In vitro Cytotoxicity and Antioxidant Study of Rhodomyrtus tomentosa (Aiton) Hassk. Ethanolic Leaf Extract on LPS-induced RAW 264.7 Macrophage Cells

Abstract

Aims: Ethanol extract of Rhodomyrtus tomentosa leaves found specifically on Malaysian soil was used to further investigate the antioxidant properties and cytotoxicity against RAW 264.7 macrophage cells in the search for a safer and effective natural antioxidant agent. Study Design: Antioxidant potential of R. tomentosa were analyzed through series of spectrometric assays and cell-based bioassays model. Place and Duration of Study: This study was carried out at Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 43200, Selangor, Malaysia from the year of 2019 to 2021. Methodology: R. tomentosa leaves were subjected to extraction with 95% ethanol. The extracts were then denoted as ethanolic leaves extract of R. tomentosa or EtRT extract. EtRT extract were then screen for its antioxidant activity (AOA) and total antioxidant capacity (TAC) through DPPH radical scavenging assay and ferric reducing antioxidant power (FRAP) assay. After that, EtRT extract were brought to observe its toxicity against RAW 264.7 macrophage cells in MTT assay. Once their toxicity was obtained, EtRT extracts were finally tested for their ability to inhibit intracellular reactive oxygen species (ROS) and nitric oxide (NO) inhibition in RAW 264.7 macrophage cells to further analyze their antioxidant properties. Results: In this study, EtRT extracts dose dependently showed the ability to scavenge DPPH radicals and reduce ferric ions during DPPH radicals scavenging activity assay and ferric reducing antioxidant power assay (FRAP), respectively. In DPPH radical scavenging activity assay, EtRT extracts showed EC50 value at 12.37 ± 1.73 µg/mL with ARP value of 0.08 almost as near as ascorbic acid’s ARP value which is 0.09. Further into the study, EtRT extract were not cytotoxic to RAW 264.7 macrophage cells at concentrations 3.91 µg/mL and lower which showed more than 86.4% cell viability with IC50 value at 204.70 ± 5.30 µg/mL. EtRT extract possessed the ability to inhibit ROS production on LPS-induced RAW 264.7 macrophage cells at 7.813 µg/mL and lower, with the highest concentration can reduce up to 30.20% ± 1.01 out of the total ROS produced by the induced cells. Furthermore, EtRT extract also have evidenced that it is able to significantly inhibit NO production by the LPS-induced RAW 264.7 macrophage cells at 7 µg/mL and lower being the highest at 56.73% ± 0.11 inhibition of the highest concentration tested. Conclusions: This study suggests that EtRT extracts have the potential to reduce LPS-induced oxidative stress due to the antioxidant activities of phenolic compounds in the extracts, and that at low doses, EtRT extracts had low to no cytotoxicity on RAW 264.7 macrophage cells. As a result, EtRT extract could be a promising natural medicinal agent for the treatment of oxidative stress.