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.     

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 © 2011 John Wiley & Sons, Ltd.     

Hirsutane sesquiterpenes from the fungus Lentinus connatus BCC 8996

Two new hirsutane sesquiterpenes, connatusins A (1) and B (2), were isolated from the fungus Lentinus connatus BCC 8996. The structures, closely related to hypnophilin, were elucidated on the basis of the spectroscopic data. An X-ray analysis was performed to confirm the structure of 1. Six known compounds were also obtained. Panepoxydone (5), panepoxydione (6), and dihydrohypnophilin (8) exhibited significant antimalarial and cytotoxic activities.     

Risk factors of helminthiases among schoolchildren in southern Thailand

School-age children are particularly at risk of soil-transmitted helminthiasis (STH), which affects their growth and development. Hence, school-based helminthiasis control has been discussed and conducted as one of cost-effective ways in developing countries. A parasite control program is to be planned and conducted in an evidence based way as one of practical medical fields. However, a prevalence is likely to be influenced by various factors such as local environment and residents' behavior and so on. As few reports mentioning the relation of the prevalence and children's behavior and practice have been published, we conducted a survey to investigate such relation by asking children directly. Two hundred and eighty pupils in grade 3 to grade 5 were enrolled, who were in the two schools in Nakhon Si Thammarat Province, southern Thailand. A cross-sectional study was conducted in February and in November 2001, on the relationship between helminthiasis and children's knowledge and practice by using Kato-Katz method and a questionnaire, respectively. Hookworm (Hw) was the most predominant helminth followed by Trichuris. Boys had more intensive Hw infection than girls (p = 0.022), and wore shoes less frequently than girls (p < 0.001). The pupils who nearly acquired Hw infection after the first stool examination had lower levels of the knowledge of STH comparing to those who did not (p = 0.011). This study suggests that a prior survey can identify unique local factors as a part of diagnostic process, the results of which are useful for teachers to understand some points of health education at schools and can be applied as indicators for monitoring and evaluation.     

Anti‐inflammatory effect of isopimarane diterpenoids from Kaempferia galanga

Two new isopimarane diterpenes, 1α‐hydroxy‐14α‐methoxyisopimara‐8(9),15‐diene ( 7 ) and 1α,14α‐dihydroxyisopimara‐8(9),15‐diene ( 9 ) and eight known isopimarane diterpenes including (‐)‐sandaracopimaradiene ( 1 ), 6β‐acetoxysandaracopimaradiene‐9α‐ol ( 2 ), sandaracopimaradiene‐7β,9α‐diol ( 3 ), sandaracopimaradiene‐1α,9α‐diol ( 4 ), 6β‐acetoxysandaracopimaradiene‐9α‐ol‐1‐one ( 5 ), 6β‐acetoxysandaracopimaradiene‐1α,9α‐diol ( 6 ), 6β,14α‐dihydroxyisopimara‐8(9),15‐diene ( 8 ), and 6β,14β‐dihydroxyisopimara‐8(9),15‐diene ( 10 ) were isolated from hexane fraction of Kaempferia galanga ethanol extract. Compounds 5 , 6 , 8 , and 9 exerted the good anti‐inflammatory effect on lipopolysaccharide‐stimulated nitric oxide production from RAW264.7 cells with IC₅₀ of 11.2, 7.7, 14.3, and 12.1 μM, respectively. These four compounds inhibited nitric oxide synthase (iNOS) mRNA expression. Compounds 5 and 6 also suppressed cyclooxygenase 2 (COX‐2) mRNA expression; in addition, compound 6 had mild inhibitory effect on TNF‐α mRNA. Among these compounds, 5 dramatically inhibited iNOS and COX‐2 mRNA expression. The influential structures were proposed to be oxygen substitute at C‐1, C‐6, and α‐OH at C‐14.     

Anti-inflammatory mechanism of Kaempferia parviflora in murine macrophage cells (RAW 264.7) and in experimental animals

Ethnopharmacological relevance

The rhizomes of Kaempferia parviflora Wall. ex Baker have been used in Thailand for treatment of gout, apthous ulcer, peptic ulcer and abscesses.

Aim of the study

In our previous study, the crude ethanol extract of Kaempferia parviflora and its compound (5, 5-hydroxy-3,7,3′,4′-tetramethoxyflavone), was reported to show nitric oxide (NO) inhibition in RAW 264.7 cells. The present study is thus investigated the anti-inflammatory mechanism of Kaempferia parviflora extract and compound 5 against inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expressions.

Materials and methods

The extract of Kaempferia parviflora and its compound were tested against NO and prostaglandin E₂ (PGE₂) releases using RAW264.7 cells as well as studied on anti-inflammatory activity in carrageenan-induced rat paw edema and acute toxicity in mice.

Results

The results revealed that the ethanol extract of Kaempferia parviflora markedly inhibited PGE₂ release with an IC₅₀ value of 9.2 μg/ml. This plant extract and compound 5 also suppressed mRNA expression of iNOS in dose-dependent manners, whereas COX-2 mRNA expression was partly affected. According to the in vivo study, chloroform and hexane fractions greater decreased rat paw edema than ethanol, ethyl acetate and water fractions.

Conclusion

The mechanisms for anti-inflammatory activity of Kaempferia parviflora and compound 5 are mainly due to the inhibition of iNOS mRNA expression but partly through that of COX-2 mRNA.     

Dietary Composition and Its Association with Newly Diagnosed Nonalcoholic Fatty Liver Disease and Insulin Resistance

Dietary modification is essential for treating nonalcoholic fatty liver disease (NAFLD); however, the dietary components are less well defined. We enrolled 252 adults with no history of liver disease and excessive alcohol use to evaluate the relationship between macronutrients and NAFLD and insulin resistance. Participants took photographs of their meals and documented their food intake in a food diary for seven consecutive days. A dietitian estimated the type and portion size of food items and analyzed nutrients with INMUCAL-Nutrients software. Later, participants underwent transient elastography to diagnose NAFLD and blood tests to measure insulin resistance using the homeostasis model. Total energy intake and the proportion of carbohydrate, fat, and protein consumption did not differ between participants with NAFLD (n = 41) and those without NAFLD (n = 211). Using multiple logistic regression analysis, daily intake of protein < 1.0 g/kg (OR: 3.66, 95% CI: 1.41–9.52) and full-fat dairy product ≥ 50 g (OR: 0.42, 95% CI: 0.18–0.99) were associated with NAFLD. Insulin resistance was associated with a daily intake of protein < 1.0 g/kg (OR: 3.09, 95% CI: 1.59–6.05), full-fat dairy product ≥ 50 g (OR: 0.46, 95% CI: 0.25–0.82), and dietary fiber ≥ 8 g (OR: 0.41, 95% CI: 0.22–0.74). Our data show that a low protein intake increases the odds for NAFLD and insulin resistance. Contrarily, a high intake of full-fat dairy products and dietary fiber has been associated with a potential protective effect against NAFLD and insulin resistance.     

In vitro activity of colistin or sulbactam in combination with fosfomycin or imipenem against clinical isolates of carbapenem-resistant Acinetobacter baumannii producing OXA-23 carbapenemases

This study investigated the in vitro activity of colistin or sulbactam in combination with fosfomycin or imipenem against eight strains of carbapenem-resistant A. baumannii (CRAB). The eight CRAB clinical isolates were collected from hospitalized patients admitted to Songklanagarind Hospital in southern Thailand during January-December 2008. The isolates were divided into 4 different patterns of clonal relationships using the Repetitive Extragenic Palindromic-Polymerase Chain Reaction method (REP-PCR). The in vitro activity of combination antibacterial agents against theses isolates were determined by chequerboard and time-kill methods. All isolates producing OXA-23 carbapenemases were universally susceptible to colistin but intermittently susceptible to other antimicrobial agents. A chequerboard assay showed the synergistic effects of sulbactam plus fosfomycin and colistin plus fosfomycin in 75% and 12.5% of isolates, respectively. Sulbactam at a concentration of 1 x MIC plus fosfomycin at 1 x MIC or at 1/4 x MIC showed synergism in 75% and 37.5% of clinical isolates, respectively. Bactericidal activity was observed for up to 12 hours of incubation. There was no synergism between colistin and sulbactam, sulbactam and imipenem, and colistin and imipenem, against the tested isolates. Combined use of sulbactam and fosfomycin may provide an alternative therapeutic option for CRAB infections.