTS: Morin has a negligible cytotoxic effect at doses of 20, 40, 60, 80, and 100 µM concentration according to cell viability assay. Morin revealed that the levels of the antioxidant enzymes SOD and CAT in L6 myotubes had increased. When the cells were subjected to the nitro blue tetrazolium assay, morin lowered reactive oxygen species (ROS) formation at 60 µM concentration displaying 39% ROS generation in oxidative stress condition. Lesser NO activity and a drop in green fluorescence emission in the DCFDA assay, demonstrating its anti-oxidative nature by reducing ROS formation in vitro. Glucose uptake by the L6 myotube cells using 2-NBDG, and with IRTK and PI3K inhibitors (genistein and wortmannin) showed a significant increase in glucose uptake by the cells which shows the up regulated GLUT-4 movement from intracellular pool to the plasma membrane. Morin (60 µM) significantly enhanced the expression of antioxidant genes GPx, GST and GCS as well as insulin signalling genes IRTK, IRS-1, PI3K, GLUT-4, GSK-3β and GS in L6 myotubes treated cells. CONCLUSION: Morin has the ability to act as an anti-oxidant by lowering ROS levels and demonstrating insulin mimetic activity by reversing insulin resistance associated with oxidative stress.
BACKGROUND: Plant-derived phytochemicals such as flavonoids have been explored to be powerful antioxidants that protect against oxidative stress-related diseases. In the present study, Morin, a flavonoid compound was studied for its antioxidant and antidiabetic properties in relation to oxidative stress in insulin resistant models conducted in rat skeletal muscle L6 cell line model. METHODS: Evaluation of antioxidant property of morin was assayed using in vitro methods such as cell viability by MTT assay, estimation of SOD and CAT activity and NO scavenging activity. The anti-oxidative nature of morin on L6 cell line was conducted by the DCF-DA fluorescent activity. Glucose uptake in morin treated L6 myotubes are accessed by 2-NBDG assay in the presence or absence of IRTK and PI3K inhibitors. Further glycogen content estimation due to the morin treatment in L6 myotubes was performed. Antioxidant and insulin signaling pathway gene expression was examined over RT-PCR analysis. RESULTS: Morin has a negligible cytotoxic effect at doses of 20, 40, 60, 80, and 100 µM concentration according to cell viability assay. Morin revealed that the levels of the antioxidant enzymes SOD and CAT in L6 myotubes had increased. When the cells were subjected to the nitro blue tetrazolium assay, morin lowered reactive oxygen species (ROS) formation at 60 µM concentration displaying 39% ROS generation in oxidative stress condition. Lesser NO activity and a drop in green fluorescence emission in the DCFDA assay, demonstrating its anti-oxidative nature by reducing ROS formation in vitro. Glucose uptake by the L6 myotube cells using 2-NBDG, and with IRTK and PI3K inhibitors (genistein and wortmannin) showed a significant increase in glucose uptake by the cells which shows the up regulated GLUT-4 movement from intracellular pool to the plasma membrane. Morin (60 µM) significantly enhanced the expression of antioxidant genes GPx, GST and GCS as well as insulin signalling genes IRTK, IRS-1, PI3K, GLUT-4, GSK-3β and GS in L6 myotubes treated cells. CONCLUSION: Morin has the ability to act as an anti-oxidant by lowering ROS levels and demonstrating insulin mimetic activity by reversing insulin resistance associated with oxidative stress.