Phytochemical Markers of Diospyros paniculata Dalzell: HPLC Validation for Juglone and Betulin with HPTLC Quantification of Juglone

Introduction

A lesser-known medicinal plant Diospyros paniculata has potential therapeutic compounds. Among these, two significant bioactive compounds are Juglone and Betulin. The pharmacological assessment of plant extracts and quality control depend on the precise and effective quantification of these substances. Nevertheless, there aren’t many approved analytical techniques for estimating these markers simultaneously in different parts of D. paniculata.

Objectives

This study aimed to develop and validate straightforward, accurate, and economical HPLC and HPTLC procedures for the simultaneous estimation of Juglone and Betulin in the bark and leaves of Diospyros paniculata.

Materials and Methods

To create improved samples, plant materials (bark and leaves) were extracted using methanol as a hot solvent and then further fractionated. A Waters Alliance system with a PDA detector was used for HPLC analysis in isocratic settings that were optimised for efficient separation. Toluene and ethyl acetate in an 8:2 (v/v) ratio was used as the mobile phase for HPTLC, which produced the best resolution. Both the techniques were evaluated for linearity, accuracy, precision and robustness in accordance with the ICH guidelines.

Results

With an Rf value of 0.88 and a strong correlation coefficient (r²=0.9917), the HPTLC technique produced clear and repeatable peaks, demonstrating exceptional linearity. The quantification of juglone produced consistent findings using both HPLC and HPTLC techniques. The reliability and reproducibility of the suggested analytical processes were demonstrated by the validation parameters for both methods falling within acceptable ICH limits.

Conclusion

For the quantification of Juglone and Betulin in Diospyros paniculata, the suggested HPLC and HPTLC techniques are accurate, precise, cost-effective and repeatable. These proven methods work well with variety of plant extracts and herbal formulations that contain these phytoconstituents.