Inhibition of hematopoietic prostaglandin D2 Synthase (H-PGDS) by an alkaloid extract from Combretum molle

Background: Hematopoietic prostaglandin D-2 synthase (H-PGDS, GST Sigma) is a member of the glutathione S-transferase super family of enzymes that catalyses the conjugation of electrophilic substances with reduced glutathione. The enzyme catalyses the conversion of PGH(2) to PGD(2) which mediates inflammatory responses. The inhibition of H-PGDS is of importance in alleviating damage to tissues due to unwarranted synthesis of PGD(2). Combretum molle has been used in African ethno medicinal practices and has been shown to reduce fever and pain. The effect of C. molle alkaloid extract on H-PGDS was thus, investigated. Methods: H-PGDS was expressed in Escherichia coli XL1-Blue cells and purified using nickel immobilized metal affinity chromatography. The effect of C. molle alkaloid extract on H-PGDS activity was determined with 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate. The effect of C. molle alkaloid extract with time on H-PGDS was determined. The mechanism of inhibition was then investigated using CDNB and glutathione (GSH) as substrates. Results: A specific activity of 24 mu mol/mg/min was obtained after H-PGDS had been purified. The alkaloid extract exhibited a 70% inhibition on H-PGDS with an IC50 of 13.7 mu g/ml. C. molle alkaloid extract showed an uncompetitive inhibition of H-PGDS with K-i = 41 mu g/ml towards GSH, and non-competitive inhibition towards CDNB with K-i = 7.7 mu g/ml and K-i = 9.2 mu g/ml. Conclusion: The data shows that C. molle alkaloid extract is a potent inhibitor of H-PGDS. This study thus supports the traditional use of the plant for inflammation.