Extraction and identification of bioactive compounds from anisophyllea disticha using supercritical fluid extraction

Abstract

Anisophyllea disticha is one of the underexplored species from genus Anisophyllea despite its high utilization by the folklore. Till today conventional extraction methods are generally employed to obtain bioactive compounds from the plant; while often introduces contaminants that must be removed later. Supercritical fluid extraction (SFE) offers rapid, selective, environmental-friendly and green chemical processed products. In this study, A. disticha was extracted using soxhlet and the highest total yield was obtained by leaf followed by root and stem extracts with average values of 23.97 ± 0.19, 9.57 ± 1.16 and 8.75 ± 1.54%, respectively. Comparatively, total yield that ranged between 0.65-4.14% was observed by stem extracted from SFE. In phytochemical screening, steroids and flavonoids were detected in leaf while stem and root gave positive tests for steroids, terpenoids, flavonoids and saponins. Subsequent antimicrobial capability test using disc diffusion assay revealed positive growth inhibitory activity, but considered weak, for all extracts against gram-positive bacteria namely Staphylococcus epidermidis and Staphylococcus aureus with leaf extract displayed the highest zone of inhibition with values of 10.67 ± 0.58 and 8.67 ± 0.58 mm, respectively. On the other hand, at sample concentration of 3 mg/mL, stem showed the highest amount of total phenolic content (TPC) followed by root and leaf with mean value of 27.73 ± 4.10, 19.50 ± 0.87 and 12.19 ± 1.77 mg GAE/g extract, respectively. The similar trend was also observed in antioxidant activities where the highest scavenging activity (66.20 ± 11.74%) and reducing power (203.63 ± 16.03 mg Fe(II)/g extract) were found in stem at sample concentration of 0.6 mg/mL. Stem was chosen and subsequently extracted using SFE and further analyzed. Box-Behnken Design (BBD) was used to optimize and examine the effect of independent variables of SFE such as temperature (50-70˚C), pressure (20-30 MPa) and concentration of co-solvent (10-20%) on TPC and antioxidant activities of A. disticha. The model suggested that the optimal conditions of independent variables to be 50˚C, 21 MPa and 20% with predicted TPC of 77.06 mg GAE/g extract, scavenging activity of 109.51% and reducing power of 585.36 mg Fe(II)/g extract. Reverse-phase high performance liquid chromatography (RP-HPLC) was developed to identify and quantify phenolic compounds (gallic acid, p-coumaric, ferulic acid and quercetin) presented in A. disticha. SFE extracts that demonstrated the highest responses (SFE 7) was chosen and used to compare with those from soxhlet. The results showed that the extract from SFE possessed remarkable TPC, antioxidant activities and concentration of phenolic compounds identified by HPLC, indicating its superior recovery of compounds. Further study on A. disticha will be of great medicinal importance to treat diseases associated with bacteria and oxidative stress.