High-performance thin-layer chromatographic method development and determination of bio-enhancer from Piper trichostachyon: an ethnomedicinal plant

Abstract

Rare endemic ethnomedicinal plants that are indigenous to the Piperaceae family have an ancient history as traditional medicine in the treatment of diverse ailments like gastrointestinal disorders. The present research work highlights a digitally optimized, rapid, accurate and highly sensitive high-performance thin-layer chromatography (HPTLC) method for the simultaneous screening and quantification of piperine from ex-situ cultured callus, a kind of endangered Piper trichostachyon species. Analysis of piperine was performed on thin-layer chromatography (TLC) aluminum plates pre-coated with silica gel F₂₅₄ as the stationary phase. Linear ascending development was carried out in a twin-trough glass chamber with a saturation pad. Developing phase comprising of n-hexane‒ethyl acetate (5:15, V/V) was put to task. Spectrodensitometric analysis of the plate was performed in absorbance mode at 342 nm using a CAMAG TLC Scanner IV. Compact bands for piperine with R_F value of 0.50 ± 0.010 were recorded exhibiting a good linear relationship with R = 99.98% in the concentration range of 100‒700 ng/spot with respect to the peak area. The proposed HPTLC method was validated in accordance with the International Council for Harmonization (ICH) guidelines for linearity, accuracy, precision, repeatability, reproducibility, limit of detection, limit of quantification, sensitivity, and specificity. The total piperine content was found to be 62.39 μg/mL in the wild plant and 41.57 μg/mL in the callus of P. trichostachyon. The chromatographic analysis concluded that the method developed and validated as a novel effort for the concurrent evaluation and comparative quantification of piperine in species of P. trichostachyon is reproducible and selective for the analysis of piperine.