TLC–Bioautography-Guided Isolation, HPTLC and GC–MS-Assisted Analysis of Bioactives of Piper betle Leaf Extract Obtained from Various Extraction Techniques: In vitro Evaluation of Phenolic Content, Antioxidant and Antimicrobial Activities

Abstract

The polyphenolic content, antioxidant and antibacterial activities of the ethanolic extracts of Piper betle leaf obtained from soxhlet (PBSx), sonication (PBSn) and maceration (PBMn) extraction methods were studied. GC–MS analysis was carried out to determine the variation in the phytoconstituents in these extracts. Whereas, thin-layer chromatography (TLC)–bioautography was conducted to localise, separate, and identify antioxidants, and their amount was determined by the newly developed high-performance thin-layer chromatography (HPTLC) method. The results of polyphenolic content, antioxidant assays and antimicrobial assay showed that PBSn contained significant amount of polyphenolics, antioxidant and antimicrobial activity followed by PBMn and PBSx. Moreover, the obtained antioxidant activity of PBSn was significant even in comparison with butylated hydroxytoluene (BHT) and commercially available grape seed extract (GRSx). In addition, GC–MS analysis shown marked variations in the amount of the phytoconstituents among all these extracts with PBSn containing higher amount followed by PBMn and PBSx. TLC bioautography resulted in the separation of three compounds which are identified as eugenol, allylpyrocatechol, and eugenyl acetate. The HPTLC densitometric determination was also supported the results of antioxidant assays by revealing the presence of higher amount of identified antioxidants in PBSn followed by PBMn and PBSx. Since, P. betle leaf extract has been used as one of the ingredients in several herbal formulations, results of this study will not only help the herbal industries in choosing the appropriate extraction technique but also the developed HPTLC method was simple, precise, sensitive and accurate hence can be utilised for the routine quality control and standardisation of formulations containing P. betle leaf extract.