Phytochemical production and antioxidant activity improvement of Rhinacanthus nasutus (L.) Kurz calli by in vitro polyploidization

Background Rhinacanthus nasutus (L.) Kurz is a multipurpose ethnomedicinal shrub containing various bioactive compounds and phytochemicals. Inducing polyploidy is an alternative way to enhance the production of secondary metabolites in medicinal plants. The main objectives of this research were to study the effect of polyploidization on the phytochemical content and antioxidant activity of R. nasutus calli. Methods In vitro polyploidy was induced by soaking calli in colchicine at different concentrations and for different exposure times. To determine callus polyploidy, the relative DNA contents of each sample were examined using flow cytometry. Diploid, tetraploid, and mixoploid calli were extracted to determine the total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity. Results and conclusion Results showed that the callus survival rate decreased with increasing colchicine concentration and exposure time. The highest percentage of induced tetraploid (66.67%) and mixoploid (66.67%) calli were obtained at 0.05% and 0.2% colchicine with exposure times of 48 and 24 h, respectively. Tetraploid calli showed the highest TPC (81.28 mg GAE/g extract), TFC (35.33 mg QE/g extract), and antioxidant activity compared to diploid and mixoploid calli. Additionally, tetraploid calli demonstrated an approximately twofold greater increase in TPC and TFC compared to diploid calli. The analysis of polyploid callus samples revealed that tetraploid calli exhibited the highest antioxidant activity, whereas diploid calli demonstrated the lowest antioxidant activity across all applied assays. Therefore, inducing a tetraploid of R. nasutus calli plays a critical role in modifying phytochemical content and antioxidant activity.