Identification of Bioactives in Clinacanthus Nutans Leaf Extract with ROS Reducing and GSH Enhancing Properties on Liver Cells

Ng, Kai Song-Aaron (2023) Identification of Bioactives in Clinacanthus Nutans Leaf Extract with ROS Reducing and GSH Enhancing Properties on Liver Cells. Masters thesis, Tunku Abdul Rahman University of Management and Technology.

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Abstract

Oxidative stress is defined as a condition characterized by an imbalance between the protective actions of antioxidants and the production of reactive oxygen species (ROS) and oxidants. Clinacanthus nutans Lindau (C. nutans), a perennial shrub belonging to the Acanthaceae family, has been recognized for its antioxidant and anti-inflammatory qualities. Consequently, the main objectives of this research are: (1) to compare the antioxidative and hepatoprotective properties of C. nutans leaves (CNL) and C. nutans stem (CNS) extracts, (2) to identify the most potent active fraction(s) and the specific plant compounds responsible for the beneficial effects observed in C. nutans leaves. In this research, chemical-based and cell-based assays as well as metabolomics approach utilizing NMR along with molecular docking techniques were employed to aid in the identification of the key phytocompounds involved. Based on DPPH and ABTS radical scavenging activities, CNL demonstrated stronger radical scavenging potential as compared to CNS. The leaves extract also recorded slightly higher reducing power properties. A HepG2 cell model system was used to investigate the ROS reduction potential of these extracts. It was shown that cells treated with CNL and CNS reduced innate ROS levels as compared to untreated control. Interestingly, cells pre-treated with both extracts were also able to decrease ROS levels in cells induced with oxidative stress. CNL was again notably the better antioxidant than CNS. According to the multivariate data analysis of the 1H NMR results, the main metabolites postulated to contribute to the antioxidant and hepatoprotective abilities of leaves were clinacoside B, C and isoschaftoside. This extract was further subjected to liquid-liquid fractionation. All fractions (hexane, n-butanol, ethyl-acetate and water) displayed non-toxicity towards HepG2 cell lines when subjected to testing at concentrations of up to 100 μg/mL. It was shown that cells treated with hexane and ethyl acetate fractions were able to reduce ROS levels by 36% and 27.56% respectively, during oxidative stress as compared to untreated cells. Furthermore, hexane increased glutathione (GSH) levels in HepG2 cells by approximately 3-fold whereas ethyl acetate treated cells showed only 2.37-fold increment of GSH in comparison to untreated control. Interestingly, during NMR analysis, phytocompounds; clinacoside B, clinacoside C and isoschaftoside which were postulated to contribute in C. nutans leaves antioxidative properties were detected in hexane fractions. Therefore, molecular docking was conducted to investigate the binding orientations and interactions of these three molecules in the binding site of Keap1. The compounds mainly engaged in H-bonding and hydrophobic interactions with adjacent binding site residues. Amongst them, isoschaftoside attained the highest docking score and formed interactions with important binding site residues, such as Ser555, Tyr572, Ser363, Arg415, Ser508, Gln530, and Tyr334, suggesting its -tighter binding within the binding pocket of Keap1. However, more comprehensive investigations should be carried out to confirm and validate these findings.