Extraction, fractionation, and isolation of hydroxychavicol from piper betle leaves

INTRODUCTION Piper betle are commonly used as traditional plant remedy. Betel leaves contain hydroxychavicol (HC), a phenolic compound that has properties such as antioxidant, antifungal, antimicrobial and tyrosinase inhibitor [1]. The aims of this study are to extract and isolate HC from P. betel leaves. MATERIALS AND METHODS 5kg of betel leaves were dried in drying oven overnight at 60°C [2], which later were ground into powder form (800g). The powder was soaked in 2.5L n-hexane for 48 hours followed by suction through a Buchner funnel. The filtrate was evaporated to dryness. Then, the betel powder was soaked in 2.5L DCM for 48 hours, followed by suction through a Buchner funnel. The maceration process using DCM solvent was repeated twice to obtain the crude extract. 49g of crude extract were subjected to column chromatography. 2% methanol in DCM was used as eluting solvent.. Fractions were subjected to thin layer chromatography (TLC) with DCM:ethyl acetate (EtOAc) (8:2) as mobile phase to detect HC. Later, the fractions were compared to HC standard (Rf value). Presence of HC was determined using ferric chloride reagent. Preparative TLC was used to isolate and collect pure HC. RESULTS AND DISCUSSION First maceration process was done using n-hexane to remove non-polar compounds from the betel powder. Second maceration process using DCM solvent was to obtain semi-polar compounds primarily the HC compound. 49g (6.125%) of DCM extract was yielded from 800g of betel powder. Fractions were collected in 100 mL beaker and subjected to TLC plate developed in a mobile phase 2% EtOAc in DCM. Fraction no. 87-108 showed bands of compounds with spot that similar to HC standard single spot with Rf value of 0.64±0.03 (Fig. 1) after observation under UV light and sprayed with FeCl3 reagent, DPPH scavenging reagent, and vanillin. Fractions with major HC compound were pooled and weighed (5g, 10.204% yield). Pure HC obtained by preparative TLC from 2.5g of HC-rich fraction was 0.027g (1.08%). Fig. 1. TLC observation of pure HC at (A) shortwave UV light (B) longwave UV light (C) ferric chloride reagent (D) vanillin/H2SO4 reagent (E) DPPH. TLC used was silica gel 60 aluminium sheets with 200 µm thickness. Solvent system was DCM:EtOAc (ratio 8:2). Rf value for HC was 0.64±0.03. CONCLUSIONS n-Hexane and DCM was used as solvents and this method yielded high extract with major HC compound (49g). Pure HC obtained from 2.5g of HC-rich fraction using preparative TLC was 0.027g (1.08%)