Quantitative Estimation and Cytotoxicity Studies of Quercetin & Kaempferol in Passiflora quadrangularis L.

Background Standardization of medicinal plants requires analytically validated marker quantification coupled with biological relevance. Passiflora quadrangularis L. is a traditionally used medicinal plant rich in flavonoids; however, matrixspecific analytical validation and selectivity-based cytotoxic evaluation of its key flavonol markers remain limited.
Objective The present study aimed to develop and validate a matrix-specific reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of quercetin and kaempferol in P. quadrangularis leaves
and to evaluate their selective cytotoxic potential against human breast cancer cells (MCF-7) relative to normal human
endothelial cells (HUVECs).
Methods An RP-HPLC method employing a binary mobile phase system with UV detection at 350 nm was optimized and
validated in accordance with ICH guidelines for specificity, linearity, precision, repeatability, robustness, and system suitability. Quantitative analysis of quercetin and kaempferol was performed in plant extracts and test samples. The biological
relevance of the quantified markers was assessed using an MTT-based cytotoxicity assay on MCF-7 and HUVEC cell lines,
supported by morphological evaluation and selectivity index determination.
Results The validated method provided well-resolved, symmetrical peaks with consistent retention times for quercetin
(~15.5 min) and kaempferol (~16.5 min) and showed no interference from blank or matrix components. Quantitative analysis confirmed reproducible estimation of both flavonoids across sample matrices. In vitro cytotoxicity studies revealed that
kaempferol exhibited greater antiproliferative activity against MCF-7 cells (IC₅₀ = 72.54 µg/mL) compared to quercetin (IC₅₀
= 148.5 µg/mL), while both compounds demonstrated substantially lower toxicity toward HUVECs, resulting in favorable
selectivity indices. Morphological observations corroborated the cytotoxic and selective effects.
Conclusion This study provides a validated, regulatory-relevant RP-HPLC method for the simultaneous quantification of
quercetin and kaempferol in Passiflora quadrangularis, supporting reliable quality control and standardization. The demonstrated selective cytotoxicity, particularly of kaempferol, establishes biological justification for these markers and highlights
their potential relevance in phytopharmaceutical development. The integrated analytical–biological approach strengthens the
translational value of P. quadrangularis as a standardized herbal resource for further anticancer research.
Keywords Passiflora quadrangularis · RP-HPLC · Quercetin · Kaempferol · Method validation · Cytotoxicity ·
Selectivity index · Phytopharmaceutical standardization