Optimized Isolation and RP-HPLC Standardization of Karanjin From Pongamia Pinnata Seeds

Abstract

Background: The transition from traditional herbal extracts to clinically viable therapeutic leads requires rigorous analytical validation and physical characterization. Karanjin, a bioactive furanoflavonoid present in Pongamia pinnata seed oil, have shown strong potential for treating neurological disorders. However, a reliable, validated method for its high-purity isolation and quantitative analysis is necessary to support its development as a standardized pharmaceutical agent.

Objective: This study establishes an optimized protocol for isolating high-purity Karanjin, characterizes its physical and structural profile, develops and validates a robust RP-HPLC method for its quantification, and evaluates it’s in vitro acetylcholinesterase (AChE) inhibitory kinetics.

Methods: Pure Karanjin was isolated using Pongamia pinnata seeds, was optimized using Soxhlet extraction process followed by liquid-liquid partitioning and silica gel column chromatography. Further monitored by using TLC. The isolated crystals were characterized by physical constants such as melting point, solubility, and partition coefficient and structural Fourier-transform infrared (FT-IR) spectroscopy. Further, quantitative determination was achieved using a validated reversed-phase high-performance liquid chromatography (RP-HPLC) method on a C₁₈ column with an isocratic mobile phase of Methanol: Water: Glacial Acetic Acid (85: 13.5: 1.5 v/v/v) detected at 300 nm. In vitro enzymatic activity was evaluated using Ellman's acetylcholinesterase inhibition assay.

Results: Column purification yielded highly pure, off-white needle-shaped crystals with a sharp melting point matching standard specifications. FT-IR confirmed key functional markers, including the characteristic furan ring and methoxy configurations. The RP-HPLC method showed excellent linearity (r² > 0.999) over a concentration range of 1^–10 μg/mL, along with high precision and accuracy. In the enzymatic assay, isolated Karanjin demonstrated significant, concentration-dependent in vitro inhibition of acetylcholinesterase, comparable to standard Galanthamine.

Conclusion: This study provides a validated analytical and standardization profile for isolated Karanjin, establishing a reliable quality control framework for its development as a high-purity phytopharmaceutical lead.