Comprehensive Phytochemical Characterization and Molecular Insights into the Anticancer Activity of Itrifal-e-Aftimoon: An Integrated HPLC-DAD, LC-MS/MS, and Mechanistic Approach

Abstract:

Itrifal-e-Aftimoon is a classical Unani polyherbal formulation documented in the National Formulary of Unani Medicine, comprising twelve ingredients traditionally used as a rejuvenator, skin disorders and cancer preparation. Its constituent herbs principally the Triphala group (Terminalia chebula, Emblica officinalis, Terminalia belerica), Plumbago zeylanica and Cuscuta reflexa are individually documented to contain gallic acid, ellagic acid, plumbagin, quercetin and related polyphenols with antioxidant, anti-inflammatory, immunomodulatory and cytotoxic activity relevant to oncology. Objective: This study aimed to chemically fingerprint an ethanolic extract of the formulation using HPLC-DAD and LC-MS/MS, and to integrate the identified marker compounds with the documented multi-pathway anticancer mechanisms of Itrifal-e-Aftimoon. Methods: The powdered sample was Soxhlet-extracted in ethanol (6 h), concentrated to dryness and reconstituted in methanol:water (50:50 v/v). HPLC-DAD analysis was performed on a Shimadzu Shim-pack C18 column (methanol:water, 65:35, 235 nm, 15 min run). LC-MS/MS confirmation used a Waters Acquity UPLC–Xevo TQ-XS tandem quadrupole system with a formic acid–methanol gradient (m/z 100–1000). Results: LC-MS/MS identified three prominent constituents: gallic acid (retention time [RT] 2.43 min, m/z 171), gallocatechol (RT 10.22 min, m/z 307) and quercetin (RT 11.78 min, m/z 303). These compounds correspond to marker phytochemicals previously implicated in Nrf2-ARE-mediated antioxidant defence, NF-κB and COX-2 suppression, mitochondrial apoptosis induction and PI3K/Akt/mTOR pathway modulation mechanisms consistent with the FAK/STAT/Akt/ERK modulation reported for the complete formulation in chronic myeloid leukaemia cells. Conclusion: Chromatographic fingerprinting confirms the presence of pharmacologically relevant polyphenolic markers in Itrifal-e-Aftimoon, providing an analytical basis for standardisation and supporting its rationalised development as an adjuvant integrative cancer therapeutic. Further quantification against reference standards and correlation with in vitro cytotoxicity are warranted.