Application of Box-Behnken design for optimization of Vernonia amygdalina stem bark extract in relation to its antioxidant and anti-colon cancer activity

Abstract

Vernonia amygdalina is a popular plant used both in traditional medicine and as a culinary herb, one of which is anticancer. Microwave-assisted extraction (MAE) is reported to have the advantage of producing extracts with better phenol and flavonoid content so that the anticancer potential of the extract increases. This study was conducted to produce the polyphenol-rich extract of V. amygdalina stem bark as an anticancer. Responses Surface Methodology (RSM) was used to determine the optimum conditions for extracting phenolics and flavonoids from V. amygdalina stem bark. The total phenolic and flavonoid contents were carried out using a colorimetry assay. The antioxidant activity of the extract from optimal conditions (VABEEo) and extract from non-optimum conditions (VABEE) was determined using the DPPH method to support its anticancer activity. Meanwhile, the anticancer activity of the VABEEo was established through several tests which as cytotoxic test using MTT assay, cell cycle arrest and apoptosis activity using flow cytometer, and protein expression with immunocytochemical analysis. This research informs that the highest yield, total phenolic content (TPC), and total flavonoid content (TFC) were obtained respectively at 7.402 g, 73.634 mg GAE/g, and 64.466 mg QE/g at a microwave power of 450 W for 3 min and using a solvent with a concentration of 76.19 %. In addition, the antioxidant and cytotoxic activity of the VABEEo was 25.413 ± 0.525 µg/mL, higher than VABEE was 41.254 ± 0.614 µg/mL. In vitro studies show that VABEEo inhibits colon cancer growth by inducing cell cycle arrest at G1 stages, increasing apoptosis, and decreasing Cyclin D1/p53 levels. This study showed the application of RSM is successful for VABEEo preparation using MAE. Moreover, the VABEEo indicates promising potential for further research as a colon cancer treatment via in vitro tests.