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

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-1ß and IL-6 were assessed. In addition, the histopathology and immunohistochemistry (NF-Ò¡B, 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; 3ß-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), 3ß-O-dihydrocinnamyl betulinic acid (Compound 4), 3ß-O-trans-coumaryl-lup-6, 9(11), 20(29)-triene-27, 28-dioic acid (Compound 5), 16ß-hydroxy-2, 3-seco-lup-5, 20(29)-dien-2, 3, 28-trioic acid (Compound 6). Among the all isolated compounds 3ß-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-Ò¡B, 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.

Inhibition of α-glucosidase by new prenylated flavonoids from euphorbia hirta L. herb.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia hirta L. (Euphorbiaceae) is a pantropical medicinal rhizomatous herb, traditionally used in the treatment of diabetes, respiratory and gastro-intestinal disorders. AIM OF THE STUDY: To isolate and characterize the constituents of Euphorbia hirta and evaluate their in-vitro α-glucosidase inhibitory activity. The study is also aimed at describing structural activity relationship, type of α-glucosidase inhibition and in-vivo potential to regulate post prandial hyperglycemia in Wistar rats. MATERIALS AND METHODS: Methanolic extract of whole plant 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 under the following experimental conditions, pressure (up to 5 kg/cm(2)) and flow rate (2 in./min). The structural elucidation of isolated compounds was done on the basis of detailed spectral analysis. The α-glucosidase inhibitory potential of isolated compounds was evaluated and compared with standard drug acarbose. In addition, type of inhibition was dwelled by Lineweaver-Burk plot analysis. Further, sucrose tolerance test was performed in Wistar rats pre-treated with the isolated compounds and acarbose (0.015 mM) followed by a sucrose load (2g/kg, p.o.) and blood glucose level was measured up to 120 min by the glucose oxidase method. RESULTS: The ethyl acetate fraction afforded quercetrin (1), dimethoxy quercetrin (2), along with two new prenylated flavonosides designated as hirtacoumaroflavonoside (3) and hirtaflavonoside-B (4) characterized as 7-O-(p-coumaroyl)-5,7,4'-trihydroxy-6-(3,3-dimethyl allyl)-flavonol-3-O-ß-D-glucopyranosyl-(2" → 1"')-O-α-L-rhamnopyranoside and 5, 7, 3', 4'-trihydroxy-6-(3, 3-dimethyl allyl)-8-(iso-butenyl)-flavonol-3-C-ß-d-glucopyranoside, respectively. All the isolated compounds showed dose dependent inhibition of α-glucosidase which was found to be comparable to acarbose. Maximum α-glucosidase inhibition was achieved with compound 3 (IC50 0.022 mM) followed by 4 (IC50 0.071 mM) in comparison to acarbose (IC50 0.092 mM). The results revealed that 5,7,4'- trihydroxyflavone structure is imperative for the inhibitory activity. The prenylation in the flavonoids increase the potency and p-coumaroyl substitution at C-7 further enriched the α-glucosidase inhibition. Compound 3 exhibited non-competitive inhibition while compounds 1, 2 and 4 showed mixed non-competitive inhibitory pattern. The results of sucrose tolerance test corresponded well with the in vitro studies. CONCLUSION: α-Glucosidase inhibitory activity and sucrose tolerance test demonstrated by the prenylated flavonoids present in E. hirta provide credence to the ethnomedicinal use of the plant in the management of diabetes in folk medicine.

Inhibitory effect of Careya arborea on inflammatory biomarkers in carrageenan-induced inflammation.

CONTEXT: Careya arborea Roxb. (Lecythidaceae) has multiple applications in traditional medicine; it exhibits analgesic, antibacterial, anti-inflammatory, antiulcer, and protective effects. However, the effect of C. arborea on biochemical and immmunological inflammatory mediators has not been explored. OBJECTIVE: The present study investigates the anti-inflammatory potential of the methanol extract of C. arborea stem bark and further assesses its possible mechanism on the modulation of inflammatory biomarkers. MATERIALS AND METHODS: Anti-inflammatory activity of C. arborea methanol extract (CAME) was evaluated (100 and 200 mg/kg, p.o) using indomethacin (10 mg/kg, p.o) as the standard drug in Wistar albino rats. Inflammation was induced by injecting 0.1 ml carrageenan (1% w/v) into the left hind paw. The anti-inflammatory mechanism was studied by measuring malondialdehyde (MDA), C-reactive protein (CRP), nitric oxide (NO), myeloperoxidase (MPO), TNF-α, and IL-1ß levels in both control and treated groups. A protocol has also been established to quantify quercetin and betulinic acid content in CAME using HPTLC fingerprint. RESULTS: Careya arborea significantly (p < 0.001) decreased carrageenan-induced paw edema, showed a reduction of 48.87 and 65.53% at doses of 100 and 200 mg/kg, respectively. Moreover, CAME significantly decreased the MDA, CRP, NO, and MPO levels, elevated by carrageenan induced inflammation. CAME also markedly down-regulated serum TNF-α and IL-1ß levels. These findings were further supported by the histological study. The content of quercetin and betulinic acid in CAME was found to be 0.177 and 3.14%, respectively. CONCLUSION: Several mechanisms, including the inhibition of pro-inflammatory cytokines, enzymes and mediators release, appear to account for the anti-inflammatory potential of C. arborea.