Identification, Quantification, Validation and Characterization of Stigmasterol Using HPTLC Method and Its Anti-arthritic Activity

Abstract

Objective: Alpinia calcarata Roscoe (Family: Zingiberaceae) rhizomes are often used in Indian traditional systems of medicine as a remedy for bronchitis, cough, respiratory ailments, diabetes mellitus, asthma and arthritis. The Present study was designed to investigate the anit arthritic property of Stigmasterol isolated from Alpinia calcarata rhizomes of methanolic extract by In-vitro studies.

Methods: As per International Conference on Harmonization (ICH) guidelines we have applied different concentrations of Stigmasterol as standard on HPTLC plates for the quantification of Stigmasterol from the Alpinia calcarata rhizomes and the concentration of standard Stigmasterol was 1mg/ml. Before that it was purified by HPLC method and characterized by UV, FTIR, ¹H and ¹³C NMR and Mass spectroscopic methods. It was evaluated by in-vitro anti arthritic activities using percentage of protein denaturation and HRBC membrane stabilization method.

Results: The retention factor of Stigmasterol was 0.58. Linearity was obtained in the range of 50 mg - 250 mg for Stigmasterol. The developed and validated HPTLC method was employed for Stigmasterol in methanolic rhizomes extract of Alpinia calcarata for standardization of the content of the marker. The linear regression data for the calibration plots showed a good linear relationshipwith r=0.99977 for Stigmasterol, respectively Satisfactory recoveries of 99.77% were obtained for Stigmasterol. UV, FTIR and NMR values were remarkably correlated with systhesised stigmasterol. It shows 99.4% of protein denaturation at 500 \(\mu\)g/mL. The isolated compound shows higher activity with the IC50 values of 280.1 \(\mu\)g/mL than standard Diclofenac sodium with the IC50 value of 298.34 \(\mu\)g/mL respectively. It shows 75.59% of HRBC membrane stabilization at 500 \(\mu\)g/mL. The isolated compound stigmasterol shows higher activity with the IC50 values of 355.71 than standard Diclofenac sodium with the IC50 value of 351.15 \(\mu\)g/mL respectively. The isolated compound stigmasterol displayed remarkable anti arthritic activity.

Conclusion: The results obtained in validation indicate the accuracy and reliability of the developed HPTLC method for the quantification of Stigmasterol in methanolic rhizomes extract of Alpinia calcarata. The characterization of stigmasterol confirm the structure and its biological studies prove that it is an anti-arthritic agent for the first time.