Assessment of Phytochemicals and Characterisation of a Polyherbal Formulation

Objectives: The present study aims to isolate and characterize bioactive compounds from a polyherbal formulation (PHF). Methodology: A PHF was prepared by homogenously mixing specific proportions of the following medicinal plants: Justicia adhatoda, Glycyrrhiza glabra, Withania somnifera, Cassia angustifolia, Zingiber officinale, Elettaria cardamomum, Terminalia chebula, Nigella sativa, Curcuma longa, Piper nigrum, Moringa oleifera, Cuminum cyminum, Phyllanthus emblica, Tribulus terrestris, Andrographis paniculata, Cinnamomum verum, Tinospora cordifolia, Gymnema sylvestre, Abies webbiana, Vetiveria zizanioides, Ocimum sanctum, Centella asiatica, Trigonella foenum-graecum, Solanum trilobatum, and Azadirachta indica. Preliminary phytochemical screening of the PHF was conducted using both qualitative and quantitative analyses to identify key phytoconstituents. The optimization of solvent systems was performed using Thin Layer Chromatography (TLC), followed by compound separation via column chromatography using a gradient of high to low polarity solvents. The eluted fractions were further analysed using a series of advanced characterization techniques like GC-MS to identify bioactive compounds based on retention time, peak area, and molecular weight. LC-MS to determine the composition and specific compounds present in the fractions. FT-IR analyses to detect the functional group present in the compound, H1NMR analyses to know the type of proton present. Findings: Phytochemical analysis confirmed the presence of tannins, steroids, alkaloids, flavonoids, quinones, and saponins. Quantitative analysis of the methanolic extract revealed 46.3 mg AE/g of alkaloids, 45.3 mg QE/g of flavonoids, and 47.5 mg GAE/g of phenolic compounds. GC-MS analysis identified 1,1-bis(hydroxymethyl)cyclopropane as a major component. LC-MS analysis revealed the presence of 3-hydroxy-N-methylcoclaurine, an isoquinoline alkaloid. FT-IR to detect functional groups, where the highest peak at 1020.34 cm⁻¹ indicated a C–N (amine) stretch. ¹H-NMR to identify proton environments, with the highest peak at δ 3.227 ppm corresponding to alkyl protons. Novelty and Conclusion: These findings highlight the pharmacological potential of the PHF, suggesting it is a rich source of bioactive natural compounds. The identified constituents could serve as promising candidates for the development of novel drug delivery systems and therapeutic agents. Keywords: Polyherbal formulation, GC-MS, LC-MS, FT-IR, TLC, Column Chromatography, 1H-NMR, Phytochemical Screening