To examine the immunoregulatory activity of a chrysanthemum flower (CF), Shiranui Himekiku (Chrysanthemum indicum × Erigeron annuus), we studied the effect on tumor necrosis factor α (TNF-α) production by a mouse macrophage-like cell line RAW264.7. The production of TNF-α was enhanced upon treatment with CF water extract. We tried to characterize the active component of this crude extract. To determine whether polyphenols are involved in the promotive effect of TNF-α production by CF extract, we prepared a polyphenol-excluded CF extract. This exclusion did not affect the CF's ability to promote the release of TNF-α. Generally, it has been suggested that polyphenol be dissolved in an alcoholic solvent. We dissolved the CF extract in methanol and were able to separate the extract into both soluble and insoluble fractions. The activity to enhance the release of TNF-α was observed only in the methanol-insoluble fraction. These results imply that the bioactive compounds may not be polyphenols. Next, we examined the effect of heat stability on the CF extract. Boiled-water extract of CF maintained its activity to promote the TNF-α production, indicating that the active component in CF extract is heat-stable. Based on this result and the fact that the UV-Vis absorption spectrum of CF extract did not exhibit the typical features of proteins, it was suggested that the active compound may not be a protein. In order to purify the substance enhancing TNF-α secretion, we subjected the methanol-insoluble fraction of the boiled-water extract to size-separation gel-filtration chromatography. The obtained chromatogram detected carbohydrate, one of the major compounds of chrysanthemum, as a candidate for the active substance other than polyphenol and protein. The promotive effect of TNF-α production was observed in only one fraction that contained carbohydrates with the molecular weight over 66,000 Da. These results suggest that polysaccharide in the CF extract enhances the production of TNF-α by RAW264.7 cells.
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