Silymarin as a phyto-pharmaceutical: Isolation, simultaneous quantification of four biomarkers and in-silico anti-inflammatory activity

Abstract

CDSCO has introduced a new class of drug, phytopharmaceutical, in the year 2014. It includes the purified and standard fractions with a minimum of four bioactive compounds of an extract of the medicinal plant. We have isolated four flavonolignans from the silymarin extract. As the isolated biomarkers are stereoisomers of each other, thus, we have qualitatively characterized them by their optical rotation and H1-NMR. Validated, sensitive, and highly selective RP-HPLC method was developed for the simultaneous quantitative assessment of the isolated compounds. The RP-HPLC quantification was achieved on C-18 Nucleosil 100-5 and the isolation of compounds were done on a C18 LiChrospher100 using a mobile phase of methanol: water (45:55 v/v) at a flow rate of 1.0 mL/min. The injection volume was 10 μL and analysed by UV detector set at a wavelength of 288 nm. In addition, the in silico molecular docking study has been performed evaluating the potential anti-inflammatory activity of silymarin. The calibration curve was observed to be linear in the range of 10-120 μg/ml with r 2 =0.998. The estimated percentage of silychristin, silybin A, silybin B, and mixture of isosilybin A and B was found to be 12.64%, 8.71, 8.71, and 10.02%, respectively in the extract. The proposed method was compared statistically with the reported HPLC methods which showed no significant difference in accuracy and precision. Binding affinity was found to be in the range of -5.29 to -9.18 kcal/mol for four enzymes, i.e., NF-kB, MAP Kinase, Cyclooxygenase -2 (COX-2) and Phospholipase A2 (PLA2). Till date, no paper has been published regarding the preparation of ‘Phytopharmaceuticals’.