活血方与活血加味方对小鼠月经模型干预作用及与阿魏酸煎出量的相关性实验研究
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摘要
月经是在雌激素和孕激素序贯性作用下，子宫内膜发生增殖、分化以及崩解、出血等现象。子宫形态和功能经历着周期性特征改变，构成了完整的月经周期。月经占据了女性一生中较长的时间，对妇女的生殖健康影响巨大。中医学认为月经的周期性出现是“肾—天癸—冲任—胞宫”之间相互作用与平衡的结果[1]。妇人以血为本，以气为用，气调血畅，下养胞宫，血海充盛，则血满而溢出现月经。因此，气血调畅是使月经顺利来潮的必要条件。关于月经，临床研究多于实验性研究，其原因是缺少动物模型。本实验根据国内外文献[2-5]报道，采用序贯激素处理和人为诱导子宫内膜蜕膜化，成功复制了km小鼠月经出血模型，为研究中医药对月经发生发展的影响提供了基础。并在此模型基础上，以川芎为主药，自拟活血方、活血加味方，观察比较两方对月经模型的干预作用；同时以川芎的主要成分阿魏酸为指标，检测活血方与活血加味方中阿魏酸的煎出量变化，并进行阿魏酸的含量与其干预月经模型作用的相关性的研究，旨在为临床正确使用中药提供科学依据。
第一部分活血方与活血加味对小鼠月经模型的干预比较实验研究
目的：应用km小鼠复制生理性孕酮撤退的月经出血模型，并以此小鼠月经模型为基础，揭示活血方与活血加味方对小鼠月经模型的干预作用。
方法：将雌性km小鼠试养一周后，随机分为模型（A）组、活血方（B）组、活血加味方（C）组。采用乙醚麻醉，摘除卵巢，恢复一周以清除内源激素，后进行激素序贯处理。各组小鼠在实验第1~3天皮下注射雌二醇工作液A（15μg/kg）；第7天皮下注射孕酮工作液（7.5μg/kg）和雌二醇工作液B（0.75μg/kg），同时将处理好的孕酮硅胶管皮埋于小鼠背部；第8~9天皮下注射雌二醇工作液B（0.75μg/kg）。其中A组每天灌胃生理盐水；B组灌胃活血方药液；C组灌胃活血加味方药液。后于第9天将20μL的无菌花生油注射到小鼠右侧子宫角，以诱导蜕膜化，以另一侧子宫角做对照，第11天取出小鼠背部皮埋管，分别于小鼠生理性孕酮撤退后0h、4h、6h、8h、12h、16h对小鼠进行阴道细胞学涂片检查，摘眼球取血并处死，测定血清中E2与P的含量，观察子宫形态，并将双侧子宫角称重后，固定于福尔马林溶液中24h以上，石蜡包埋，HE染色，在光镜下进行组织学观察。
结果：⑴阴道涂片显示A组在12h-16h有少数（2-3只）小鼠的阴道分泌物中可检测到红细胞。B组在8h有1只小鼠检测到红细胞，16h检测到红细胞的小鼠数目增至6只。C组小鼠8h有2只可检测到红细胞，至16h有9只均可检测到红细胞。
⑵雌孕激素变化情况①血清E2含量变化A组小鼠0h点含量约为37.63pg，4-16h E2含量显著降低（P＜0.01）；4-8h含量最低，12-16h渐回升（P＜0.01）。B组小鼠4-12h血清E2明显低于0h、16h点（P＜0.01，P＜0.05），16h有明显回升与0h点相比无显著差异；与A组同时间点相比8-16h血清E2含量均高（P＜0.05，P＜0.01）。C组小鼠4-8h血清E2含量较0h、16h低（P＜0.01， P＜0.05），12-16h升高明显与0h相比无显著差异；与A、B组同时间点相比，6-16h血清E2含量均高（P＜0.05，P＜0.01）。②血清P含量变化A组小鼠0h点血清P含量约为50.28ng，4-16h明显降低（P＜0.01），6-16h含量维持在较低水平，与16h无显著差异。B组0h点血清P含量约为47.56ng，4-16h水平较低（P＜0.01），与16h无显著差异；与A组同时间点相比，B组4h含量较低（P＜0.01），6-16h含量渐高（P＜0.05，P＜0.01）。C组0h点血清P含量约为48.67ng，4-16h含量均显著降低（P＜0.01），4-8h含量最低；与A组同时间点相比，4h含量较低（P＜0.01），6-16h明显升高（P＜0.05，P＜0.01）；与B组相比，12-16h含量渐升高（P＜0.05，P＜0.01）。
⑶子宫形态三实验组小鼠子宫对照侧均呈浅粉色。A组8-16h实验侧子宫角肿大、充血现象逐渐明显；至16小时呈深红色，充血肿大呈结节状，欠均匀。B组小鼠6h实验侧子宫角出现肿大及部分轻微的充血现象；随时间延长其充血、肿大现象加重，较A组显著；16h充血最为明显，呈暗红色，均匀肿大。C组小鼠6h实验侧子宫角出现较明显的充血肿大现象；12h充血肿大现象最显著，呈均匀紫红色；16h有明显回落趋势。
⑷子宫角重量与对照侧子宫角相比，A、B、C3组小鼠实验侧子宫角重量均有明显增重（P＜0.01）。B组4-16h实验侧子宫明显较A组同时间点重（P＜0.05，P＜0.01）；C组实验侧子宫0-16h较A组重（P＜0.01），与B组相比0-12h较重（P＜0.01），16h与B组无显著差异（P＞0.05）。
⑸组织学观察组织学观察见A组0h-6h子宫内膜逐渐增厚，被覆上皮完整，上皮细胞呈柱状；8h-12h子宫腔内出现细胞碎屑，上皮有局部崩解脱落；16h宫腔被覆上皮坏死脱落，蜕膜化间质小部分与基底层分离。B组0h-6h子宫腔由于间质细胞蜕膜化内膜增厚使宫腔狭小，被覆上皮完整，上皮细胞呈柱状；8h子宫腔内出现细胞碎屑，上皮有局部崩解脱落；12h宫腔被覆上皮坏死脱落；16h蜕膜化间质出现大面积坏死并伴有出血，部分与基底层界限渐清晰。C组0h-4h，子宫内膜增厚使宫腔狭小，被覆上皮完整，上皮细胞呈柱状；6h-8h子宫腔内出现细胞碎屑，上皮有局部崩解脱落；12h宫腔被覆上皮坏死脱落，蜕膜化间质出现大面积坏死并伴有出血，16h蜕膜化区域与基底层几乎全部分离，界限清晰。
结论：⑴结果显示km小鼠月经出血模型复制成功；⑵活血方及活血加味方均对km小鼠月经模型有显著的干预作用，其中活血加味方组的干预作用更显著。其作用机理可能是通过调节雌孕激素水平，从而影响子宫内膜变化来实现对月经的干预作用。
第二部分活血方与活血加味方阿魏酸煎出量的比较研究
目的：研究活血方与活血加味方中阿魏酸的煎出量的变化，从而考察中医配伍用药的科学性、严谨性。
方法：采用高效液相法检测其阿魏酸的煎出量。①色谱条件：选择合适的固定相，调整流动相的组成、配比、流速以使阿魏酸色谱峰与其它峰完全分离。②标准曲线的绘制：配制系列浓度的阿魏酸对照品溶液，分别进样，按含量测定方法测定峰面积，以阿魏酸对照品浓度为横坐标，相应峰面积为纵坐标，绘制标准曲线。③精密度试验：精密量取阿魏酸对照品溶液，重复进样5次，在色谱条件下测定峰面积。④稳定性试验：精密量取同一份供试品溶液，在48小时内于不同时间注入高效液相色谱仪，分别测定峰面积。⑤重复性试验：精密量取同一份供试品溶液，平行进样5次，分别测定峰面积。⑥回收率试验：采用加样回收率测定法，取已知含量的样品溶液适量，每组分别加入适量的阿魏酸对照品，对所得样品分别进样，在上述色谱条件下测定峰面积，计算回收率。⑦样品含量测定：分别精密量取供试品溶液，在上述色谱条件下测定峰面积，计算其中的阿魏酸含量。
结果：①色谱条件：色谱柱为迪马公司Diamonsil C18柱(4.6×200mm,5μm)；流动相：甲醇-0.1%磷酸（40:60）；检测波长：320nm；柱温：25℃；流速：1.0ml/min；进样量：10μl；在此色谱条件下，阿魏酸保留时间约为12min，分离度大于1.5。②标准曲线的绘制：阿魏酸在0.0209～0.1881mg/ml范围内线性关系良好，回归方程为y=5×107x+493701，R2=0.9997，③精密度试验：仪器的精密度良好，RSD值为0.62％（n=5）。④稳定性试验：样品在48小时内稳定，RSD值为0.70％（n=6）。⑤重复性试验：本方法重现性良好，RSD值为0.64％（n=5）。⑥回收率试验：阿魏酸的平均回收率为99.10％，RSD值为0.96％（n=9），表明回收率较高，方法准确可靠。⑦样品含量测定：活血方中阿魏酸煎出量为0.803mg/g，活血加味方中阿魏酸煎出量为1.089mg/g，与活血方相比，其阿魏酸煎出量明显提高35%以上。
结论：⑴高效液相色谱（HPLC）法所建立的方法精密度、稳定性、重复性良好，回收率高，可以用于活血方与活血加味方中阿魏酸的煎出量的检测。⑵活血加味方配伍菟丝子有助于提高阿魏酸的煎出量。⑶与动物实验结果相互印证表明，阿魏酸的煎出量与其对小鼠月经模型的干预作用呈正相关。
Menstruation is a phenomenon that endometrial proliferation,differentiation, crumbling, and bleeding with sequential function of estrogenand progesterone. The cyclical characteristics change of uterine form and thefunction make up Menstrual cycle. Menstruation occupied a long time inwoman's lifetime, and have enormous influence to women's reproductivehealth. TCM holds that menstruation comes up periodically is results ofinteraction and balance between "kidney-TianGui-ChongRen-uterus"[1].Woman take blood as root, the gas for use. Qi and blood was regulated, makeuterus good nutrition, so the blood is full and overflow appearmenstruation.Therefore, regulate qi and blood is to make the necessaryconditions of menstrual comes smoothly.The clinical studies of menstruationis more than laboratory study, the reason is lack of animal model. Thisexperiment replicated the km mice menstrual bleeding model successfully bysequential hormone treatment and man-made decidua according to thedomestic and international literature[2-5], and provides the basis for theresearch on the influence of Chinese medicine on the origin and developmentof menstruation.Mainly chuanxiong based.Make Huoxue Recipe andModified Huoxue Recipe，and observe the affect to menstrual model. At thesame time,make ferulic acid which is the major components of thechuanxiong as the index, detect the content changes of ferulic acid in HuoxueRecipe and Modified Huoxue Recipe.And research the relativity betweenferulic acid content and its influence to menstruation role model. With theview of provide the scientific basis for clinical use of Chinese medicine.
Part One Study of the influence Huoxue Recipe and ModifiedHuoxue Recipe upon mice menstruation model
Objective: To copy the physiological progesterone retreating menstrualbleeding model with km mice,and reveals the influence Huoxue Recipe andModified Huoxue Recipe on mice menstruation model.
Methods: Take the female km mice to trying raised for a week，and thenrandomly divided into three groups, the model(A)group, Huoxue Recipe(B)group and Modified Huoxue Recipe(C)group. Narcotize the mice with ethylether, and excise ovarian. Restore a week to remove endogenous hormones,then treat with hormone sequential method. The mice was subcutaneousinjected estradiol solution A(15μg/kg)in the1~3days. On the seventh day themice was subcutaneous injected progesterone solution(7.5μg/kg)and estradiolsolution B(0.75μg/kg), and at the same time, to lay the worked progesteronesilicon tube in the subcutaneous tissue of the mice’s back. The mice wassubcutaneous injected estradiol solution B(0.75μg/kg)in the8~9days. Themice of group A were given a gavage of physiological saline, groupB weregiven a gavage of Huoxue Recipe, groupC were given a gavage of ModifiedHuoxue Recipe. On the ninth day, inject20μL aseptic peanut oil into the rightside of uterine to induce decidualization,the left side is controlled. On the11thday, take out the progesterone silicon tube which buried in the back of themice. After physiological progesterone retreat0h,4h,6h,8h,12h and16h,inspect the vaginal smear test respectively,and then pick the eyeball to takeblood and put them to death, determine the content of E2and P, observationthe form of uterus, and weigh the bilateral uterine horn respectively. Fix theuterus in formalin solution for more than24h, paraffin, HE dyed, and proceedhistopathologic study by light microscopy.
Results:⑴Vaginal smear In12h-16h,there were only2-3mice ofgroup A can be detected the red blood cells in vaginal secretions. Group Bhave one detect the red blood cells in8h, in16h, the amount of mice which can be detected the red blood cells increased to six. Group C have2detect thered blood cells in8h, and in16h have9can detect the red blood cells.
⑵Changes of the content of E2and P①Changes of the content of E2The content of E2in serum of mice in group A was37.63pg in0h,significantly reduced in4-16h(P＜0.01),and minimum in4-8h, pick up in12-16h(P＜0.01). In4-12h the content of E2in serum of mice in groupBobviously lower than0h(P＜0.01,P＜0.05).The content obviously reboundedin16h,and had no significant difference compared with0h. The content of E2in serum were high compared with group A in8-16h(P＜0.05,P＜0.01).In4-8h the content of E2in serum of mice in group C obviously lower than0hand16h(P＜0.01,P＜0.05),in12-16h the content rise significantly andcompared with0h had no significant differences. In6-16h the content of E2inserum were high compared with group A and B at the same time points(P＜0.05,P＜0.01).②Changes of the content of P The content of P in serum ofmice in group A was50.28ng,significantly reduced in4-16h(P＜0.01),and thecontent maintain at very low levels in6-16h,had no significant differencecompared with16h. The content of P in group B was47.56ng in0h, keepinglow level in4-16h(P＜0.01)and had no significant difference compared with16h (P <0.01). Compared with group A, the content of P in group B were lowin4h(P＜0.01),and high in6-16h(P＜0.05,P＜0.01). The content of P ingroup C was48.67ng in0h, significantly reduced in4-16h(P＜0.01),and thecontent in4-8h were lowest. Compared with group A, the content were low in4h(P＜0.01),and observably high in6-16h(P＜0.05, P＜0.01),Compared withgroup B, the content were observably high in12-16h(P＜0.05,P＜0.01).
⑶Uterine form The uterine horn of the controlled side of the3groupswere all light pink. In8-16h,the phenomenon of swollen and congestion ofexperimental side uterine horn of group A were increasingly obvious, until16h, the experimental side uterine horn were deep red, swollen and congestionappear nodular and nonuniform. The experimental side uterine horn of groupB represent some slight enlargement and congestion phenomenon in6h, withthe time going the congestion and enlargement were more and more obvious, and more evident than group A, until16h the experimental side uterine hornwere dark red, congestive obviously and uniform enlargement. Theexperimental side uterine horn of group C appeared congestive and enlargeobviously in6h, the phenomenon of congestion and enlargement were mostobvious and appeared homogeneous purple in12h,and in16h present weak.
⑷The weight of uterine horn Compared with the controlled side, theweight of experimental side of group A, B and C were obviously increased(P＜0.01). In4-16h, the weight of experimental side uterine horn of group Bsignificantly better than group A(P＜0.01). In0-16h, the weight ofexperimental side uterine horn of group C significantly better than group A(P＜0.01).The weight of experimental side uterine horn of group C increasedheavily compared with group B in0-12h(P＜0.01),and in16h, there are nosignificant difference between group B and C(P＞0.05).
⑸Histopathologic study Histopathologic study showed the mice’sendometrium of group A were gradually incrassated, and the epithelium werecomplete with the cells were columnar in0h-6h. The uterine cavity appeareddebris of cells, and partial epithelial were disintegrated and fall off in8h-12h.The epithelial of uterus were necrotic and fall off, a little of decidualmesenchyme were separated from stratum basale in16h. Endometrium ofgroup B were incrassated due to the decidua of interstitial cell make theuterine cavity narrow, and the epithelium were complete with the cells werecolumnar in0h-6h. There were debris of cells in uterine cavity, partialepithelium were disintegrated and fall off in8h. Uterine epithelium werenecrotic and fall off in12h.The decidual mesenchyme were massive necrosiswith bleeding, and parts of them were separated from stratum basale withclear boundaries in16h. Endometrium of group C were incrassated make theuterine cavity narrow, and the epithelium were complete with the cells werecolumnar in0h-4h. In6h-8h, in the uterine cavity appeared debris of cells,and partial epithelium were disintegrated and fall off. The uterine epitheliumwere necrotic and fall off, and decidual mesenchyme were massive necrosis with bleeding in12h.Almost all decidual area were separated from stratumbasale with clear boundaries in16h.
Conclusion:⑴Results showed that the model of km mice menstrualbleeding was made successfully；⑵Huoxue Recipe and Modified HuoxueRecipe both have significant influence on km mice menstrual model,andModified Huoxue Recipe’s intervention is the most significant. Its mechanismis probably by means of regulate sex hormone level, which affect endometrialchange to realize the influence on the menstrual.
Part two Comparative study of the content of ferulic acid in HuoxueRecipe and Modified Huoxue Recipe
Objective: To research the content change of ferulic acid in HuoxueRecipe and Modified Huoxue Recipe,thus investigate the scientificalness andpreciseness of Chinese crude drug compatibility.
Methods: Using high performance liquid method to detect the content offerulic acid.①Chromatographic conditions: Choose appropriate fixed phase,adjust different formulation and proportion of mobile phase and regulate flowrate and wavelength in order to separate the peak of Ferulic acid from otherswell.②s tandard curve:Prepare a series of the Ferulic acid reference solutionof different concentration, examine peak area, then the regress equation wasobtained with the content of Ferulic acid as abscissa, the relevant peak area asordinate.③P recision:Inject Ferulic acid reference substance solution into thechromatograph for five times, examine the peak area under thechromatographic conditions, and then calculate the precision.④Stability:Prepare the test solution inject into the chromatograph at different time in48hours, and then determine the peak area respectively.⑤Reproducibility:Prepare the test solution inject into the chromatograph five times in the sameway,to measure the peak area of Ferulic acid respectively.⑥Recovery: takinga certain amount of Ferulic acid reference substance add into the knownconcentration of sample solution,and mix. inject into the chromatograph respectively. determine peak area under the chromatographic conditions tocalculate the recovery.⑦S ample Determination:take the test solution injectinto the chromatograph,the content of Ferulic acid in sample solution wasanalyzed under above-mentioned conditions.
Results:①C hromatographic conditions:Diamonsic-C18column(200×4.6mm,5μm)was used for HPLC. The mobile phase was a mixtureconsisted of Methanol-0.1%phosphoric acid (40:60) at a flow rate of1.0ml·min-1and the detective wavelength was set at320nm. Column temperaturewas25℃. Sample size was20μl. Under the system, the peaks of sampleswere separated well with the resolution of not less than1.5. The retention timeis about12min.②Preparation of standard curve: The liner range forFerulicacid was0.0209～0.1881mg/ml.Regress equation was Y=5×107X+493701(R2=0.9997, n=5).③Precision: The precision instrument is fine. The RSDvalue in the precision test was0.62%(n＝5).④Stability: The sample wasstable in48hours. The RSD value of stability was0.70%(n＝6).⑤R eproducibility:This method has good reproducibility. The RSD valueof reproducibility was0.64%(n＝5).⑥R ecovery: Average recovery is99.10％and the value of RSD was0.96%(n＝9). That explained it had a higherrecovery rate, and the method was accurate and reliable.⑦SampleDetermination: The content of Ferulic acid in Huoxue Recipe were0.803mg/g，the content of Ferulic acid in Modified Huoxue Recipe were1.089mg/g，significantly increased35%compare with Huoxue Recipe.
Conclusions:⑴the method of High performance liquid chromatography(HPLC) has good precision, stability and reproducibility, the recovery is high.It can be used to detect the content of ferulic acid in Huoxue Recipe andModified Huoxue Recipe.⑵Modified Huoxue Recipe with the compatibilityof dodder can help to improve the content of ferulic acid.⑶Combined withanimal experimental results show that the content of ferulic acid waspositively associated with the influence to the menstrual model.
第一部分活血方与活血加味对小鼠月经模型的干预比较实验研究
目的：应用km小鼠复制生理性孕酮撤退的月经出血模型，并以此小鼠月经模型为基础，揭示活血方与活血加味方对小鼠月经模型的干预作用。
方法：将雌性km小鼠试养一周后，随机分为模型（A）组、活血方（B）组、活血加味方（C）组。采用乙醚麻醉，摘除卵巢，恢复一周以清除内源激素，后进行激素序贯处理。各组小鼠在实验第1~3天皮下注射雌二醇工作液A（15μg/kg）；第7天皮下注射孕酮工作液（7.5μg/kg）和雌二醇工作液B（0.75μg/kg），同时将处理好的孕酮硅胶管皮埋于小鼠背部；第8~9天皮下注射雌二醇工作液B（0.75μg/kg）。其中A组每天灌胃生理盐水；B组灌胃活血方药液；C组灌胃活血加味方药液。后于第9天将20μL的无菌花生油注射到小鼠右侧子宫角，以诱导蜕膜化，以另一侧子宫角做对照，第11天取出小鼠背部皮埋管，分别于小鼠生理性孕酮撤退后0h、4h、6h、8h、12h、16h对小鼠进行阴道细胞学涂片检查，摘眼球取血并处死，测定血清中E2与P的含量，观察子宫形态，并将双侧子宫角称重后，固定于福尔马林溶液中24h以上，石蜡包埋，HE染色，在光镜下进行组织学观察。
结果：⑴阴道涂片显示A组在12h-16h有少数（2-3只）小鼠的阴道分泌物中可检测到红细胞。B组在8h有1只小鼠检测到红细胞，16h检测到红细胞的小鼠数目增至6只。C组小鼠8h有2只可检测到红细胞，至16h有9只均可检测到红细胞。
⑵雌孕激素变化情况①血清E2含量变化A组小鼠0h点含量约为37.63pg，4-16h E2含量显著降低（P＜0.01）；4-8h含量最低，12-16h渐回升（P＜0.01）。B组小鼠4-12h血清E2明显低于0h、16h点（P＜0.01，P＜0.05），16h有明显回升与0h点相比无显著差异；与A组同时间点相比8-16h血清E2含量均高（P＜0.05，P＜0.01）。C组小鼠4-8h血清E2含量较0h、16h低（P＜0.01， P＜0.05），12-16h升高明显与0h相比无显著差异；与A、B组同时间点相比，6-16h血清E2含量均高（P＜0.05，P＜0.01）。②血清P含量变化A组小鼠0h点血清P含量约为50.28ng，4-16h明显降低（P＜0.01），6-16h含量维持在较低水平，与16h无显著差异。B组0h点血清P含量约为47.56ng，4-16h水平较低（P＜0.01），与16h无显著差异；与A组同时间点相比，B组4h含量较低（P＜0.01），6-16h含量渐高（P＜0.05，P＜0.01）。C组0h点血清P含量约为48.67ng，4-16h含量均显著降低（P＜0.01），4-8h含量最低；与A组同时间点相比，4h含量较低（P＜0.01），6-16h明显升高（P＜0.05，P＜0.01）；与B组相比，12-16h含量渐升高（P＜0.05，P＜0.01）。
⑶子宫形态三实验组小鼠子宫对照侧均呈浅粉色。A组8-16h实验侧子宫角肿大、充血现象逐渐明显；至16小时呈深红色，充血肿大呈结节状，欠均匀。B组小鼠6h实验侧子宫角出现肿大及部分轻微的充血现象；随时间延长其充血、肿大现象加重，较A组显著；16h充血最为明显，呈暗红色，均匀肿大。C组小鼠6h实验侧子宫角出现较明显的充血肿大现象；12h充血肿大现象最显著，呈均匀紫红色；16h有明显回落趋势。
⑷子宫角重量与对照侧子宫角相比，A、B、C3组小鼠实验侧子宫角重量均有明显增重（P＜0.01）。B组4-16h实验侧子宫明显较A组同时间点重（P＜0.05，P＜0.01）；C组实验侧子宫0-16h较A组重（P＜0.01），与B组相比0-12h较重（P＜0.01），16h与B组无显著差异（P＞0.05）。
⑸组织学观察组织学观察见A组0h-6h子宫内膜逐渐增厚，被覆上皮完整，上皮细胞呈柱状；8h-12h子宫腔内出现细胞碎屑，上皮有局部崩解脱落；16h宫腔被覆上皮坏死脱落，蜕膜化间质小部分与基底层分离。B组0h-6h子宫腔由于间质细胞蜕膜化内膜增厚使宫腔狭小，被覆上皮完整，上皮细胞呈柱状；8h子宫腔内出现细胞碎屑，上皮有局部崩解脱落；12h宫腔被覆上皮坏死脱落；16h蜕膜化间质出现大面积坏死并伴有出血，部分与基底层界限渐清晰。C组0h-4h，子宫内膜增厚使宫腔狭小，被覆上皮完整，上皮细胞呈柱状；6h-8h子宫腔内出现细胞碎屑，上皮有局部崩解脱落；12h宫腔被覆上皮坏死脱落，蜕膜化间质出现大面积坏死并伴有出血，16h蜕膜化区域与基底层几乎全部分离，界限清晰。
结论：⑴结果显示km小鼠月经出血模型复制成功；⑵活血方及活血加味方均对km小鼠月经模型有显著的干预作用，其中活血加味方组的干预作用更显著。其作用机理可能是通过调节雌孕激素水平，从而影响子宫内膜变化来实现对月经的干预作用。
第二部分活血方与活血加味方阿魏酸煎出量的比较研究
目的：研究活血方与活血加味方中阿魏酸的煎出量的变化，从而考察中医配伍用药的科学性、严谨性。
方法：采用高效液相法检测其阿魏酸的煎出量。①色谱条件：选择合适的固定相，调整流动相的组成、配比、流速以使阿魏酸色谱峰与其它峰完全分离。②标准曲线的绘制：配制系列浓度的阿魏酸对照品溶液，分别进样，按含量测定方法测定峰面积，以阿魏酸对照品浓度为横坐标，相应峰面积为纵坐标，绘制标准曲线。③精密度试验：精密量取阿魏酸对照品溶液，重复进样5次，在色谱条件下测定峰面积。④稳定性试验：精密量取同一份供试品溶液，在48小时内于不同时间注入高效液相色谱仪，分别测定峰面积。⑤重复性试验：精密量取同一份供试品溶液，平行进样5次，分别测定峰面积。⑥回收率试验：采用加样回收率测定法，取已知含量的样品溶液适量，每组分别加入适量的阿魏酸对照品，对所得样品分别进样，在上述色谱条件下测定峰面积，计算回收率。⑦样品含量测定：分别精密量取供试品溶液，在上述色谱条件下测定峰面积，计算其中的阿魏酸含量。
结果：①色谱条件：色谱柱为迪马公司Diamonsil C18柱(4.6×200mm,5μm)；流动相：甲醇-0.1%磷酸（40:60）；检测波长：320nm；柱温：25℃；流速：1.0ml/min；进样量：10μl；在此色谱条件下，阿魏酸保留时间约为12min，分离度大于1.5。②标准曲线的绘制：阿魏酸在0.0209～0.1881mg/ml范围内线性关系良好，回归方程为y=5×107x+493701，R2=0.9997，③精密度试验：仪器的精密度良好，RSD值为0.62％（n=5）。④稳定性试验：样品在48小时内稳定，RSD值为0.70％（n=6）。⑤重复性试验：本方法重现性良好，RSD值为0.64％（n=5）。⑥回收率试验：阿魏酸的平均回收率为99.10％，RSD值为0.96％（n=9），表明回收率较高，方法准确可靠。⑦样品含量测定：活血方中阿魏酸煎出量为0.803mg/g，活血加味方中阿魏酸煎出量为1.089mg/g，与活血方相比，其阿魏酸煎出量明显提高35%以上。
结论：⑴高效液相色谱（HPLC）法所建立的方法精密度、稳定性、重复性良好，回收率高，可以用于活血方与活血加味方中阿魏酸的煎出量的检测。⑵活血加味方配伍菟丝子有助于提高阿魏酸的煎出量。⑶与动物实验结果相互印证表明，阿魏酸的煎出量与其对小鼠月经模型的干预作用呈正相关。
Menstruation is a phenomenon that endometrial proliferation,differentiation, crumbling, and bleeding with sequential function of estrogenand progesterone. The cyclical characteristics change of uterine form and thefunction make up Menstrual cycle. Menstruation occupied a long time inwoman's lifetime, and have enormous influence to women's reproductivehealth. TCM holds that menstruation comes up periodically is results ofinteraction and balance between "kidney-TianGui-ChongRen-uterus"[1].Woman take blood as root, the gas for use. Qi and blood was regulated, makeuterus good nutrition, so the blood is full and overflow appearmenstruation.Therefore, regulate qi and blood is to make the necessaryconditions of menstrual comes smoothly.The clinical studies of menstruationis more than laboratory study, the reason is lack of animal model. Thisexperiment replicated the km mice menstrual bleeding model successfully bysequential hormone treatment and man-made decidua according to thedomestic and international literature[2-5], and provides the basis for theresearch on the influence of Chinese medicine on the origin and developmentof menstruation.Mainly chuanxiong based.Make Huoxue Recipe andModified Huoxue Recipe，and observe the affect to menstrual model. At thesame time,make ferulic acid which is the major components of thechuanxiong as the index, detect the content changes of ferulic acid in HuoxueRecipe and Modified Huoxue Recipe.And research the relativity betweenferulic acid content and its influence to menstruation role model. With theview of provide the scientific basis for clinical use of Chinese medicine.
Part One Study of the influence Huoxue Recipe and ModifiedHuoxue Recipe upon mice menstruation model
Objective: To copy the physiological progesterone retreating menstrualbleeding model with km mice,and reveals the influence Huoxue Recipe andModified Huoxue Recipe on mice menstruation model.
Methods: Take the female km mice to trying raised for a week，and thenrandomly divided into three groups, the model(A)group, Huoxue Recipe(B)group and Modified Huoxue Recipe(C)group. Narcotize the mice with ethylether, and excise ovarian. Restore a week to remove endogenous hormones,then treat with hormone sequential method. The mice was subcutaneousinjected estradiol solution A(15μg/kg)in the1~3days. On the seventh day themice was subcutaneous injected progesterone solution(7.5μg/kg)and estradiolsolution B(0.75μg/kg), and at the same time, to lay the worked progesteronesilicon tube in the subcutaneous tissue of the mice’s back. The mice wassubcutaneous injected estradiol solution B(0.75μg/kg)in the8~9days. Themice of group A were given a gavage of physiological saline, groupB weregiven a gavage of Huoxue Recipe, groupC were given a gavage of ModifiedHuoxue Recipe. On the ninth day, inject20μL aseptic peanut oil into the rightside of uterine to induce decidualization,the left side is controlled. On the11thday, take out the progesterone silicon tube which buried in the back of themice. After physiological progesterone retreat0h,4h,6h,8h,12h and16h,inspect the vaginal smear test respectively,and then pick the eyeball to takeblood and put them to death, determine the content of E2and P, observationthe form of uterus, and weigh the bilateral uterine horn respectively. Fix theuterus in formalin solution for more than24h, paraffin, HE dyed, and proceedhistopathologic study by light microscopy.
Results:⑴Vaginal smear In12h-16h,there were only2-3mice ofgroup A can be detected the red blood cells in vaginal secretions. Group Bhave one detect the red blood cells in8h, in16h, the amount of mice which can be detected the red blood cells increased to six. Group C have2detect thered blood cells in8h, and in16h have9can detect the red blood cells.
⑵Changes of the content of E2and P①Changes of the content of E2The content of E2in serum of mice in group A was37.63pg in0h,significantly reduced in4-16h(P＜0.01),and minimum in4-8h, pick up in12-16h(P＜0.01). In4-12h the content of E2in serum of mice in groupBobviously lower than0h(P＜0.01,P＜0.05).The content obviously reboundedin16h,and had no significant difference compared with0h. The content of E2in serum were high compared with group A in8-16h(P＜0.05,P＜0.01).In4-8h the content of E2in serum of mice in group C obviously lower than0hand16h(P＜0.01,P＜0.05),in12-16h the content rise significantly andcompared with0h had no significant differences. In6-16h the content of E2inserum were high compared with group A and B at the same time points(P＜0.05,P＜0.01).②Changes of the content of P The content of P in serum ofmice in group A was50.28ng,significantly reduced in4-16h(P＜0.01),and thecontent maintain at very low levels in6-16h,had no significant differencecompared with16h. The content of P in group B was47.56ng in0h, keepinglow level in4-16h(P＜0.01)and had no significant difference compared with16h (P <0.01). Compared with group A, the content of P in group B were lowin4h(P＜0.01),and high in6-16h(P＜0.05,P＜0.01). The content of P ingroup C was48.67ng in0h, significantly reduced in4-16h(P＜0.01),and thecontent in4-8h were lowest. Compared with group A, the content were low in4h(P＜0.01),and observably high in6-16h(P＜0.05, P＜0.01),Compared withgroup B, the content were observably high in12-16h(P＜0.05,P＜0.01).
⑶Uterine form The uterine horn of the controlled side of the3groupswere all light pink. In8-16h,the phenomenon of swollen and congestion ofexperimental side uterine horn of group A were increasingly obvious, until16h, the experimental side uterine horn were deep red, swollen and congestionappear nodular and nonuniform. The experimental side uterine horn of groupB represent some slight enlargement and congestion phenomenon in6h, withthe time going the congestion and enlargement were more and more obvious, and more evident than group A, until16h the experimental side uterine hornwere dark red, congestive obviously and uniform enlargement. Theexperimental side uterine horn of group C appeared congestive and enlargeobviously in6h, the phenomenon of congestion and enlargement were mostobvious and appeared homogeneous purple in12h,and in16h present weak.
⑷The weight of uterine horn Compared with the controlled side, theweight of experimental side of group A, B and C were obviously increased(P＜0.01). In4-16h, the weight of experimental side uterine horn of group Bsignificantly better than group A(P＜0.01). In0-16h, the weight ofexperimental side uterine horn of group C significantly better than group A(P＜0.01).The weight of experimental side uterine horn of group C increasedheavily compared with group B in0-12h(P＜0.01),and in16h, there are nosignificant difference between group B and C(P＞0.05).
⑸Histopathologic study Histopathologic study showed the mice’sendometrium of group A were gradually incrassated, and the epithelium werecomplete with the cells were columnar in0h-6h. The uterine cavity appeareddebris of cells, and partial epithelial were disintegrated and fall off in8h-12h.The epithelial of uterus were necrotic and fall off, a little of decidualmesenchyme were separated from stratum basale in16h. Endometrium ofgroup B were incrassated due to the decidua of interstitial cell make theuterine cavity narrow, and the epithelium were complete with the cells werecolumnar in0h-6h. There were debris of cells in uterine cavity, partialepithelium were disintegrated and fall off in8h. Uterine epithelium werenecrotic and fall off in12h.The decidual mesenchyme were massive necrosiswith bleeding, and parts of them were separated from stratum basale withclear boundaries in16h. Endometrium of group C were incrassated make theuterine cavity narrow, and the epithelium were complete with the cells werecolumnar in0h-4h. In6h-8h, in the uterine cavity appeared debris of cells,and partial epithelium were disintegrated and fall off. The uterine epitheliumwere necrotic and fall off, and decidual mesenchyme were massive necrosis with bleeding in12h.Almost all decidual area were separated from stratumbasale with clear boundaries in16h.
Conclusion:⑴Results showed that the model of km mice menstrualbleeding was made successfully；⑵Huoxue Recipe and Modified HuoxueRecipe both have significant influence on km mice menstrual model,andModified Huoxue Recipe’s intervention is the most significant. Its mechanismis probably by means of regulate sex hormone level, which affect endometrialchange to realize the influence on the menstrual.
Part two Comparative study of the content of ferulic acid in HuoxueRecipe and Modified Huoxue Recipe
Objective: To research the content change of ferulic acid in HuoxueRecipe and Modified Huoxue Recipe,thus investigate the scientificalness andpreciseness of Chinese crude drug compatibility.
Methods: Using high performance liquid method to detect the content offerulic acid.①Chromatographic conditions: Choose appropriate fixed phase,adjust different formulation and proportion of mobile phase and regulate flowrate and wavelength in order to separate the peak of Ferulic acid from otherswell.②s tandard curve:Prepare a series of the Ferulic acid reference solutionof different concentration, examine peak area, then the regress equation wasobtained with the content of Ferulic acid as abscissa, the relevant peak area asordinate.③P recision:Inject Ferulic acid reference substance solution into thechromatograph for five times, examine the peak area under thechromatographic conditions, and then calculate the precision.④Stability:Prepare the test solution inject into the chromatograph at different time in48hours, and then determine the peak area respectively.⑤Reproducibility:Prepare the test solution inject into the chromatograph five times in the sameway,to measure the peak area of Ferulic acid respectively.⑥Recovery: takinga certain amount of Ferulic acid reference substance add into the knownconcentration of sample solution,and mix. inject into the chromatograph respectively. determine peak area under the chromatographic conditions tocalculate the recovery.⑦S ample Determination:take the test solution injectinto the chromatograph,the content of Ferulic acid in sample solution wasanalyzed under above-mentioned conditions.
Results:①C hromatographic conditions:Diamonsic-C18column(200×4.6mm,5μm)was used for HPLC. The mobile phase was a mixtureconsisted of Methanol-0.1%phosphoric acid (40:60) at a flow rate of1.0ml·min-1and the detective wavelength was set at320nm. Column temperaturewas25℃. Sample size was20μl. Under the system, the peaks of sampleswere separated well with the resolution of not less than1.5. The retention timeis about12min.②Preparation of standard curve: The liner range forFerulicacid was0.0209～0.1881mg/ml.Regress equation was Y=5×107X+493701(R2=0.9997, n=5).③Precision: The precision instrument is fine. The RSDvalue in the precision test was0.62%(n＝5).④Stability: The sample wasstable in48hours. The RSD value of stability was0.70%(n＝6).⑤R eproducibility:This method has good reproducibility. The RSD valueof reproducibility was0.64%(n＝5).⑥R ecovery: Average recovery is99.10％and the value of RSD was0.96%(n＝9). That explained it had a higherrecovery rate, and the method was accurate and reliable.⑦SampleDetermination: The content of Ferulic acid in Huoxue Recipe were0.803mg/g，the content of Ferulic acid in Modified Huoxue Recipe were1.089mg/g，significantly increased35%compare with Huoxue Recipe.
Conclusions:⑴the method of High performance liquid chromatography(HPLC) has good precision, stability and reproducibility, the recovery is high.It can be used to detect the content of ferulic acid in Huoxue Recipe andModified Huoxue Recipe.⑵Modified Huoxue Recipe with the compatibilityof dodder can help to improve the content of ferulic acid.⑶Combined withanimal experimental results show that the content of ferulic acid waspositively associated with the influence to the menstrual model.
引文
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