Isolation & identification of anti-inflammatory constituents of Randia dumetorum lamk. fruit: Potential beneficial effects against acute lung injury

Ethnopharmacological relevance: Randia dumetorum Lamk. is an Indian traditional medicinal plant that has been used for the treatment of various disorders including respiratory ailments. Aim of the study: In continuation of our recent report that the Ethanol soluble fraction (ESF) of Randia dumetorum fruit had potent anti-inflammatory activity against acute lung injury (ALI) in mice, the present work was un-dertaken to unveil the key bioactive constituents possessing anti-inflammatory action against ALI by employing bioactivity-guided fractionation of ESF. Material and methods: Different fractions/sub-fractions obtained by column chromatography of ESF were sub-jected to bioactivity studies by analyzing total and differential count, and protein content in broncho-alveolar lavage fluid (BALF) procured from mice. The most bioactive sub-fraction F3.2 was analyzed for the assess-ment of various inflammatory mediators using molecular techniques like ELISA, PCR, and western blotting. Further, an attempt was made to separate the key compounds in F3.2 using solvents of differential polarities; and isolated compounds were validated for their anti-inflammatory activity followed by their characterization using spectral techniques like 1HNMR, 13CNMR, FT-IR, and ESIMS Mass Spectrometry. Results: The column chromatography of ESF yielded four fractions (F1, F2, F3, and F4) and data revealed that maximum activity resides in F3. Further fractionation of F3 yielded sub-fractions F3.1, F3.2, F3.3, and F3.4 which when tested for anti-inflammatory potential, showed F3.2 as the most active one. Moreover, the effect of F3.2 on oxidative stress parameters and inflammatory mediators analyzed via biochemical assays, PCR, and ELISA revealed the proficiency of this fraction in amelioration of ALI. F3.2 was then subjected to recrystallization using different solvents and two pure compounds were isolated which were characterized as D-Mannitol and Oleanolic acid (OA). D-Mannitol did not display any bioactivity, but OA showed potent anti-inflammatory activity. Conclusion: Considering the ethnopharmacological role of R. dumetorum in respiratory ailments, OA as an aglycone moiety seems to be the main active principle possessing anti-inflammatory potential against ALI.