Antioxidant and flavonoid activities in different varieties of Ficus deltoidea jack (Mas Cotek) leaves

Ficus deltoidea Jack or known as Mas Cotek in Malaysia is one of the most promising medicinal plants to be commercialised due to its high antioxidant activities. To date, there is no information on the best variety of F. deltoidea in terms of antioxidant activity for commercialisation purposes. Hence, the general objective of this study was to evaluate the antioxidant activity and flavonoid contents of three F. deltoidea varieties that are commonly used as herbal medicines in Malaysia. Specific objectives were to determine the total antioxidant capacity in leaf extracts of F. deltoidea using different solvent extractions, to analyse and identify the flavonoid content in leaf extracts of F. deltoidea, and to examine the flavonoid production in cell suspension culture of F. deltoidea. Two accessions from each variety were chosen. They were FDK1 and FDK2 (var. kunstleri), FDT1 and FDT2 (var. trengganuensis), and FDA1 and FDA2 (var. angustifolia). From this study, leaf extracts of F. deltoidea showed different activity depending on methods and solvents used. Leaf extracts from var. trengganuensis showed the highest total protein and reducing sugar content with 30.6 and 17.9 mg/g FW in FDT2 and FDT1, respectively while total hydrolysed sugar content was highest in leaf extract of FDA2 with 30.7 mg/g FW. Total ascorbic acid content was highest in leaf extract of FDT1 with 4.12 mg/g FW. Enzymatic antioxidant activity was highest in leaf extract of FDK1 for determination of catalase, peroxidase, ascorbate peroxidase and ascorbate oxidase activity with 0.23, 0.75, 9.01 and 23.20 unit/mg protein. Aqueous extract at room temperature (25 °C) of FDK1 showed the highest activity of total antioxidant with 1.85 mg Trolox equivalent (TE)/g fresh weight (FW) using Ferric Reducing Antioxidant Potential (FRAP) method and leaf extract of FDT2 contained the highest total antioxidant using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method with 4.85 mg TE/g FW. Analysis of total antioxidant and phenolic compounds using hot and cold aqueous extracts of leaf extracts of F. deltoidea was showed that most of leaf extracts contained higher antioxidant content in hot aqueous extracts compared to that of cold aqueous extract. Furthermore, methanol and ethanol extracts of F. deltoidea were also analysed in which ethanol extracts showed more antioxidants as compared to that of methanol extracts. Leaf extract of F. deltoidea var. kunstleri (FDK1) contained the highest activity of total polyphenol content for both methanol and ethanol extracts with 1.12 and 1.13 mg GAE/g FW, respectively. The highest total phenolic acid content was found in leaf extract of accession FDK1 using methanol extraction with 4.54 mg GAE/g FW while from ethanol extraction, leaf extract of FDK2 showed the highest activity with 4.00 mg GAE/g FW. All six accessions, which represented three varieties, were analysed for specific flavonoid content using high performance liquid chromatography (HPLC). From the chromatograms, two flavonoid compounds; rutin and naringin were detected in all the intact plants where rutin was more abundant than naringin. Leaf extract of FDK1 showed the highest rutin content with 12.83 μg/g DW, while FDT2 contained the highest naringin content with 3.04 μg/g DW. Cell suspension culture was established from leaf-derived callus of accession FDK1. Cell suspension culture was initiated by culturing the callus liquid MSB5 basal media on an orbital shaker at 120 rpm. Cell suspension of FDK1 at day 12 was chosen for subsequent experiments due to its highest flavonoid production with 2.10 mg RE/g DW. An initial inoculum size of 2 g/culture was found to produce the highest biomass with 0.65 g/25 mL of media while the highest flavonoid production was found with an initial inoculum size of 0.5 g/culture with 3.3 mg RE/g DW. The biomass and flavonoid production of cell suspension culture of FDK1 was proportional with cell aggregate size with 0.28 g/25 mL of media and 3.3 mg RE/g DW, respectively using 750 μm mesh size compared to that of other cell aggregate sizes. Initial pH value of control (pH 5.75) was found to be the best for biomass and flavonoid production with 0.28 g/25 mL of media and 3.3 mg RE/g DW. Total antioxidant content of intact plant using DPPH method was 3-folds higher compared to that of cell suspension culture while 6-folds for FRAP method. At the same time, total flavonoid was 7-folds higher in intact plant compared to that of cell suspension culture of FDK1. Rutin production in intact plant was 4-folds higher compared to that of cell suspension culture of FDK1 while no naringin production was detected in cell suspension culture of FDK1 compared to 0.89 μg/g DW which was detected in intact plant. In conclusion, it is proven that F. deltoidea contains antioxidant activity and flavonoid contents