Anti-inflammatory effect of Etlingera pavieana (Pierre ex Gagnep.) R.M.Sm. rhizomal extract and its phenolic compounds in lipopolysaccharide-stimulated macrophages

Abstract

Background:In our continuing search for anti-inflammatory agents from Thai herbs, Etlingera pavieana (Pierre ex Gagnep.) R.M.Sm. showed potent inhibition on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced macrophages. However, the mechanism behind its inhibitory effect has not been yet explored, and little is known regarding its bioactive compounds responsible for the anti-inflammatory effect.Objective:In the present study, anti-inflammatory effect of hexane, ethyl acetate, and water fractions of rhizomal ethanol extracts of E. pavieana was evaluated for their inhibition on NO production and mechanism in LPS-stimulated macrophages. Active compounds responsible for such anti-inflammatory activity were identified.Materials and Methods:Inhibitory activities on NO production were performed in LPS-stimulated RAW264.7 macrophage. Cytotoxicity of plant extracts was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, mRNA and protein expressions by reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. Anti-inflammatory compounds were isolated by activity-guided isolation technique using column chromatography.Results:Ethyl acetate fraction of E. pavieana (EPE) showed the most potent inhibitory effect on NO production in macrophages. EPE significantly decreased NO production and inhibited inducible nitric oxide synthase (iNOS) protein and mRNA expression in a dose-dependent manner. Furthermore, the level of nuclear factor-kappa B p65 subunit was markedly reduced in activated cells treated with EPE. Four phenolic compounds, 4-methoxycinnamyl alcohol (1), trans-4-methoxycinnamaldehyde (2), 4-methoxycinnamyl p-coumarate (3), and p-coumaric acid (4), were obtained from bioactivity-guided isolation technique.Conclusions:The anti-inflammatory property contained in E. pavieana rhizome extract and conferred through inhibition of iNOS expression, and NO formation provides scientific evidence and support for the development of new anti-inflammatory agents based on extracts from this plant.SUMMARY Ethyl acetate fraction (EPE) of Etlingera pavieana showed the most potent inhibitory effect on NO production in LPS-induced macrophagesFour phenolic compounds, 4-methoxycinnamyl alcohol (1), trans-4-methoxycinnamaldehyde (2), 4-methoxycinnamyl p-coumarate (3) and p-coumaric acid (4), responsible for the anti-inflammatory effect of EPE were isolated. Abbreviations used: EPE: Ethyl acetate fraction of Etlingera pavieana; EPH: Hexane fraction of Etlingera pavieana; EPW: Water fraction of Etlingera pavieana; NO: Nitric oxide (NO); LPS: Lipopolysaccharide; iNOS: Inducible nitric oxide synthase (iNOS); MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB: Nuclear factor-kappa B; DMSO: Dimethyl sulfoxide; EtOAc: Ethylacetate; MeOH: Methanol; AG: Aminoguanidine; DCM: Dichloromethane; MCA: 4-methoxycinnamyl alcohol; MCD: trans-4-methoxycinnamaldehyde; MCC: 4-methoxycinnamyl p-coumarate; CM: p-coumaric acid.

Cytotoxic effects of Etlingera pavieana rhizome on various cancer cells and identification of a potential anti-tumor component

“…A high singlet signal at δ3.80 ppm indicated a methoxy group on an aromatic ring. By comparison with a reported chemical shift analysis (Battistuzzi et al, ; Srisook et al, ), it was found that SF1 fraction contained trans ‐4‐methoxycinnamaldehyde (4‐MCA) or (2E)‐3‐(4‐methoxyphenyl)prop‐2‐enal as a major component (Figure B). The presence of 4‐MCA as a major compound in the SF1 fraction was confirmed by HPLC analysis (http://onlinelibrary.wiley.com/doi/10.1111/jfbc.12540/suppinfo).…”

“…This process was shown to be involved in inflammation, delayed gastric emptying, cold sensation, and chemosensation (Moon et al, ). Recently, anti‐inflammation activity of 4‐MCA has been recognized (Srisook et al, ). Although anticancer effect of several cinnamaldehyde derivatives have been noticed (Han et al, ; Jeong et al, ; Lin, Wu, & Lin, ; Perng et al, ; Wong et al, ), this is the first report on in vitro antitumor activity of 4‐MCA.…”

“…Due to small amount of SF1 sub‐fraction, identification of containing compounds in this sub‐fraction was primarily evaluated by 1 H‐NMR spectroscopy (Bruker AVANCE 400, Switzerland) in CDCl 3 solvent and compared with published data (Battistuzzi, Cacchi, & Fabrizi, ; Srisook et al, ). The major phytochemical compound in SF1 was confirmed by HPLC using a Waters 600 HPLC pump and controller in‐line degasser coupled to a Waters 996 photodiode array detector, utilizing a RP‐18 analytical column 250 mm × 4.6 mm Luna, Phenomenex, Inc. (Waters, The United States).…”

“…Recently, an anti‐inflammatory activity of the ethyl acetate (EA) fraction from its ethanolic extract has been revealed. The extract has been shown to prevent the LPS‐stimulated inflammation in RAW264.7 macrophage by inhibiting NF‐κB signaling pathway, leading to down‐regulated iNOS expression and decreased NO production (Srisook, Palachot, Mankhong, & Srisook, ). Four bioactive phenolic compounds (4‐methoxycinnamyl alcohol, trans ‐4‐methoxycinnamaldehyde, p ‐coumaric acid, and 4‐methoxycinnamyl p ‐coumarate) were isolated from this EA fraction (Srisook et al, ).…”

Pharmacological Activities and Phytochemicals of Etlingera pavieana (Pierre ex Gagnep) R.M.Sm

“…It has been recently reported that the investigation of rhizomes of Etlingera pavieana from Thailand resulted in isolation of an uncommon compound which is (E)-((E)-3-(4-methoxyphenyl)ally) 3-(4-hydroxyphenyl) acrylate or 4-methoxycinnamyl p-coumarate (MCC) (1) and a series of phenylpropanoids such as 7, from the rhizome of E. pavieana by activity-guided isolation [8]. The other three common phenylpropanoids, (E)-4methoxycinnamaldehyde (3), trans-anethole (8), and trans-methyl isoeugenol (9), were also extracted from the plant by Srisook and colleagues (unpublished data).…”