2. Ritsumeikan Global Innovation Research Organization (R-GIRO),
3. Research Organization of Science and Technology,
4. Department of Surgery, Kansai Medical University, Hirakata, Osaka, Japan;
5. Department of Food Sciencesand Nutritional Health, Kyoto Prefectural University, Kyoto, Japan. and
6. Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan;
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.