Optimisation of asarone removal from Piper sarmentosum Roxburgh leaves using supercritical carbon dioxide extraction: the Box-Behnken design

Abstract Piper sarmentosum is a herbaceous shrub with numerous pharmacological benefits. However, the presence of two toxic phenylpropanoids (α- and β-asarone) limits the medicinal usage of the plant. In this study, the extraction of three asarone isomers, namely α-, β-, and -asarone was optimised using supercritical carbon dioxide extraction (SC-CO2) combined with Box-Behnken experimental design. Comparison of asarone contents in different conventional solvent extracts of P. sarmentosum leaves prior to and after SC-CO2 extraction was performed. The SC-CO2 method successfully maximised the extraction of α-, β-, and ɣ-asarone at P = 81.16 bar, T = 50.11°C, and t = 80.90 min, yielding 13.91% α-asarone, 3.43% β-asarone, and 14.95% ɣ-asarone. The SC-CO2 residue of the leaves re-extracted with conventional solvents showed a significant decrease of asarone ranging from 45% to 100% (p<0.001) compared to their counterparts without SC-CO2 treatment. α-, β-, and ɣ-asarone were completely removed in the ethanol extract of the residue. These findings suggested that the optimised SC-CO2 extraction parameters may serve as a quick treatment step for the selective removal of asarone from P. sarmentosum to develop safer extracts for the food and nutraceutical industries applications.

Selected metabolites profiling of Orthosiphon stamineus Benth leaves extracts combined with chemometrics analysis and correlation with biological activities.

BACKGROUND: Studies on selected metabolites profiling of Orthosiphon stamineus extracts using chromatographic and spectroscopic techniques combined with chemometric tools have not been fully elucidated. Thus present study was performed to profile selected metabolites in O. stamineus leaves extracts using HPLC and FTIR combined with chemometric tools and correlated with biological activities. METHODS: Five different extracts were prepared using three methods; maceration, soxhlet and reflux. The extracts were analyzed using UV-Vis, HPLC and FTIR techniques. Analysis of selected primary and secondary metabolites was also evaluated. The antioxidant and cytotoxic activities of the extracts were evaluated. Chemometric tools were employed to classify the extracts based on HPLC analysis and FTIR fingerprints. RESULTS: The ethanolic extract using maceration characterized high content of phenolics and flavonoids, (rosmarinic acid and eupatorin) with high antioxidant activity. Ethanolic (50%) and methanolic extracts using soxhlet showed high proteins and glycosaponins. Water extracts using reflux and maceration showed high polysaccharides. Methanolic extract (50%) using soxhlet and methanolic extract using maceration showed strong cytotoxic effect against MCF7 and HCT116 cell lines, respectively. Antioxidant and cytotoxic activities showed significant correlation with selected primary and secondary metabolites. HPLC fingerprints combined with chemometrics showed the extracts have been clustered based on selected major peaks profile. FTIR fingerprints combined with chemometrics showed that the extracts have been clustered based on protein and polysaccharide contents. CONCLUSION: Ten different extracts of O. stamineus have showed significant differences in the content of selected primary and secondary metabolites as well as the biological activities. Chemometric tools were able to classify and discriminate the distinctive features of extracts thus can be correlated with the biological activities.

A novel reverse phase high-performance liquid chromatography method for standardization of Orthosiphon stamineus leaf extracts.

BACKGROUND: Orthosiphon stamineus Benth. (Lamiaceae) is a traditional medicinal plant which has been used in treating various ailments such as kidney diseases, bladder inflammation, arthritis and diabetes. The leaves contain high concentration of phenolic compounds, thus, rosmarinic acid (RA), 3'-hydroxy-5, 6, 7, 4'-tetramethoxyflavone (TMF), sinensetin (SIN) and eupatorin (EUP) were chosen as a marker compounds for standardization of various O. stamineus leaf extracts. OBJECTIVE: The aim was to develop and validate a new high-performance liquid chromatography (HPLC) method for quantification of 4 marker compounds (RA, TMF, SIN, EUP) in various O. stamineus leaf extracts. MATERIALS AND METHODS: The method was developed and validated using RP-HPLC-diode-array detection at 320 nm for accuracy, precision and limits of detection and was applied for quantification of it markers in five different extracts prepared in solvents with increasing polarity, using a gradient mobile phase 0.1% formic acid: Acetonitrile at a flow rate of 1 ml/min on reverse phase acclaim polar advantage II C18 column (3 µm, 3 × 150 mm) with 18 min separation time. RESULTS: The developed method provided satisfactory precision, and the accuracy of this method was in the range of 90.2% to 105.5%. All of 4 compounds showed good linearity at R2 > 0.999. CONCLUSION: The developed method is a simple, cost effective with shorter run time (18 min) in comparison to previous methods (30 min) and utilization of environmental-friendly solvents system. Therefore, this method has the potential to replace currently used methods in the routine standardization work of O. stamineus extracts, raw materials and its commercial products.