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Four water extracts of Kampo formulae (Yi-kkan-sen, Dai-ho-in-gan, Ni-chi-gan, Tsu-kan-gan) were screened for their inhibitory activities on bone resorption induced by parathyroid hormone (PTH) in organ culture using neonatal mouse parietal bones. Among the Kampo formulae, Tsu-kan-gan (TKG) showed the most potent inhibitory activity. We further fractionated the TKG water extract by monitoring the inhibitory activity on bone resorption stimulated by PTH in vitro. The MeOH fraction of the water extract inhibited PTH-stimulated bone resorption, and its inhibitory activity was more potent than those of other fractions. The MeOH fraction was then subjected to Sephadex LH-20 column chromatography to give fractions I, II and III, which were examined for bone resorption activity. Fraction I inhibited PTH-stimulated bone resorption, and its inhibitory activity was more potent than those of the other fractions. Upon oral administration of the three fractions (100 mg/kg/d) to ovariectomized (OVX) mice, fractions I and III prevented the decrease of bone mineral density (BMD) of the lumbar vertebra. Eleven compounds isolated from the MeOH fraction were examined for their inhibitory effect on PTH-stimulated bone resorption. Among them, berberine (1), syringin (3), limonin (4) and mangiferin (10) showed a significant inhibitory effect on bone resorption. In the formation assay of osteoclast-like cells, these compounds decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs). The inhibitory effect of TKG on bone resorption may be at least partly due to the inhibitory action of these compounds.

The Effect of Kampo Formulae on Bone Resorption in Vitro and in Vivo. I. Active Constituents of Tsu-kan-gan.