| 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 modelObjective: 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 RecipeObjective: 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. |
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