Establishment Of In Vitro Culture Techniques For Clinacanthus Nutans And The Evaluation On Its Antioxidant Potential
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Date
2021-03
Authors
Bong, Fui Joo
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Publisher
Universiti Sains Malaysia
Abstract
Clinacanthus nutans (Burm.f.) Lindau is a valuable traditional medicinal plant that has gained interest as an alternative side treatment for cancer, particularly in South East Asia. Phenolic and flavonoid compounds identified in this plant have been linked to its anti-cancer properties due to their antioxidant activity in scavenging radical species. The present study aims to establish callus and cell suspension cultures of C. nutans and to evaluate the accumulation of phenolics, flavonoids and antioxidant activity from extracts of in vitro cultures (callus and cell suspension cultures) and intact plants parts (leaf and stem). Different plant growth regulators (2, 4-D and BAP, picloram) and combinations of auxin (2, 4-D or NAA) and cytokinin (BAP, TDZ or KIN) were used for the induction of friable callus. Besides, different proliferation medium and sucrose concentrations were tested on callus proliferation. Growth kinetics of two suspension cell lines was studied. The total phenolic and total flavonoid contents as well as the antioxidant activity of the extracts from intact plants and in vitro cultures were evaluated using spectrophotometric analysis. High-performance liquid chromatography (HPLC) analysis was performed to detect the selected flavonoids. Friable and pale-yellow callus was induced from young leaf explants of C. nutans on MS medium augmented with 2, 4-D and BAP whereas compact and brownish callus was induced on medium supplemented with picloram. Friable callus with maximum biomass (0.623 ± 0.016 g) was produced on MS medium supplemented with a combination of 0.25 mg/L 2, 4-D and 0.25 mg/L BAP. This medium was also found to be optimum for callus proliferation. Results indicated that sucrose at the concentration of 30 g/L was optimum for callus proliferation. The friable callus showed a stable growth pattern after four subculture cycles where cell line CN2 showed a faster growth rate than cell line CN1. The suspension culture of C. nutans was maintained up to 10 subcultures with small aggregates and fine cells. The highest total phenolic content was obtained in cell line CN2 with 55.35 mg GAE/g dry weight (DW) whereas the highest flavonoid content obtained in leaf with 25.13 mg QE/g DW. The leaf exhibited the highest antioxidant activity with the lowest IC50 value (117.42 μg/mL) followed by cell line CN2. HPLC analysis revealed the presence of catechin, luteolin, quercetin and kaempferol in the cell line CN2 and the leaf samples at varying amounts indicating the presence of bioactive compounds linked to anti-cancer properties. The present study successfully established cell suspension cultures of C. nutans with the present of selected flavonoid compounds as the intact plant which can be further manipulated as an alternative strategy in the production of valuable phenolics and flavonoids under controlled conditions for various pharmaceutical purposes.
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Natural history