HPTLC Method Development and Validation for Simultaneous Quantification of Purpurin and Alizarin in Rubia cordifolia L. Roots and their Marketed Preparations

Background: Rubia cordifolia L. a spiky perennial climber of Rubiaceae family, is a rich reservoir of anthraquinone containing compounds purpurin and alizarin. It emerges as a versatile powerhouse possessing diverse pharmacological properties which includes antibacterial, antitumor, antioxidant, antiplatelet, potent blood purifier, diuretic, anti-stress, neuroprotective, anti-mutagenic, anticancer, anti-inflammatory, antidepressant, antidiabetic and vasodilation. Thereby it offers a holistic approach for disease prevention through its pharmacological properties. Objectives: However due to lack of standardization for R. cordifolia roots and its derived marketed herbal products, its quality control remains a daunting task..The present study aims to develop a simple, novel, reliable and specific HPTLC method for quantifying purpurin and alizarin in R. cordifolia roots as well as in its marketed preparations. Materials and Methods: The separation and quantification of purpurin and alizarin has been carried out on HPTLC plates pre-coated with Silica gel 60 F₂₅₄ as stationary phase and toluene, ethyl acetate, glacial acetic acid (6:3.5:0.5; v/v/v) as mobile phase. Determination and quantification were performed by densitometric scanning at 254 nm. Results: The developed method gave compact spots at Rf 0.57±0.007 for purpurin and 0.73±0.008 for alizarin. The method was validated as per International Council for Harmonization Q2(R1) guidelines for specificity, precision, robustness, accuracy and recovery. Linearity range for purpurin and alizarin was 100-2000 ng/spot, which showed good regression coefficient for purpurin and alizarin; R²=0.9967 and R²=0.9941 at 254 nm respectively. The LOD and LOQ for validated marker compounds purpurin and alizarin at 254 nm were found as 23.87±0.52, 71.61±0.84 ng/spot and 18.36±0.72, 55.08±0.26 ng/spot respectively. The content of purpurin and alizarin in R. cordifolia plant extract was found as 17.960±0.658, 34.645±1.153 μg/mg, marketed preparation A, contains 21.741±0.746 and 6.824±0.485 μg/mg while marketed preparation B, contains 18.289±1.014 and 3.031±0.234 μg/mg of the sample. Conclusion: The developed method was found to be simple, precise, accurate, economical and convenient for rapid screening of bioactive marker purpurin and alizarin present in methanolic extracts of R. cordifolia roots and its marketed preparations.