RESUMO
We previously isolated berberine from aqueous extracts of tsu-kan-gan, a Kampo formula used for the treatment of osteoporosis. Berberine caused an inhibitory effect on parathyroid hormone (PTH)-stimulated bone resorption in neonatal mouse bone. In this report we describe the inhibitory effect of berberine on the formation of osteoclast-like multinucleated cells (OCLs) in the co-culture of mouse osteoblastic cells and bone marrow cells in the presence of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], PTH and interleukin-1alpha (IL-1alpha). Berberine dose-dependently inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive OCLs induced by 1alpha25(OH)2D3, PTH and IL-1alpha. We prepared OCLs in the co-culture of osteoblastic cells and bone marrow cells. The effect of berberine on pit formation by OCLs was examined using dentin slices. As OCLs are terminally differentiated multinucleated cells, the survival of OCLs affects the bone-resorbing activity of OCLs. This prompted us to count the number of TRAP-positive OCLs on the slices. Berberine dose-dependently inhibited pit formation and caused a decrease in the number of TRAP-positive OCLs. Calcitonin (CT) inhibited pit formation without affecting the number of OCLs. Berberine accelerated the cell death in OCLs cultivated on a culture plate, but CT did not affect the cell death of OCLs. This suggests that the decrease in the number of OCLs on dentin slices may be due to apoptotic cell death in OCLs. In fact, Hoechst 33258 staining revealed that the treatment of OCLs with berberine resulted in condensed nuclei and a decrease in cell size. Oral administration of the berberine (30 and 50 mg/kg/d) to ovariectomized rats prevented a decrease in bone mineral density (BMD) of the lumbar vertebra without affecting the weight of the uterus and plasma concentration of estradiol. These results suggested that berberine prevented a decrease in BMD in vivo by inhibiting osteoclastic bone resorption.
Assuntos
Berberina/farmacologia , Reabsorção Óssea , Medicamentos de Ervas Chinesas/farmacologia , Macrolídeos , Animais , Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Calcitonina/farmacologia , Calcitriol/farmacologia , Feminino , Interleucina-1/farmacologia , Masculino , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Ovariectomia , Hormônio Paratireóideo/farmacologia , Ratos , Ratos Wistar , Útero/efeitos dos fármacosRESUMO
We previously found that a methanolic extract of the stems of Sambucus sieboldiana inhibited bone resorption in organ culture. In this study, we further fractionated the methanol extract guided by the activity towards bone resorption stimulated by parathyroid hormone (PTH) in vitro. The ethyl acetate fraction (EtOAc Fr.) of the methanolic extract inhibited PTH-stimulated bone resorption of neonatal mouse bones, and the inhibitory activity was more potent than those of other fractions. Oral administration of the EtOAc Fr. (50 and 100 mg/kg/d) to ovariectomized (OVX) rat prevented the decrease in bone mineral density (BMD) of the lumbar (L2-4) vertebra, indicating that the EtOAc Fr. is effective in vivo. Furthermore, the EtOAc Fr. (50, 100 and 150 mg/kg/d) decreased the serum calcium level elevated in low calcium dietary rats. The phenolic constituents of the EtOAc fraction were examined for their inhibitory effect on bone resorption stimulated by PTH in neonatal mouse bone. Among them, vanillic acid, vanillin and coniferyl alcohol showed significant inhibitory effects on bone resorption. Of the compounds examined, vanillic acid was found to have a significant inhibitory effect on the decrease of BMD in OVX mice. Therefore, the EtOAc Fr. of S. sieboldiana showed a suppressive effect on bone resorption in vitro and in vivo. In addition, the inhibitory effects of the EtOAc Fr. on bone resorption may be at least partly due to the inhibitory action of vanillic acid.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Plantas Medicinais/química , Acetatos , Animais , Benzaldeídos/farmacologia , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/induzido quimicamente , Cálcio/sangue , Cálcio/deficiência , Cálcio da Dieta/administração & dosagem , Camundongos , Ovariectomia , Hormônio Paratireóideo/toxicidade , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Ratos , Ratos Wistar , Solventes , Fatores de Tempo , Ácido Vanílico/farmacologiaRESUMO
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.