Method development and validation for quantification of six bioactive compounds (andrographolide, columbin, piperine, gallic, paracoumaric and oleanolic acids) by HPTLC.

Abstract

During the spread of pandemic diseases, immunity boosting herbal drugs are taken as a preventive medicine. Kapacurak Kuṭinīr Cūraṇam is a Siddha drug used for flu like viral infections, cold and fever. Developing an analytical method to estimate the content of active phytoconstituents in such antiviral immune boosting drug will be useful in the phyto pharmaceutical industry. A precise, reliable and sensitive ordinary phase high performance thin layer chromatography (HPTLC) method has been developed and validated for identification and simultaneous estimation of six bioactive components namely like andrographolide, columbin, gallic acid, ρ-coumaric acid, piperine and oleanolic acid from any Indian traditional medicine, medicinal plant, drugs and food materials etc. The separation was achieved on silica gel 60F254 TLC plates using toluene: ethyl acetate: formic acid (7:3:0.5, v/v) as mobile phase. The gallic acid, ρ-coumaric acid, piperine markers were estimated using the densitometric scanning in absorption mode at 254nm. The densitometric scanning was done after derivatization (vanillin-sulphuric acid reagent) at λ=520nm for andrographolide, columbin and oleanolic acid. The linear regression analysis data for the calibration plots showed a correlation coefficient in the concentration range 1-5μg per band for the bioactive markers with respect to area. The method was validated for accuracy, precision, limit of detection (LOD), and quantitation of limit (LOQ). Developed method was accurate, precise and fast to ensure the quality of Kapacurak Kuṭinīr Cūraṇam.