Klaokwan Srisook

Anti-inflammatory effect of ethyl acetate extract from Cissus quadrangularis Linn may be involved with induction of heme oxygenase-1 and suppression of NF-κB activation

AIM OF THE STUDY Cissus quadrangularis (family: Vitaceae) has been widely used in traditional herbal medicine for the treatment of hemorrhoids, gastric ulcers and bone healing. In the present study, we determined the anti-inflammatory activity and the molecular mechanism of the ethyl acetate extract of Cissus quadrangularis stem (CQE) in LPS-stimulated RAW 264.7 macrophage cells. MATERIALS AND METHODS The inhibitory effect of CQE on LPS-induced nitric oxide (NO) production was evaluated in conditioned media. Cell viability was monitored by MTT assay. Protein and mRNA expressions were determined by RT-PCR and Western blotting analysis, respectively. RESULTS CQE potently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells in a dose-dependent manner. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS) were suppressed also by CQE as was p65 NF-κB nuclear translocation. Further study demonstrated that CQE by itself induced heme oxygenase-1 (HO-1) gene expression at the protein and mRNA levels in dose- and time-dependent manner. In addition, the inhibitory effects of CQE on NO production were abrogated by a HO-1 inhibitor, zinc protoporphyrin IX (ZnPP). CONCLUSIONS Collectively, these results suggest that CQE exerts an anti-inflammatory effect in macrophages, at least in part, through the induction of HO-1 expression. These findings provide the scientific rationale for anti-inflammatory therapeutic use of Cissus quadrangularis stem.

Antioxidant and anti-inflammatory activities of hot water extract from Pluchea indica Less. herbal tea

In this study, antioxidant activity of the hot water extract of Pluchea indica tea leaves (HWEP) was measured by assays for radical scavenging against 1,1-diphenyl-2-picrylhydrazyl, superoxide and hydroxyl radical, ferric ion reducing power, as well as ferrous ion chelating. Results showed that HWEP exhibited good antioxidant activity in all test systems in a concentration-dependent manner. Furthermore, HWEP has potent inhibitory effects against lipopolysaccharide-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophages. The hot water extract of P. indica leaves contains a source of antioxidants and inhibitors of NO and PGE2 production that can be used as dietary supplements containing good health promoting effect.

Bioassay-guided isolation and mechanistic action of anti-inflammatory agents from Clerodendrum inerme leaves

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 PGE2 production as well as iNOS and cyclooxygenase-2 expressions via the blockade of NF-κB DNA-binding 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.

Non-cytotoxic hybrid bioscaffolds of chitosan-silica: Sol-gel synthesis, characterization and proposed application

Biohybrid chitosan-silica scaffolds were synthesized through the sol-gel and the freeze drying processes. Hydrolysis and condensation of chitosan with tetraethylorthosilicate (TEOS) in the presence of 3-isocyanatopropyl triethoxysilane (ICPTES) were successfully carried out. Results obtained from FTIR, swelling test and pyrolysis confirmed that the hybrid scaffolds containing covalent coupling between the organic and inorganic networks were formed with high crosslink density of SiOSi bridging and could be classified as the class II material. The hybridization also resulted in improvements on mechanical strength and stability comparing to the pure chitosan. In vitro investigations on the guided bone regeneration and the cytotoxicity were also performed. SEM-EDS was used to examine the proliferation of calcium phosphate mineral at the scaffold surface after an immersion in simulated body fluid. The results revealed that the hybrid scaffolds exhibited a rapid induction of calcium phosphate mineral without cytotoxicity effect, reflecting an excellent in vitro bone bioactivity which was superior to the pure chitosan scaffold.

Involvement of p38 MAPK and ATF-2 signaling pathway in anti-inflammatory effect of a novel compound bis[(5-methyl)2-furyl](4-nitrophenyl)methane on lipopolysaccharide-stimulated macrophages

Abstract Activated macrophages produce various pro‐inflammatory mediators such as inducible nitric oxide synthase (iNOS)‐derived nitric oxide (NO) and cyclooxygenase (COX)‐2‐derived prostaglandin E2 (PGE2) during inflammatory response. However, overproduction of NO and PGE2 appears to be involved in pathogenesis of various inflammatory diseases. Therefore, inhibition of NO and PGE2 production might be useful for the treatment of inflammatory‐related diseases. In this study, the bis[(5‐methyl)2‐furyl](4‐nitrophenyl)methane or BFNM was evaluated for the anti‐inflammatory activity and mechanism of action in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage. BFNM inhibited NO and PGE2 production in a concentration‐dependent manner and down‐regulated the expression of iNOS and COX‐2 at mRNA and protein levels. BFNM suppressed nuclear translocation of NF‐&kgr;B p65 subunit only very slightly, and failed to decrease NF‐&kgr;B DNA binding activity. In contrast, the compound significantly reduced phosphorylation of p38 MAPK and ATF‐2, a component of AP‐1 known to be involved in the transcriptional regulation of iNOS and COX‐2, in a dose‐dependent manner in LPS‐induced cells. Collectively, these results suggest that BFNM has an anti‐inflammatory effect in RAW 264.7 macrophages, at least in part, by suppression of NO and PGE2 production. The inhibitory effect of BFNM is mediated mainly via the p38 MAPK/ATF‐2 signaling pathway. Thus, BFNM would be a lead compound for the development of novel anti‐inflammatory agents. HighlightsBis[(5‐methyl)2‐furyl](4‐nitrophenyl)methane (BFNM) inhibited NO and PGE2 production in a concentration‐dependent manner.BFNM suppressed the expression of iNOS and COX‐2 at mRNA and protein levels.BFNM slightly suppressed nuclear translocation of NF‐&kgr;B p65 subunit but failed to decrease NF‐&kgr;B DNA binding activity.BFNM reduced phosphorylation of p38 MAPK and ATF‐2, but not ERK and JNK.

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

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. 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.

4-methoxycinnamyl p-coumarate isolated from Etlingera pavieana rhizomes inhibits inflammatory response via suppression of NF-κB, Akt and AP-1 signaling in LPS-stimulated RAW 264.7 macrophages.

BACKGROUND 4-methoxycinnamyl p-coumarate (MCC) was isolated from rhizomes of Etlingera pavieana by bioactivity-guided isolation, however, the molecular mechanism underlying its anti-inflammatory activity remains inadequately understood. PURPOSE In this study, we elucidated the suppressive effect of MCC on LPS-induced expression of inflammatory mediators and the molecular mechanisms responsible for anti-inflammatory activities in RAW 264.7 macrophages. METHODS Cell viability of MCC-treated RAW 264.7 macrophage was measured by MTT assay. Anti-inflammatory activity was evaluated by measurement of NO, PGE2, and cytokine production in LPS-stimulated cells. qRT-PCR and Western blotting analysis were used to investigate mRNA and protein levels of inflammatory responsive genes. NF-κB activation and transactivation activity were determined by immunofluorescence and reporter gene assay, respectively. RESULTS MCC considerably suppressed both the production of NO, PGE2, IL-1β as well as TNF-α and their expression. MCC inactivated NF-κB by reducing phosphorylation of IκBα and inhibiting NF-κB p65 nuclear translocation. Also, MCC significantly inhibited NF-κB transactivation activity. However, the inhibitory effect of MCC was independent of the MAPK signaling pathway. Furthermore, MCC significantly decreased phosphorylation of Akt and c-Jun, a main component of AP-1. CONCLUSION These findings suggest that the anti-inflammatory effect of MCC could be mediated by the inhibition of LPS-induced expression of inflammatory mediators by down-regulation of the NF-κB, Akt and AP-1 signaling pathways in murine macrophages.