| In recent years, the research of controlled-release and prolonged-release drugs in preparation and dinical is active, and is becoming an important direction of the development in domestic and foreign pharmaceutical industry. Because of the effects on enhancing biological activity, prolong effecting time of drugs and the character of highly-bioavailable, implants, as an important part of the sustained-release preparation, are developing and applied more and more widely.Implants, as the controlled-release and prolonged release system, are aimed to reduce the side effects of the drugs, to stabilize blood level, so as to achieve the best efficacy in the body. This experimental research is progesterone subdermal implants. Necessary basic experiment data from the research are provided through the observation of implants in vitro and in vivo of the ovariectomized mice and verifying its release degree of stability. Progesterone norpalnts implants are widely used in the fields of birth control, that most common clinical side effect is the change of endometrial, which leads to bleeding and menstrual disorder and severely affects the quality of life of women on implants contraception, and there is no effective drug at present. The traditional Chinese medicine believes that the kidney is the congenital foundation. "kidney stores essence and dominates reproductive system". Kidney is the driving force of human activities. Through applying castrated mice with Recipe, observating the effects of the recipe on progesterone content and changes of endometrium of castrated mice and discussing the effects of Bushen Recipe on mice ovariectomized by being implanted with progesterone, this research is hoped to provide scientific data for clinical application of kidney-tonifying medicine and improving and treating endometrial disease changes. Part One Research on stability of progesterone implant tubeObjective: To study the release of progesterone implants in vitro and in vivo objective. Evaluate the implant stability and uniformity of the drug release.Methods:1 Prepare progesterone tube implant.2 Use ultraviolet spectrophotometry method to measure content of drugs and research the vitro release level of the drug.3 Choose clean level KM female mice in vivo study, try to raise the mice for one week before bilateral ovariectomy, restore one week, then implant the progesterone implant tube in the loose skin of the necks of the mice.Then removed after 96h(96h×1)、192h(96h×2)、288h(96h×3), and use the electronic balance to measure the weight loss and calculate the drug release level in vivo.Results:1 Vitro experiments showed that at 96h(96h×1) 、 192h(96h×2) 、288h(96h×3)after removed, the range of absorption value of the released liquide are 0.52- 0.79, 0.53-0.76,0.53- 0.78.2 The amount of progesterone release showed in the vitro experiments : ①at 96h(96h×1),1.12 mg,1.08 mg, 1.13 mg, 1.06 mg, 1.14mg; ②at 192h(96h×2), 1.89 mg, 1.93 mg, 2.02 mg, 2.00 mg, 1.96mg; ③at 288h(96h×3), 2.72 mg, 2.81 mg, 2.79 mg, 2.90 mg, 2.86 mg.Conclusions:1 The experiment in vitro showed that at 96h(96h×1)、192h(96h×2)、288h(96h×3), the drug release rate remained constant, which suggested that progesterone implants release is uniform and stable;2 The release rate of progesterone implants in 96h(96h×1)、192h(6h×2)、288h(6h×3)are basically same in vivo, which suggested that the progesterone implants have a constant release rate and accords to the zero order release;3 The progesterone implant tube release is stable. It can be used for animal experiment of progesterone constant with drug design. Part Two Study on Bushen Recipe’s effects on endometrium of the castrated mice.Objective: Through observing the intervention of the Bushen Recipe impacts, menstrual-like model with progesterone implant, study the effects of progesterone impant at 96h(96h×1) 、 192h(96h×2) 、 288h(96h×3)on the progesterone contents and uterine endometrium in mice.Methods:1 The establishment of menstrual-like mice model: The female KM mice, was divided the mice into the normal group, model group, Bushen group randomly after a week probation any period. Then carry bilateral ovariectomy on the mice and keep for a week to clear the interference of hormone remained in the body. The normal group mice were given water, fed. The implants were implate in the model group and the bushen group. Bushen group were given with 20g/(kg·d) at 8 to 9 in the morning, and the model group was gavaged with saline.2 The three groups of mice were sacrificed when the progesterone implant effected after 96h(96h×1)、192h(96h×2)、288h(96h×3), take blood samples, separate serum, and detect the progesterone content by RIA method.3 At the same time, the uterus of the mice, were collected observing the morphological change of the uterus, take photoes and weight, calculate the uterus index. And observe the structure under microscope.Results: 1 Changes of progesterone content in serumthe serum progesterone levels of normal group at 96h(96h×1) was 0.82 ±0.92 pg/m L, 192h(96h×2)was 0.79±0.93 pg/m L, 288h(96h×3) was 0.80±0.95 pg/m L. The progesterone content had no significant change as the time passed. The serum progesterone levels of model group mice at 96h(96h×1) was 21.10±0.90 pg/m L, 192h(96h×2)was 35.23±1.32 pg/m L, 288h(96h×3) was 45.51±1.03 pg/m L. Compared to the normal group it showed a significantly rising trend with time going on(P<0.05). The serum progesterone levels of bushen group at 96h(96h×1) was 18.22±0.97 pg/m L, 192h(96h×2)was 16.93±1.21 pg/m L, 288h(96h×3)was 14.69±0.93 pg/m L, compared to the model group, obvious change, except at the 96 h there were significantly decreased(P<0.05). 2 Observation on endometrium of the castrated mice 2.1 Morphological observation of the uterinenormal group mice’s uterine horn was milky white, both sides uniformed in size and had no obvious change with the extension of time. The uterine horn of model group mice at 96 h was slightly swollen, the color was light pink; at 192 h it changed little. The color was light pink. At 288 h they swelled obviously, and were coated by capillary. Bushen group mice uterine horn at 96 h was slightly swollen, with liquid filling, the color was pale pink; at 192 h the uterus was enlarged, capillary gradually got rich, the color was pink; at 288 h it was swelling significantly, and a lot of red capillary filling can be seen. 2.2 The changes of the uterus indexthe uterus index of the normal group at 96h(96h×1)was 0.16±0.04 g/g, at 192h(96h×2) was 0.15±0.04 g/g, at 288h(96h×3) was 0.16±0.03g/g, with the time changes, the uterus index had no obvious change; The uterus index of model group at 96h(96h×1) was 0.16±0.04 g/g, at 192h(96h×2) was 0.21±0.05 g/g, at 288h(96h×3) was0.26±0.04g/g, compared with the normal group, while the 96 h group had no obvious change, the other two groups were significantly increased(P<0.05); The uterus index of bushen group at 96h(96h×1)was 0.15±0.03g/g, at 192h(96h×2)was 0.22±0.05 g/g, at 288h(96h×3)was 0.19±0.03g/g, compared with the model group, 96 h and 192 h had no obvious change, the index of uterus at 288 h was decreased obviously(P<0.05). 2.3 The morphological observation of the endometriumNormal group’s endometrial stromal compacted and were simple columnar epithelium. The nucleoplasm is high. The nucleus was oval shaped. Model group’s endometrial were thickened. The number of glands appeared in the interstitial 96 h in endometrium spiral arterioles gradually developed, the change at 192 h was more obvious than at 96 h. And spiral arterioles showed further proliferation. The endometrium at 288 h was thick, stroma rarefaction was showed. Spiral arterioles decreased. Bushen group uterine horn in the endometrium was thick at 96 h, spiral arteriolar increased in number and were slightly curved. At 192 h the thickening was obvious. The palace cavity volume reduced. Stroma rarefaction was showed. Spiral arterioles decreased. At 288 h the endometrial thinned, glandular interstitial in quantity and spiral arterioles decreased obviously.Conclusions:1 The Bushen Recipe can reduce the serum content of progesterone in mice.2 The Bushen Recipe has an inhibitory effect on the mice’s uterine morphologicalchanges3 The results suggest that Bushen Recipe can adjust the progesterone levels, improve the endometrial changes, and prevent the disorder bleeding. |
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