Bioassay-guided isolation and mechanistic action of anti-inflammatory agents from Clerodendrum inerme leaves (original) (raw)

2015, Journal of Ethnopharmacology

Ethnopharmacological relevance: The leaves of Clerodendrum inerme (L.) Gaertn. have commonly been used in Thai traditional medicine for treatment of inflammatory diseases. However, the bioactive compounds responsible for the anti-inflammatory effect of leaves have not been yet determined. The objective of the present study was to isolate these bioactive compounds by bioassay-guided isolation technique and to determine the mode of action of isolated compounds in LPS-induced macrophages. Materials and methods: Anti-inflammatory effect of various fractions (hexane, ethyl acetate and water) of ethanol extract of C. inerme leaves was determined from the production of nitric oxide (NO) in RAW 264.7 macrophage stimulated with LPS. The mRNA and protein levels were determined also by real-time reverse transcription-polymerase chain reaction and western blot analysis, respectively. Leaf bioactive compounds were isolated by bioassay-guided fractionation technique using column chromatography. Results: The ethyl acetate fraction (EA) among solvent extracts provided the most potent inhibitory activity on NO production. Also, EA reduced the mRNA and protein expressions of inducible nitric oxide synthase (iNOS) in LPS-stimulated macrophages. Three known flavones, acacetin (1), hispidulin (2) and diosmetin (3), were isolated based on inhibition of NO production. Furthermore, hispidulin also inhibited PGE 2 production as well as iNOS and cyclooxygenase-2 expressions via the blockade of NF-κB DNAbinding activity and JNKway. Conclusions: Our results found acacetin (1), hispidulin (2) and diosmetin (3), were responsible for the anti-inflammatory properties of C. inerme leaves. We provide scientific evidence to support the usefulness of C. inerme leaves in traditional medicine for the treatment of inflammation-related diseases. & 2015 Elsevier Ireland Ltd. All rights reserved. of these mediators causes a harmful effect to tissues and organisms as well as has been associated with the pathogenesis of various inflammatory-related diseases, including rheumatoid arthritis, diabetes, inflammatory bowel disease, atherosclerosis, and cancer (Yang et al., 2013). Clerodendrum inerme (L.) Gaertn. (Verbenaceae family) known also as Sam-Ma-Nga in Thai or, seaside clerodendrum in English, occurs widely in coastal mangrove forests of Thailand (Office of Mangrove Resources Conservation, 2009) and other South Asian countries and is used in traditional medicine for the treatment of skin diseases, rheumatic pain and arthritis, fever, cough, hepatitis, and other inflammatory diseases (Shrivastava and Patel, 2007a; Chethana et al., 2013). The major chemical constituents of C. inerme are flavonoids, terpenes, steroids and phenolic compounds (Shrivastava and Patel, 2007b; Parveen et al., 2010; Shahabuddin et al., 2013). C. inerme possesses a number of biological activities including antimicrobial, anti-hepatotoxic, anti-oxidant, analgesic and anti-inflammatory activities (Gopal Contents lists available at ScienceDirect