Chlorogenic acid from the Japanese herbal medicine Kinginka (Flos Lonicerae japonicae) suppresses the expression of inducible nitric oxide synthase in rat hepatocytes
Flos Lonicerae japonicae (FLJ; Kinginka) is the dried flowers and buds of the Japanese honeysuckle Lonicera japonica Thunberg. FLJ has been used as a Japanese Kampo medicine to treat infectious and inflammatory diseases. However, it is not clear which constituent of FLJ is responsible for its pharmacological effects.
FLJ was extracted with methanol and fractionated by hydrophobicity. We measured the effects of each fraction on the induction of the inflammatory mediator nitric oxide (NO), which was induced by interleukin 1β in primary cultured rat hepatocytes. To estimate cytotoxicity, the activity of lactate dehydrogenase released from the hepatocytes was measured. The expression of inducible nitric oxide synthase (iNOS) was analyzed by Western blot analysis and reverse transcription-polymerase chain reaction.
The methanol extract was fractionated into hydrophobic (11.1%), butanol-soluble (16.4%), and water-soluble fractions (72.5%). These three fractions dose-dependently suppressed the induction of NO and reduced the level of iNOS protein in interleukin 1β-stimulated hepatocytes. Chlorogenic acid, a major constituent of the water-soluble fraction, significantly reduced the levels of NO production, iNOS protein, and iNOS mRNA. Chlorogenic acid also decreased the levels of mRNAs encoding cytokines and chemokines that are involved in inflammatory disease. Caffeic acid, which is formed by the hydrolysis of chlorogenic acid, markedly reduced the induction of NO, although it did not exist at a detectable level in the water-soluble fraction. In contrast, other constituents of the water-soluble fraction, such as inositol fructose, glucose, and sucrose, did not affect the induction of NO.
The anti-inflammatory effects of the FLJ extract and its constituents were analyzed by measuring the induction of NO and iNOS in hepatocytes. We demonstrated that chlorogenic acid, one of the main constituents of FLJ, is involved in the anti-inflammatory effect of the FLJ extract, suggesting its therapeutic potential.
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ABSTRACT: Gomishi is the dried fruit of Schisandra chinensis Baillon (Fructus Schisandrae chinensis, FSC) and has been used in Japanese Kampo medicine to treat inflammatory and liver diseases. However, it is unclear which constituent of FSC is primarily responsible for its pharmacological effects. FSC was extracted with methanol, fractionated by hydrophobicity, and further purified. We measured the effects of each fraction or constituent thereof on the induction of the inflammatory mediator nitric oxide (NO), which was induced by interleukin 1β in primary cultured rat hepatocytes. The hydrophobic fraction markedly suppressed NO induction and reduced the expression of inducible nitric oxide synthase (iNOS) in interleukin 1β-treated hepatocytes. Gomisin N and γ-schizandrin, two major constituents of the hydrophobic fraction, significantly reduced NO production and the levels of the iNOS protein, mRNA, and antisense transcript. Gomisin N and γ-schizandrin also decreased the transcription of interleukin 1β and inflammatory chemokines. The overexpression of the p65 subunit of nuclear factor κB or CCAAT/enhancer-binding protein β increased the promoter activity of the iNOS gene in the firefly luciferase assay, whereas gomisin N decreased the promoter activity. The anti-inflammatory activity of FSC and its constituents were analysed, and we demonstrated that gomisin N and γ-schizandrin are involved in the hepatoprotective effect of the FSC extract, which has therapeutic potential for liver disease.Nitric Oxide 10/2012; 28. DOI:10.1016/j.niox.2012.10.003 · 3.52 Impact Factor
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ABSTRACT: Flavanol (flavan-3-ol)-rich lychee fruit extract (FRLFE) is a mixture of oligomerized polyphenols primarily derived from lychee fruit and is rich in flavanol monomers, dimers, and trimers. Supplementation with this functional food has been shown to suppress inflammation and tissue damage caused by high-intensity exercise training. However, it is unclear whether FRLFE has in vitro anti-inflammatory effects, such as suppressing the production of the proinflammatory cytokine tumor necrosis factor α (TNF-α) and the proinflammatory mediator nitric oxide (NO), which is synthesized by inducible nitric oxide synthase (iNOS). Here, we analyzed the effects of FRLFE and its constituents on the expression of inflammatory genes in interleukin 1β (IL-1β)-treated rat hepatocytes. FRLFE decreased the mRNA and protein expression of the iNOS gene, leading to the suppression of IL-1β-induced NO production. FRLFE also decreased the levels of the iNOS antisense transcript, which stabilizes iNOS mRNA. By contrast, unprocessed lychee fruit extract, which is rich in flavanol polymers, and flavanol monomers had little effect on NO production. When a construct harboring the iNOS promoter fused to the firefly luciferase gene was used, FRLFE decreased the luciferase activity in the presence of IL-1β, suggesting that FRLFE suppresses the promoter activity of the iNOS gene at the transcriptional level. Electrophoretic mobility shift assays indicated that FRLFE reduced the nuclear transport of a key regulator, nuclear factor κB (NF-κB). Furthermore, FRLFE inhibited the phosphorylation of NF-κB inhibitor α (IκB-α). FRLFE also reduced the mRNA levels of NF-κB target genes encoding cytokines and chemokines, such as TNF-α. Therefore, FRLFE inhibited NF-κB activation and nuclear translocation to suppress the expression of these inflammatory genes. Our results suggest that flavanols may be responsible for the anti-inflammatory and hepatoprotective effects of FRLFE and may be used to treat inflammatory diseases.PLoS ONE 04/2014; 9(4):e93818. DOI:10.1371/journal.pone.0093818 · 3.23 Impact Factor
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ABSTRACT: A new flavanone, shisoflavanone A (1), and several flavonoids were purified from the ethyl acetate-soluble fraction of green perilla leaves (Perilla frutescens Britton var. crispa form viridis), and their structures were identified. Shisoflavanone A was elucidated as 8-hydroxy-6,7-dimethoxyflavanone based on its spectral data. Other constituents of the ethyl acetate-soluble fraction, i.e. 5,8-dihydroxy-7-methoxyflavanone (2), negletein (5,6-dihydroxy-7-methoxyflavone) (3), luteolin (4), apigenin (5), esculetin (6), and protocatechuic acid (7), were identified. This is the first time that constituents 2, 3, and 6 have been found in green perilla. Shisoflavanone A and the other constituents (except 7) significantly inhibited nitric oxide production in interleukin 1β-stimulated rat hepatocytes, which have been used to monitor the anti-inflammatory effects of herbal constituents. The present findings suggest that these constituents, including shisoflavanone A, may be involved in the anti-inflammatory effects of green perilla leaves.Bioscience Biotechnology and Biochemistry 09/2014; 79(1):1-9. DOI:10.1080/09168451.2014.962474 · 1.06 Impact Factor