| Daidzein is a natural isoflavone found in Leguminosae with structural similarities to natural and synthetic estrogens and anti-estrogens. Studies suggest that daidzein exhibits a variety of biological effects on human health in cardiovascular disease, cancer chemoprevention and especially in estrogen replacement therapy (ERT) to prevent and treat osteoporosis with good therapy and low side effect. In order to further learn isoflavones, the effect of daidzein on osteoporosis was studied in cells and animals. Moreover, daidzein loading microspheres were prepared to obtain long sustained release using chitosan as carrier and evaluated in vitro and in vivo.MC3T3-E1 osteoblastic-like cells were cultured for 10, 20 or 30 days in the presence of P-GP and L-AA. The effect of daidzein on the differentiation and mineralization in MC3T3-E1 cells was investigated. The MTT assay, the measurement of alkaline phosphatase (ALP) activity, the measurement of calcium (Ca) and phosphorus (P) concentrations, the alizarin red S and von Kossa staining were used to investigate the effect of different concentrations of daidzein from 10-9 M to 10-5M on the cells. The results showed that daidzein prompted the proliferation, differentiation and mineralization of MC3T3-E1 cells. In the study, ovariectomized mice osteoporosis model was built to investigate the effect of daidzein solution through intraperitoneal administration (i.p.) on osteoporosis. Compared with 17β-estradiol, daidzein meliorated the appearance of shaggy coat, tardive action, slow response and hyponoia, due to ovariectomization and increased the uterus index, density of trabecular bone and thickness of cortical bone. Moreover, dadizein regulated the ratio of osteoprotegerin (OPG) to receptor activator of nuclear factor kB ligand (RANKL) mRNA expression level to modulate the balance between osteoblasts and osteoclasts in the bone remodeling process, which showed significance to learn the action of daizein on osteoporosis.In the pre-formulation study, high performance liquid chromatography (HPLC) was used to determine the content of daidzein. The stability of daidzein solution, solubility, partition coefficient in oil/water, and stability of daidzein powder were investigated by measuring the relative change of daidzein content. Using Caco-2 cells model, transport characteristics of daidzein across the monolayer in the different pH medium and the different concentration were studied. The apparent permeability coefficient (Papp) of daidzein, independent to the pH medium and concentration, was similar to that of transcellcular marker--propranolol, suggesting the good absorption of daidzein in vivo. By hydrolysis withβ-glucuronidase, low metabolites were detected in monolayer and transport medium, verifying the existence of glucuronides and sulfates.The emulsification/glutaraldehyde crosslinking method was used to prepare daidzein loading chitosan micropsheres. Micronized daidzein was dispersed in chitosan solution by ultrasonication and dropped into liquid paraffin containing span-80 to form emulsion droplets under stirring. The following crosslink with glutaraldehyde produced the spherical appearance, good dispersing and high drug loading microspheres. After the single factor investigation for the effe, ct on the particle size, particle size distribution, drug loading and release rate, a spherical symmetric design-response surface methodology was applied to optimize the formulation and preparing condition of microspheres. The medium size, span and drug loading of the optimized microspheres were 70.40μm, 0.526, 30.48％, respectively. The release of daidzein from microspheres was long sustained with dialysis bag release method. The experimental values were close to the predicted values stimulated by Statistics softare Statistica version 6.0, with low percentage bias, suggesting that the optimized formulation was reliable and reasonable. The repeatability of three batches and stability experiments of daidzein-loaded chitosan microspheres suggested that the quality of preparation was stable and the purposed was obtained.The assay method of determining the residue amount of daidzein after intramuscular injection of microspheres was built to study drug release in vivo. The results showed that the release of drug was sustained for 35 days and the burst effect was not clearly in the release profile. Micrography of the retrived microspheres in muscle using a scanning electron microscope and pathological section of muscle by HE staining showed good biocompatibility and weak degradation in the upper release period. Plasma concentrations of daidzein were determined using time-resolved fluoroimmunoassay. The intravenous injection of a daidzein solution in rats was best fitted to two-compartment model. The pharmacokinetic parameters k10,t1/2(α),t1/2(β) and Vc were 1.17h-1,0.30h,18.94h,0.46 L/kg, respectively. The mean plasma concentration-time profile of daidzein after intramuscular injection of the daidzein-loaded chitosan microspheres showed that the total daidzein concentration showed fluctuation and lasted for about 35 days in plasma. The absolute bioavailability (F) of the daidzein-loaded chitosan microspheres was 39.02％. Compared with the low absolute bioavailability of oral formulations, our result indicated that intramuscular injection of the daidzein-loaded chitosan microspheres significantly improved the bioavailability of daidzein. The relation between the absorption percent in vivo and the accumulative release percent with dialysis bag release method in vitro showed linear (the relative coefficient was 0.9975). The release of daidzein from the chitoan microspheres was the diffused manner, companied with degradable erosion in the end.In the study, ovariectomized mice osteoporosis model was built to investigate the effect of daidzein loading chitosan microspheres through i.m. route on osteoporosis. The pathological section of femur by HE staining showed that compared with daidzein i.g. group, daidzein microspheres i.m. groups, especially high dose microspheres i.m. group, increased the uterus index, density of and thickness of cortical bone, suggesting the increase of bioavailability in vivo.
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