Suppressive effects of methoxyflavonoids isolated from Kaempferia parviflora on inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells.
BACKGROUND
The rhizomes of Kaempferia parviflora Wall. ex Baker have been traditionally used in Thailand to treat abscesses, gout, and peptic ulcers.


OBJECTIVE
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.


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).


CONCLUSIONS
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.
Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla, Thailand.
Chutha
C
Sae-Wong
Hisashi
H
Matsuda
Supinya
S
Tewtrakul
Pimpimon
P
Tansakul
Seikou
S
Nakamura
Yukiko
Y
Nomura
Masayuki
M
Yoshikawa
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000302:isolation & purification / Q000494:pharmacology* / Q000627:therapeutic use; D047310:Apigenin / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D047309:Flavones / Q000302:isolation & purification / Q000494:pharmacology* / Q000627:therapeutic use; D005419:Flavonoids / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use; D007249:Inflammation / Q000188:drug therapy / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008070:Lipopolysaccharides; D047311:Luteolin / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D008517:Phytotherapy; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D027343:Rhizome; D000072377:Syk Kinase; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D030521:Zingiberaceae / Q000737:chemistry*
D000893:Anti-Inflammatory Agents; D047309:Flavones; D005419:Flavonoids; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D010936:Plant Extracts; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C060298:5,7-dimethoxyflavone; D009569:Nitric Oxide; D047310:Apigenin; D052247:Nitric Oxide Synthase Type II; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606897:Syk protein, mouse; C000606898:Syk protein, rat; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D047311:Luteolin