Pharmacodynamics Study Of Different Polar Components Of Optimized Formula Of Zuogui Pill On Immune Premature Ovarian Failure Mice

Objective:To screen effective parts of optimized formula of Zuogui Pill by establishing immune premature ovarian failure(POF) mice. Methods:Aqueous extraction was used to extract soak dried anointing of Zuogui Pill(Zq) and optimized formula of Zuogui Pill(Zx). Alcohol precipitation of optimized formula of Zuogui Pill(Zx1) was obtained using water-extraction and alcohol-precipitation method from optimized formula of Zuogui Pill. After concentrating alcohol soluble part, alcohol soluble part of optimized formula of Zuogui Pill(Zx2) was obtained. Using cyclohexane and ethyl acetate extracting alcohol soluble part in sequence, then hexamethylene extraction component(Zx3) and ethyl acetate extraction component(Zx4) were received.Immune POF mice were established by subcutaneous injecting Zona Pellucida 3(ZP3), and divided into model control group, positive control group, Zuogui Pill group, optimized formula of Zuogui Pill group and its four different components groups according to weight. Besides, negative control group was also setted. Including to negative control group, all groups were dosed corresponding drugs for 6 days every week, 4 weeks successively. Each group had 10 mice. Vaginal exfoliated cells smear was conducted to analysis estrus cycle of mice every day. Weight was analyzed every week. After the experiment ended, blood was collected from orbital venous plexus, centrifuged and detected FSH and E2 level of serum using radioimmunoassay. Ovaries were dissected, fixed in 10% neutral formaldehyde, and dehydrated, embedded, section, HE stained to observe pathology change. Apoptosis rate of granule cells and apoptin(Fas, Fas L, GDF-9, BMP-15) of ovary were analyzed using TUNEL method and immunohistochemical method respectively. Results: 1. Extraction yield: The extraction yield of Zq, Zx, Zx1, Zx2, Zx3 and Zx4 were 67.48%, 57.75%, 20.90%, 26.08%, 0.13% and 0.34% severally. 2. Estrus cycle: Compared with model control group, estradiol valerate group, Zq group, Zx group, Zx1 group had significant differences on estrous cycle(P<0.01, P<0.05). 3. Weight: Weight of each group had no difference(P>0.05). 4. Levels of FSH and E2 in serum: Zx4 and estradiol valerate both obviously reduced FSH of POF mice(P<0.01); Zx, Zx1 and estradiol valerate increased E2 of POF mice(P<0.05, P<0.01). 5. HE pathology of ovary:The number of primordial follicle and mature follicle in model control group are less than those in negative control group. Granular layer in model control group are arranged in disorder. Follicles are in irregular forms, extending to adjacent mesenchyme. There are more broken apoptotic bodies of granulosa cell under 400 times magnification. The number of primordial follicle and mature follicle in treatment groups are more than those in model control group, and follicles are in regular forms. 6. Apoptosis rates of granule cells: Zx, Zx2 and estradiol valerate effectively decreased apoptosis rates of granule cells(P<0.05). 7. Apoptin(Fas, Fas L, GDF-9, BMP-15) of ovary:Compared with negative control group, the expression of Fas, GDF-9 and BMP-15 protein of model control group decreased(P<0.05, P<0.01), while the expression of Fas L and of model control group increased(P<0.05). Except the expression of BMP-15 in groups of Zx1、Zx2 decreased(P<0.01,P<0.05), and the other treatment groups have no statistical difference(P>0.05).Compared with model control group, the expression of Fas in groups of estradiol valerate, Zq, Zx, Zx1, Zx3 and Zx4 increased(P<0.05, P<0.01). The expression of Fas L in groups of positive control, Zx, Zx1, Zx2, Zx3 and Zx4 all declined(P<0.01, P<0.05). The expression of GDF-9 in groups of positive control, Zq, Zx and Zx4 all increased(P<0.05, P<0.01). The expression of BMP-15 in groups of positive control, Zq, Zx, Zx2, Zx3 and Zx4 all increased(P<0.01, P<0.05). Conclusion:Optimized Formula of Zuogui Pill is superior to Zuogui Pill in the treatment of immune POF mice. And its therapeutic effect is the result of comprehensive function of four different polar components, which are all effective parts.