Validation and Development of HPTLC Method for Simultaneous Estimation of Apigenin and Luteolin in Selected Marketed Ayurvedic Formulations of ‘Dashmula’ and in Ethyl Acetate Extract of Premna integrifolia L.

Abstract

Dashmula are specific ayurvedic combination of ten roots used for various disorders of liver, kidney, uterus. Standardisation of herbals are necessity for efficacy and quality parameter as per WHO guidelines. Therefore, through this original research attempt was made to standardise one of ‘Dashmula’, P. integrifolia and selected batches of marketed formulation using Apigenin and Luteolin as active biological marker for simultaneous quantification and fingerprinting through developed and validated HPTLC techniques. Developed mobile phase Toluene: Ethyl acetate: Formic acid (6:4:0.15) gave Rf (Retention factor) 0.39 and 0.29 for Standard Apigenin and Luteolin respectively at 347 nm iso absorptive wavelength. The ethyl acetate extract of P. integrifolia (PI-ET), ‘Dashmularishtha’: Manufactuer 1; 3 coded batches as DF1, DF2, DF3, Manufacturer 2; 3 coded batches -as BF4, BF5, BF6, ‘Dashmulkadha’: Manufacturer 3; 3 coded batches as KF, KF8, KF9 were found to contain : 12.8% w/w, 0.294 mg/ml%, 0.429 mg/ ml%, 0.314 mg/ml%, 0.077 mg/ml%, 0.071 mg/ml%, 0.145 mg/ml%, 0.176 mg/ml%, 0.242 mg/ml%, 0.098 mg/ml% of Apigenin and 4.7% w/w, 0.542 mg/ml%, 0.365 mg/ml%, 0.569 mg/ml%, 0.343 mg/ml%, 0.311 mg/ml%, 0.607 mg/ ml%, 0.812 mg/ml%, 0.828 mg/ml%, 0.439 mg/ml% of Luteolin respectively. In stem powder of P. integrifolia 19.84 mg/gm% Apigenin and 7.433 mg/gm% Luteolin was calculated. The estimation shows variance in manufacturers and even within batches, but will be quality control parameter. The method was validated for specificity, linearity, accuracy, precision, and robustness. It was found to be linear in range of 40-120 ng/band with regression coefficient 0.9983, 0.9997 for Apigenin and luteolin. Percentage recovery study carried out for extract PI-ET and DF1 with spike of Apigenin and Luteolin at 80, 100 and 120% level, carried out for inter and intraday precision, subjected for one way ANOVA and found F value is below tabulated F(2,6) value 5.14, therefore there is no significance variance of obtained values.