Inhibition of proinflammatory mediators by coumaroyl lupendioic acid, a new lupane-type triterpene from Careya arborea, on inflammation-induced animal model

Abstract

Ethnopharmacological relevance: Careya arborea Roxb. (Lecythidaceae) is a large tree found throughout India in deciduous forests and grasslands. C. arborea is traditionally used in tumors, inflammation, anthelmintic, bronchitis, epileptic fits, astringents, antidote to snake-venom, skin disease, diarrhea, dysentery with bloody stools, dyspepsia, ulcer, tooth ache, and ear pain.

Aim of the study: In our previous work, the methanolic extract of Careya arborea stem bark showed significant anti-inflammatory activity. As a continuity of that work, this study aimed at the isolation and evaluation of the anti-inflammatory effect of coumaroyl lupendioic acid, a new lupane-type triterpene from Careya arborea stem bark. Further, to give an insight into the underlying mechanism of action of the compound on the modulation of proinflammatory mediators.

Materials and methods: Methanolic extract of Careya arborea stem bark was suspended in water, and sequentially fractionated with n-hexane and ethyl acetate. Further ethyl acetate fraction was subjected to medium pressure liquid chromatography (MPLC) to isolate the active molecules. The isolated compounds were characterized by the various spectral techniques namely UV, IR, ¹H NMR, ¹³C NMR, DEPT, ¹H-¹H COSY, HMBC and Mass spectral techniques. In vitro COX-1 and COX-2 enzyme inhibition assays using human whole blood was performed to investigate the inhibitory effect of the isolated compounds. The resulted potent COX-2 inhibitor of the isolated constituents compound 5, designated as coumaroyl lupendioic acid (CLA), was investigated in carrageenan induced inflammation and its effect was also compared with betulinic acid (BA) at the doses of 10 and 20mgkg^-1, p.o. using indomethacin and celecoxib (10 and 20mgkg^-1, p.o., respectively) as reference drugs. The effect of CLA on the production of NO, MPO, PGE2, TNF-α, IL-1β and IL-6 were assessed. In addition, the histopathology and immunohistochemistry (NF-ҡB, COX-2 and TNF-α protein expression) in paw tissues were also carried out.

Results: The chromatographic fractionation of the methanolic extract resulted in isolation of six new derivatives of lupane type triterpenes for the first time from the stem bark of C. arborea; 3β-hydroxy-lup-5,20 (29),21-trien-28-oic acid (Compound 1), 1, 3, 13, 16-tetrahydroxy-lup-9(11), 20(29)-diene-28-oic acid (Compound 2), 1, 7-di hydroxy betulinic acid (Compound 3), 3β-O-dihydrocinnamyl betulinic acid (Compound 4), 3β-O-trans-coumaryl-lup-6, 9(11), 20(29)-triene-27, 28-dioic acid (Compound 5), 16β-hydroxy-2, 3-seco-lup-5, 20(29)-dien-2, 3, 28-trioic acid (Compound 6). Among the all isolated compounds 3β-O-trans-coumaryl-lup-6, 9(11), 20(29)-triene-27, 28-olioic acid designated as coumaroyl lupendioic acid (CLA) showed higher COX-2 selectivity which is comparable to reference drug (celecoxib). CLA significantly reduced carrageenan induced inflammation whereas CLA revealed greater effect as compared to BA at the similar corresponding doses. Moreover, CLA significantly inhibited pro-inflammatory mediators elevated by carrageenan. CLA also preserved the tissue architecture as evidenced by the histopathology. Furthermore, immunohistochemical studies revealed that CLA significantly down regulated NF-ҡB, COX-2 and TNF-α protein expression.

Conclusion: The study gives an insight into the molecular mechanisms of coumaroyl lupendioic acid and suggests that the down-regulations of proinflammatory mediators provide credence to the ethno botanical use of the plant in the management of inflammation.

Keywords: Betulinic acid; COX-2; Carrageenan; Coumaroyl lupendioic acid; NF-ҡB; PGE(2); TNF-α.