Comparative Characterization of Four Medicinally Important Phyllanthus Species Using HPTLC, RP-HPLC, GC–MS/MS, ICP- OES, and Antioxidant Assays

Abstract Phyllanthus species are widely recognized for their medicinal importance due to their wide range of therapeutic applications and diverse phytochemical composition, Nevertheless, there are still few comprehensive comparison studies that integrate elemental analysis, metabolite profiling, chromatographic fingerprinting, and antioxidant evaluation across different Phyllanthus species. Four medicinally important Phyllanthus species ( P. sikkimensis, P. urinaria, P. amarus , and P. rheedei ) were comparatively evaluated using physicochemical and phytochemical analyses, HPTLC, RP-HPLC, GC–MS/MS, ICP-OES, TPC, TFC, and DPPH and ABTS antioxidant assays. Reproducible chromatographic fingerprints with both shared and species-specific bands were produced by HPTLC analysis, revealing unique phytochemical traits among the studied species. RP-HPLC analysis demonstrated that P. sikkimensis contained the highest concentrations of gallic acid (32.412 ± 0.002 mg/g) and ellagic acid (7.786 ± 0.005 mg/g), whereas P. rheedei exhibited the lowest concentrations of both phenolic markers among the four Phyllanthus species. GC–MS/MS detected 20, 30, 25, and 24 metabolites in P. sikkimensis , P. urinaria , P. amarus , and P. rheedei , respectively. All four species shared neophytadiene, phytol, squalene, 2-hexadecen-1-ol acetate, ascorbic acid 2,6-dihexadecanoate, linolenic acid and the chemotaxonomic markers lignans hypophyllanthin and niranthin. ICP-OES analysis demonstrated marked interspecific variation in mineral composition, with potassium being the predominant element. P. sikkimensis exhibited the strongest antioxidant activity and the highest phenolic and flavonoid concentrations among the taxa under investigation. These findings offer thorough phytochemical, chemotaxonomic, and elemental evidence for distinguishing pharmaceutically significant Phyllanthus species and establish solid quality control parameters that will assist with their standardization, validity, and future pharmacological application.