Phytochemical Screening and High-performance Thin-layer Chromatography Quantification of Vitex trifolia Leaves Hydro-alcoholic Extract: Potential Anti-inflammatory Properties

Abstract

Aims: The present study aims to evaluate the phytochemical composition of Vitex trifolia (V. trifolia) leaves hydro-alcoholic extract and to report for the first time, its phenolic content using a validated high-performance thin-layer chromatography (HPTLC) method. Study Design:  Qualitative phytochemical analysis and HTLC densitometric quantitative analysis. Place and Duration of Study: The study was carried out at the Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Malaysia, from March 2020 to December 2020. Methodology:  The preliminary phytochemical screening was carried out qualitatively. The HPTLC analysis was performed on glass-backed 60 F254 silica gel plates using a two steps gradient elution method of the mobile phase. In the first step, methanol was used to develop the plates until 40 mm of developing distance, while in the second step, plates were developed with n-hexane:ethyl acetate:acetic acid (20:9:1, v/v/v) until 80 mm of developing distance. Detection and quantification were performed by densitometric analysis at 254 nm. The method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline in terms of linearity, precision, accuracy, the limit of detection (LOD), and the limit of quantification (LOQ). Results: The preliminary phytochemical screening of V. trifolia leaves hydro-alcoholic extract showed the presence of alkaloids, flavonoids, phenols, phytosterols, and terpenoids. The developed HPTLC method was proved to be linear, precise, and accurate. The LOD and LOQ of the method were determined to be 2.01 µg/band and 6.08 µg/band, respectively. The total phenolic content of the extract was calculated from the standard gallic acid calibration plot and found to be 136.94 ± 4.02 mg gallic acid equivalent (GAE)/g of dried extract. Conclusion:  This preliminary study revealed that V. trifolia has a considerable amount of phenolic compounds, which can potentially contribute to its anti-inflammatory, antioxidant, and anticancer activities. Further pharmacological investigations are being carried out to support the folkloric claims.