Acute and subchronic oral toxicity assessment of extract from Etlingera pavieana rhizomes.

In Southeast Asia, the rhizome of Etlingera pavieana is commonly consumed and parts of the rhizomes have been used as a medicine for the treatment of several disorders. Its pharmacological effects have previously been reported. However, its potential toxicity has not been described. This study aimed to evaluate in vivo toxicity of E. pavieana rhizome extract (EPE) in Sprague Dawley rats. Acute toxicity testing of EPE at a single dose of 2,000 mg/kg produced no toxic effects in female rats after 14 days of treatment. Subchronic toxicity testing showed that all doses of EPE (500, 1,000, and 2,000 mg/kg/day) produced no sign of toxicity during 90 days of treatment. All biochemical and hematological values were within normal ranges. There were no significant histopathological differences in the internal organs among the tested groups. Therefore, the no-observed-adverse-effect level of EPE was 2,000 mg/kg/day in both male and female rats, thereby confirming the safety of EPE for use in traditional medicines.

Etlingera pavieana extract attenuates TNF-α induced vascular adhesion molecule expression in human endothelial cells through NF-κB and Akt/JNK pathways.

The aim of this study was to determine whether ethanol extracts of Etlingera pavieana rhizomes (EPE) can inhibit the expression of ICAM-1 and VCAM-1 in TNF-α-stimulated human vascular endothelial cells. EPE significantly reduced ICAM-1 and VCAM-1 expression in a concentration-dependent manner. EPE also suppressed phospho-IκB level and nuclear translocation of NF-κB. EPE significantly inhibited phosphorylation of JNK and c-Jun, a major component of AP-1, but had no effects on ERK and p38 MAPK pathways. Akt phosphorylation was increased in the presence of EPE, and wortmannin and SP600125 reversed the inhibitory effects of EPE on ICAM-1 and VCAM-1 expression. Furthermore, the active EPE constituents 4-methoxycinnamyl p-coumarate and trans-4-methoxycinnamaldehyde attenuated TNF-α-induced expression of ICAM-1 and VCAM-1. Taken together, our data indicate that EPE protects against vascular inflammation in endothelial cells, in part via NF-κB and Akt/JNK signalings. In future studies, E. pavieana may be developed as a therapeutic agent or dietary supplement for treating and preventing inflammatory diseases.

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-1ß 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.

Inhibition of human cytochromes P450 2A6 and 2A13 by flavonoids, acetylenic thiophenes and sesquiterpene lactones from Pluchea indica and Vernonia cinerea.

The human liver cytochrome P450 (CYP) 2A6 and the respiratory CYP2A13 enzymes play role in nicotine metabolism and activation of tobacco-specific nitrosamine carcinogens. Inhibition of both enzymes could offer a strategy for smoking abstinence and decreased risks of respiratory diseases and lung cancer. In this study, activity-guided isolation identified four flavonoids 1-4 (apigenin, luteolin, chrysoeriol, quercetin) from Vernonia cinerea and Pluchea indica, four hirsutinolide-type sesquiterpene lactones 5-8 from V. cinerea, and acetylenic thiophenes 9-11 from P. indica that inhibited CYP2A6- and CYP2A13-mediated coumarin 7-hydroxylation. Flavonoids were most effective in inhibition against CYP2A6 and CYP2A13, followed by thiophenes, and hirsutinolides. Hirsutinolides and thiophenes exhibited mechanism-based inhibition and in irreversible mode against both enzymes. The inactivation kinetic KI values of hirsutinolides against CYP2A6 and CYP2A13 were 5.32-15.4 and 0.92-8.67 µM, respectively, while those of thiophenes were 0.11-1.01 and 0.67-0.97 µM, respectively.

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. 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.

Inhibition effects of Vernonia cinerea active compounds against cytochrome P450 2A6 and human monoamine oxidases, possible targets for reduction of tobacco dependence.

The human cytochrome P450 2A6 (CYP2A6) and monoamine oxidases (MAO-A and MAO-B), catalyzing nicotine and dopamine metabolisms, respectively, are two therapeutic targets of nicotine dependence. Vernonia cinerea, a medicinal plant commonly used for treatment of diseases such as asthma and bronchitis, has been shown reducing tobacco dependence effect among tobacco users. In the present study, we found eight active compounds isolated from V. cinerea that comprise inhibitory activity toward CYP2A6 and MAO-A and MAO-B enzymes using activity-guided assays, with coumarin as substrate of CYP2A6 and kynuramine of MAOs. These compounds were three flavones (apigenin, chrysoeriol, luteolin), one flavonol (quercetin), and four hirsutinolide-type sesquiterpene lactones (8α-(2-methylacryloyloxy)-hirsutinolide-13-O-acetate, 8α-(4-hydroxymethacryloyloxy)-hirsutinolide-13-O-acetate, 8α-tigloyloxyhirsutinolide-13-O-acetate, and 8α-(4-hydroxytigloyloxy)-hirsutinolide-13-O-acetate). Modes and kinetics of inhibition against the three enzymes were determined. Flavonoids possessed strong inhibitory effect on CYP2A6 in reversible mode, while inhibition by hirsutinolides was mechanism-based (NADPH-, concentration-, and time-dependence) and irreversible. Inhibition by hirsutinolides could not be reversed by dialysis and by addition of trapping agents or potassium ferricyanide. Flavonoids inhibited MAOs with variable degrees and were more prominent in inhibition toward MAO-A than hirsutinolides, while two of hirsutinolides inhibited MAO-B approximately comparable to two flavonoids. These results could have implications in combination of drug therapy for smoking cessation.

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.

Structure­function relationships of inhibition of mosquito cytochrome P450 enzymes by flavonoids of Andrographis paniculata.

The cytochrome P450 monooxygenases are known to play a major role in pyrethroid resistance, by means of increased rate of insecticide detoxification as a result of their overexpression. Inhibition of detoxification enzymes may help disrupting insect detoxifying defense system. The Anopheles minimus CYP6AA3 and CYP6P7 have shown pyrethroid degradation activity and been implicated in pyrethroid resistance. In this study inhibition of the extracts and constituents of Andrographis paniculata Nees. leaves and roots was examined against benzyloxyresorufin O-debenzylation (BROD) of CYP6AA3 and CYP6P7. Four purified flavones (5,7,4'-trihydroxyflavone, 5-hydroxy-7,8-dimethoxyflavone, 5-hydroxy-7,8,2',3'-tetramethoxyflavone, and 5,4'-dihydroxy-7,8,2',3'-tetramethoxyflavone), one flavanone (5-hydroxy-7,8-dimethoxyflavanone) and a diterpenoid (14-deoxy-11,12-didehydroandrographolide) containing inhibitory effects toward both enzymes were isolated from A. paniculata. Structure­function relationships were observed for modes and kinetics of inhibition among flavones, while diterpenoid and flavanone were inferior to flavones. Docking of flavones onto enzyme homology models reinforced relationships on flavone structures and inhibition modes. Cell-based inhibition assays employing 3-(4,5-dimethylthiazol-2-y-l)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays revealed that these flavonoids efficiently increased susceptibility of CYP6AA3- and CYP6P7-expressing Spodoptera frugiperda (Sf9) cells to cypermethrin toxicity, due to inhibition effects on mosquito enzymes. Thus synergistic effects on cypermethrin toxicity of A. paniculata compounds as a result of enzyme inhibition could be useful for mosquito vector control and insecticide resistance management in the future.

Mechanism-based inactivation of cytochrome P450 2A6 and 2A13 by Rhinacanthus nasutus constituents.

  Human cytochrome P450 CYP2A6 and CYP2A13 catalyze nicotine metabolisms and mediate activation of tobacco-specific carcinogens including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). In this study, we found rhinacanthins A, B, and C isolated from Rhinacanthus nasutus potentially inhibited coumarin 7-hydroxylation mediated by reconstituted purified recombinant CYP2A6 and CYP2A13. Rhinacanthins A-C are mechanism-based inactivators of CYP2A6 and CYP2A13 as they cause concentration, time and NADPH-dependent inhibition. Among the three rhinacanthins, rhinacanthin-B possessed highest inhibitory potency against CYP2A13 with apparent KI and kinact of 0.16 µM and 0.1 min(-1), respectively, while values of 0.44 µM and 0.12 min(-1) were found against CYP2A6. Rhinacanthin-C had least inhibition potency, with apparent KI and kinact of 0.97 µM and 0.07 min(-1) for CYP2A6, respectively, and values of 1.68 µM and 0.05 min(-1) for CYP2A13. Rhinacanthin-A inhibited CYP2A6 and CYP2A13 with apparent KI values of 0.69 and 0.42 µM, respectively and apparent kinact of 0.18 and 0.06 min(-1), respectively. The inhibition of both enzymes by rhinacanthins A-C could not be prevented by addition of trapping agents or reversed by dialysis or potassium ferricyanide. These findings demonstrated that rhinacanthins A-C, which are 1,4-naphthoquinone derivatives, irreversibly inhibited CYP2A6 and CYP2A13 in a mechanism-based inhibition mode.

Molecular mechanism of anti-inflammatory activity of Pluchea indica leaves in macrophages RAW 264.7 and its action in animal models ofinflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: PLUCHEA INDICA LESS.: (Asteraceae) is a Thai medicinal plant used in traditional medicine for the treatment of hemorrhoids, lumbago, leucorrhoea and inflammation. This study investigated the molecular mechanism of anti-inflammatory activity of Pluchea indica leaf extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and also determined its action in acute inflammation animal models. MATERIALS AND METHODS: The inhibitory effect of Pluchea indica leaf extract on LPS-induced nitric oxide (NO) production was evaluated by Griess reaction. Protein and mRNA expressions were determined by real time RT-PCR and Western blotting analysis, respectively. Inducible nitric oxide synthase (iNOS) promoter activity was evaluated by iNOS promoter based reporter gene assay. In vivo anti-inflammatory effect was examined in ethylphenylpropiolate (EPP)-induced ear edema and carrageenan-induced paw edema in rat models. RESULTS: Ethyl acetate fraction of ethanol extract of Pluchea indica leaves (EFPI) exhibited the potent inhibitory effect on NO production in LPS-induced macrophages and also inhibited PGE2 release. EFPI reduced iNOS mRNA and protein expression through suppressed iNOS promoter activity and nuclear translocation of subunit p65 of nuclear factor-κB, but did not inhibit phosphorylation of the mitogen-activated protein kinases (MAPKs). Moreover, EFPI possessed anti-inflammatory activities on acute phase of inflammation as seen in EPP-induced ear edema and carrageenan-induced paw edema inrats. CONCLUSIONS: These data support the pharmacological basis of Pluchea indica plant as a traditional herbal medicine for treatment of inflammation.

Inhibition against mosquito cytochrome P450 enzymes by rhinacanthin-A, -B, and -C elicits synergism on cypermethrin cytotoxicity in Spodoptera frugiperda cells.

Rhinacanthus nasutus (Acanthaceae) is a shrub reported to contain insecticidal activities. The current study was conducted to determine whether R. nasutus constituents could inhibit benzyloxyresorufin O-debenzylation (BROD) mediated by CYP6AA3 and CYP6P7. Both enzymes have shown pyrethroid degradation activity and been implicated to play role in pyrethroid resistance in Anopheles minumus (Theobald) mosquito, a malaria vector. Three compounds, rhinacanthin-A, -B, and -C that exhibited potent inhibitory activity were isolated and purified from aerial part of R. nasutus. Their kinetic parameters and modes of inhibition were determined. Rhinacanthin-B was the most potent inhibitor in in vitro inhibition assay and exhibited mechanism-based inhibition against both CYP6AA3 and CYP6P7. Rhinacanthin-C which is a major compound of R. nasutus reversibly inhibited both enzymes in vitro with 2-4 folds less inhibitory potency than rhinacanthin-B. In contrast, rhinacanthin-A reversibly inhibited CYP6AA3, but inhibition against CYP6P7 was a mechanism-based inhibition type. Where mechanism-based inhibition was found, the inhibition showed characteristic of time-, concentration-dependence, and requirement of NADPH. Using 3-(4, 5-dimethylthiazol-2-y-l)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity assay in intact Spodoptera frugiperda (Sf9) cells, the three compounds increased susceptibility of CYP6AA3- and CYP6P7-expressing cells to cypermethrin cytotoxicity because of inhibition effect on mosquito enzymes. The combined inhibition effect on mosquito cytochrome P450 enzyme and synergistic effect on cypermethrin cytotoxicity of the three R. nasutus compounds could be beneficial for resistance management strategies in mosquito vector control.

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.

Syntheses of tetrahydroisoquinoline derivatives that inhibit NO production in activated BV-2 microglial cells.

Seventeen tetrahydroisoquinoline derivatives were designed, synthesized and evaluated for inhibition of NO production in lipopolysaccharide-stimulated BV-2 microglial cells. Compounds 5a, 9c and 11a potently attenuated NO production by >60%, and 5a and 11a inhibited BH4 production by >48% at 100 microM. In particular, N-ethylcarbonyl-7-hydroxy-6-methoxy-1,2,3,4-tetrahydroisoquinoline (11a) reduced NO production by 64% and tetrahydrobiopterin (BH4) production by 49%. Introducing longer alkyl component at C1 or N2 position led to attenuation of the inhibitory effect. It is possible that 11a inhibits NO production by blocking BH4-dependent dimerization of newly synthesized iNOS monomers.

Syntheses of NAMDA derivatives inhibiting NO production in BV-2 cells stimulated with lipopolysaccharide.

Sixteen derivatives of N-acetyl-3-O-methyldopamine (NAMDA), an inhibitor of BH4 synthesis, were designed and synthesized. The ability of these derivatives to inhibit NO and BH4 production by lipopolysaccharide-stimulated BV-2 microglial cells was determined. While NAMDA at 100 microM inhibited NO and BH4 production by only about 20%, its catecholamide 8, indole 23 derivative, 13, and N-acetyl tetrahydroisoquinoline 25 inhibited the NO production by >50% at the same concentration. In particular, 13 and 25 inhibited both NO and BH4 production to similar degrees, which suggested that these compounds might inhibit NO production by blocking BH4-dependent dimerization of the newly synthesized iNOS monomer.

Electrophilic aromatic addition reaction: electrophilic attack at an aromatic H substituent position.

[reaction: see text] We report and propose a mechanism for an unusual electrophilic aromatic addition reaction (Ad(E)()Ar). During our preparation of 5,7-dibromo-8-methoxyquinaldine as a key intermediate in the synthesis of 7-bromoquinaldine-5,8-dione, direct bromination in either acidic or neutral conditions led only to the formation of 5-bromo-8-methoxyquinaldine. Under basic methanolic conditions, however, we unexpectedly obtained the 5,7-dibromo-8,8-dimethoxy-7,8-dihydroquinaldine adduct 2a. This result not only allows for the functionalization of aromatic compounds via the addition adducts, but also introduces the possibility of an alternate mechanism for electrophilic substitution reactions.