Pimpimon Tansakul

Suppressive effects of methoxyflavonoids isolated from Kaempferia parviflora on inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells

ETHNOPHARMACOLOGICAL RELEVANCE The rhizomes of Kaempferia parviflora Wall. ex Baker have been traditionally used in Thailand to treat abscesses, gout, and peptic ulcers. AIM Previously, we reported that the chloroform fraction of a Kaempferia parviflora extract had an inhibitory effect on rat paw-edema. In the present study, we isolated the constituents of this fraction and investigated the anti-inflammatory mechanism against nitric oxide (NO) production, tumor necrosis factor-α (TNF-α) and the expression of inducible nitric oxide synthase (iNOS) as well as phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK). In addition, effects of trimethylapigenin (4) on the enzyme activities of protein kinases possibly leading to iNOS expression were examined to clarify the targets. MATERIALS AND METHODS The chloroform fraction was isolated using silica gel column chromatography and HPLC. Isolated compounds were tested against NO and TNF-α using RAW264.7 cells. Cytotoxicity and iNOS, p-ERK and p-JNK expression were also examined. RESULTS Three active components, 5,7-dimethoxyflavone (2), trimethylapigenin (4), and tetramethylluteolin (5), markedly inhibited the production of NO in lipopolysaccharide (LPS)-activated RAW264.7 cells. Compounds 2, 4, and 5 moderately inhibited production of TNF-α. Compounds 2, 4, and 5 strongly inhibited expression of iNOS mRNA and iNOS protein in a dose-dependent manner, but did not inhibit p-ERK or p-JNK protein expression. The most active compound, 4, did not inhibit the enzyme activity of inhibitor of κB kinases or mitogen-activated protein kinases, but inhibited that of spleen tyrosine kinase (SYK). CONCLUSION The mechanism responsible for the anti-inflammatory activity of methoxyflavonoids from the chloroform fraction of the rhizomes of Kaempferia parviflora is mainly the inhibition of iNOS expression, and the inhibition of SYK by 4 may be involved in the suppression of LPS-induced signaling in macrophages.

Anti-allergic principles of Rhinacanthus nasutus leaves

Three naphthoquinone derivatives, rhinacanthin-C (1), -D (2) and -N (3) were isolated from the extract of Rhinacanthus nasutus Kurz leaves and were tested for anti-allergic effect. The result indicated that all three compounds possessed very potent anti-allergic activity against antigen-induced beta-hexosaminidase release as a marker of degranulation in RBL-2H3 cells with IC(50) values of 6.9, 8.9 and 6.4 microM, respectively. In addition, the effects of rhinacanthin-C, -D and -N on antigen-induced release of TNF-alpha and IL-4 were also examined. It was found that rhinacanthin-C showed the most potent on antigen-induced TNF-alpha release with an IC(50) value of 0.7 microM, followed by rhinacanthin-D (IC(50)=3.8 microM) and rhinacanthin-N (IC(50)=10.3 microM), whereas those for IL-4 were rhinacanthin-D (IC(50)=5.4 microM), rhinacanthin-C (IC(50)=7.0 microM) and rhinacanthin-N (IC(50)=12.0 microM), respectively. The mechanisms in the late phase reaction of rhinacanthin-C, -D and -N were found to inhibit TNF-alpha and IL-4 gene expression in antigen-induced TNF-alpha and IL-4 releases on from RBL-2H3 cells as dose-dependent manners. The structure-activity trends of rhinacanthin-C,-D and-N on the inhibition of TNF-alpha release are as follow; substitution with octadienoic acid (rhinacanthin-C) conferred much higher activity than that of benzodioxo carboxylic acid ester (rhinacanthin-D) as well as naphthalene carboxylic acid ester (rhinacanthin-N). For the inhibition of IL-4 release, the substitution with octadienoic acid (rhinacanthin-C) and benzodioxo carboxylic acid ester (rhinacanthin-D) possessed the effect two-fold higher than that of naphthalene carboxylic acid ester (rhinacanthin-N). As regards active constituents for anti-allergic activity of R. nasutus, rhinacanthin-C, -D and -N are responsible for anti-allergic effect of this plant on both the early phase and late phase reactions. The finding may support the traditional use of R. nasutus leaves for treatment of allergy and allergy-related diseases.

Antiinflammatory constituents from Eclipta prostrata using RAW264.7 macrophage cells.

The whole plant extract of Eclipta prostrata and its isolated compounds were tested for their antiinflammatory effects against lipopolysaccharide (LPS)‐induced nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor‐alpha (TNF‐α) release in RAW264.7 cells, as well as for the antiinflammatory mechanism of the active compound on mRNA expression. Among the isolated compounds, orobol (5) exhibited the highest activity against NO release with an IC50 value of 4.6 μm, followed by compounds 1, 2 and 4 with IC50 values of 12.7, 14.9 and 19.1 μm, respectively. The IC50 value of compound 5 against PGE2 release was found to be 49.6 μm, whereas it was inactive towards TNF‐α (IC50 > 100 μm). The mechanism of orobol (5) was found to down‐regulate iNOS and COX‐2 mRNA expression in a concentration‐dependent manner. The present study may support the traditional use of Eclipta prostrata for the treatment of inflammatory‐related diseases. Copyright

Effects of rhinacanthins from Rhinacanthus nasutus on nitric oxide, prostaglandin E2 and tumor necrosis factor-alpha releases using RAW264.7 macrophage cells

Three naphthoquinone derivatives, rhinacanthin-C (1), -D (2) and -N (3) were isolated from the leaves of Rhinacanthus nasutus extract and were tested for anti-inflammatory activity. The result indicated that all three compounds possessed very potent anti-inflammatory activity against lipopolysaccharide (LPS)-induced nitric oxide release with IC(50) values of 1.8, 6.2 and 3.0 microM, respectively. In addition, the effects of rhinacanthin-C, -D and -N on LPS induced release of prostaglandin E(2) (PGE(2)) and tumor necrosis factor (TNF-alpha) were also examined. It was found that rhinacanthin-C exhibited the most potent on PGE(2) release with an IC(50) value of 10.4 microM, followed by rhinacanthin-D (IC(50)=14.4 microM) and rhinacanthin-N (IC(50)=52.1 microM), whereas those for TNF-alpha were inactive (IC(50)>100 microM). The mechanisms in transcriptional level of rhinacanthin-C were found to inhibit iNOS and COX-2 gene expressions in LPS-induced NO and PGE(2) releases from RAW264.7 cells in concentration-dependent manners. Regarding active constituents for anti-inflammatory activity of R. nasutus, rhinacanthins are responsible for this effect through the inhibition of NO and PGE(2) releases. The finding may support the traditional use of R. nasutus leaves for treatment of the inflammatory-related diseases.

Anti-inflammatory principles from Heritiera littoralis bark

Compounds from the hexane, dichloromethane and acetone extracts of Heritiera littoralis bark were investigated for their nitric oxide (NO) inhibitory effects using RAW264.7 macrophage cells. The result indicated that ergosterol peroxide (13) exhibited the highest activity against NO release with an IC(50) value of 2.5 microM, followed by 6-alpha-hydroxystigmast-4-en-3-one (11, IC(50)=9.5 microM) and stigmast-4-en-3-one (9, IC(50)=15.9 microM), whereas other compounds showed moderate and mild effects (25.4- > 100 microM). Ergosterol peroxide (13) and 6-alpha-hydroxystigmast-4-en-3-one (11) were also tested against prostaglandin E(2) (PGE(2)) and tumor necrosis factor alpha (TNF-alpha) releases. It was found that ergosterol peroxide (13) possessed marked activity against PGE(2) release with an IC(50) value of 28.7 microM, while 6-alpha-hydroxystigmast-4-en-3-one (11) was 86.7 microM. However, these two compounds were inactive towards TNF-alpha release (IC(50) > 100 microM). The mechanism in transcriptional level of ergosterol peroxide (13) was found to down regulate mRNA expressions of iNOS and COX-2 in dose-dependent manners.

Effect of methionine on production of naphthoquinones in Impatiens balsamina root cultures and detection of some secondary metabolites.

Context: Lawsone, lawsone methyl ether and 3,3′-methylelnebislawsone are the main active compounds of Impatiens balsamina L. (Balsaminaceae). These compounds possess various pharmacological activities that have been shown to assist with the treatment of skin diseases. Objective: This work focused on increased naphthoquinone production in I. basamina root cultures using methionine feeding. Materials and methods: I. balsamina root cultures were maintained in liquid Gamborg’s B5 medium supplemented with 0.1 mg/L α-naphthalene acetic acid, 0.1 mg/L kinetin, 1.0 mg/L 6-benzyladenine and 20 g/L sucrose. The effect of methionine concentration (50, 100, 300, 500 and 1000 mg/L) on naphthoquinone production of I. basamina root cultures was determined. Isolation of secondary metabolites from I. balsamina root cultures was also carried out. Results and discussion: Feeding of 300 mg/L methionine to the root cultures at the beginning of the growth cycle increased the production of 3,3′-methylelnebislawsone almost two-fold (0.63 mg/g dry weight, compared to the control group 0.32 mg/g dry weight). Optimization of the feeding conditions showed that adding 500 mg/L methionine to a 21-day old root cultures increased production of lawsone methyl ether and 3,3′-methylenebislawsone up to 2.6- and 3.1-fold higher, respectively, compared to the controls. In addition, various pharmacologically interesting secondary metabolites were isolated from I. balsamina root cultures, such as a flavonoid, luteolin, a naphthoquinone, 2,3-dihydroxy-1,4-naphthoquinone, and a triterpenoid, echinocystic acid. This is the first report of the occurrence of these compounds in this plant.

Antiinflammation constituents from Curcuma zedoaroides : Anti-inflammation Constituents from Curcuma zedoaroides

Curcuma zedoaroides, a Zingiberaceae species, has been used for snake bite antidote and wound care in Thailand. Seven compounds were isolated from the bioactive chloroform extract consisted of one new guaiane sesquiterpene lactone, 5‐epiphaeocaulisin A (4) and one new diarylheptanoid, 1,2,3,5‐tetrahydroxy‐1‐(4‐hydroxy‐3‐methoxyphenyl)‐7‐(4‐hydroxyphenyl) heptane (7), together with five known guaiane‐type sesquiterpene lactones including gweicurculactone (1), zedoalactone B (2), phaeocaulisin C (3), zedoalactone H (5), and zedoalactone E (6). The antiinflammation was investigated on NO and TNF‐α production using RAW264.7 cells. In addition, the expressions of genes involved in inflammation including iNOS, COX‐2, and TNF‐α were assessed. The results found that Compounds 1–7 presented their antiinflammation against NO production. The most potential effects were demonstrated on Compounds 1 and 7 with IC50 of 27.3 and 32.6 μM, respectively. Although, Compounds 1 and 7 did not inhibit TNF‐α production, their suppression on iNOS and COX‐2 mRNA expression were revealed. These results support the ability of chloroform fraction, Compounds 1 and 7 on antiinflammation, whereas others exhibited moderate and mild effect.