| Background: Hyperplasia of mammary glands(HMG) is a progressive disease and characterized by proliferation of mammary epithelial, fibrosis and connective tissue, as well as structural deterioration mammary ductal and lobule. Its possible mechanism is related to the imbalance of hormone in vivo. Mastitis is an inflammation status of breast with the features of inflammatory cells infiltration, endothelial dysfunction and over generation of proinflammatory cytokines. It is usually accompanied by mammary oxidative stress. Our study optimizes the extraction and purification process of the total polyphenols from Rosa rugosa(Thunb.)(FRR) via orthogonal design and evaluates the oral dosage and safety using acute and subacute oral toxicity experiments. Then,we investigated the potential protective effects of FRR against HMG and mastitis in rats and its possible mechanisms. This study provides a reliable basis for further clinical application and preparation development of R.rugosa.Method: The most applicable macroporous resin among HPD500(polarity), ADS-17(middle polarity+hydrogen bond), HPD826(hydrogen bond) and AB-8(low polarity)was selected by static adsorption and elution test. Then, the best dynamic adsorption and elution conditions were selected using orthogonal design method.The oral dosage and safety was evaluated using acute and subacute oral toxicity experiments. The anti-angiogenic activity of FRR was evaluated using 24hpf(hours post fertilization) Health TG(VEGFR2: GFP) department vascular fluorescence transgenic zebrafish model. The embryo was incubated with different doses of FRR 24 h. The intersegmental vessels(ISV) were observed under fluorescence microscope. The rat model of HMG was induced by intramuscularly injected with estrogen(0.5mg/kg-1) for 25 days, and followed with progestogen(5mg/kg-1) for another 5days. Meanwhile, FRR(200 or100mg/kg-1/d-1) was orally given for 30 days. The levels of estradiol and oxidative stress were assessed after the experimental period. The mammary expressions of AKT and JNK were evaluated by western blot analysis. The expressions of NFκB-p65, COX-2 and VEGF were measured by immunohistochemical analysis. The experimental mastitis model was induced by injecting lipopolysaccharide into rats. The effects of FRR on mammary morphological changes, oxidative stress and inflammatory responses were measured.Result: AB-8 was suitable for purifying total polyphenols from R. rugosa. The optimum conditions were with 0.5mg/ml-1sample, 1ml/min-1adsorptive speed,2ml/min-1eluted speed, and 4 fold of BV. Oral dose lower than 500 mg/kg-1/d-1was suitable. FRR(20 or 50 μ g/ml-1) inhibited the zebrafish angiogenesis.Additionally, Treated with 200mg/kg-1/d-1FRR obviously decreased the levels of estradiol(from 32.53±1.08 to 25.71±0.96) and malondialdehyde(MDA, from6.96±0.90 to 4.47±0.40nmol/ml-1) as well as increased the levels of total antioxidant capacity(TAC, from 4.17±0.73 to 7.71±0.67U/ml-1) and superoxide dismutase(SOD, from 84.5±6.7 to 126.5±9.1U/ml-1) when compared to the untreated HMG model. FRR reduced the mammary over expressions of VEGF, COX-2and NFκB, and decreased the phosphorylations of JNK and AKT. It was also found that FRR obviously attenuated the mammary morphology disorders, as well as decreased the mammary levels of oxidative stress and inflammatory cytokines. |
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