| Purpose:In this research, the composing and dosage of the prescription were optimized based on pharmacology and uniform design. Yueanjian’s mechanism of treating premenstrual syndrome was also studied.Material and method:1. The animal models of PMS with the syndrome of liver-depression and spleen-deficiency were established by chronic restraint stress.2. The content of Salvianolic acid B in Yueanjian was determined by HPLC.3. Yueanjian was extracted by three methods:water-extraction, steam-distillation and alcohol-extraction. Behavioral performances and the contents of estradiol and progestin in serum were analyzed after the animal models were treated with these three extracts. The contents of Salvianolic acid B in these three extracts were detected by HPLC. All these four indicators were used to optimize the extraction method of Yueanjian.4. According to U11(115) uniform design table, five drugs in Yueanjian were treated as five factors, which were divided into11levels. Behavioral performances and the contents of estradiol and progestin in serum were used to optimize the prescription of Yueanjian.5. The theoretical result was tested and verified by animal models of PMS. The levels of E2, P,5-HT, T and PRL in animal models’serum after treated with Yueanjian were analyzed. The ultrastructure and pathology of uterus were also checked to discuss Yueanjian’s mechanism of treating premenstrual syndrome.Results:1. Vaginal smear cystourethroscopy were used to determine the estrogen cycle of unpregnant rates. Non-estrus rates were chosen and binded in special plastic bottles three times a day.21days later, Estradiol benzoate was hypodermically injected500μg per day, for7days. Compared with normal animals, model animals were listlessness. Model animals’scores in open field experiments were lower, and the levels of progestin declined significantly, while the levels of estradiol and E2/P increased appreciably.2. The content of Salvianolic acid B in Yueanjian was determined by HPLC. The reference substance solutions preparation method:Precisely sampling the Salvianolic acid B7.2mg into10mL capacity bottle, ultrasonic dissolving in appropriate methanol, added methanol to scale, shook up. The sample preparation method:Taking some samples into25mL capacity bottle, added water to scale, shook up. Chromatographic condition: Synergi Polar-Rp80A (250×4.6mm,4μm) column. Mobile phase:methanol-acetonitrile-water-formic acid30:0:9:). Flow rate1.0mL·min-1.Column temperature:30℃. Detection wavelength:286nm.3. The levels of progestin in animals treated with these three extracts were higher than model animals, while the levels of E2/P were lower. Steam distillations extract could also make the level of E2reduce significantly. The behavioral performances of animals treated by steam distillations were also higher than model animals. The content of Salvianolic acid B in the extract of steam-distillation was higher than other two extracts.4. According to the result of multiple regression analysis of behavioral performances and levels of E2/P, the regression equation was:Y=0.7492+0.0180*X1-0.0031*X5-0.0005*X1*X1-0.0001*X4*X4-0.0001*X1*X3+0.0002*X1*X4+0.0001*X1*X5+0.0003*X2*X5+0.0001*X3*X4, multiple correlation coefficient was1.0000, the equation have prominent meanings. The best compatibility proportion of Yueanjian was rhizoma cyperi (18.18g), white atractylodes roots (40.00g), Salvia miltiorrhiza (10.91g) and rhizoma acori graminei (30.00g). According to the result of multiple regression analysis of extraction ratio of Salvia miltiorrhiza, the regression equation was:Y=0.085*X1-0.206*X2+0.01*X4+0.068*X5. These two result show that the seed of Chinese dodder could be removed.5.The behavioral performances of animals treated by Yuananjian were higher than model animals. Yuananjian could also raise the level of progestin and reduce the level of E2/P. The content of5-HT in serum raised significantly after treated by Yuanjian. Yuanjian showed good protection to the damage of mitochondria, rough endoplasmic reticulum and microvilli on cell surface. Conclusion:1. On the basis of comprehensive analysis, steam-distillation was chosen as Yueanjian’s best extraction method.2. The best compatibility proportion of Yueanjian was rhizoma cyperi (18.18g), white atractylodes roots (40.00g),Salvia miltiorrhiza (10.91g) and rhizoma acori graminei (30.00g).3. Yueanjian’s mechanism of treating premenstrual syndrome maybe related with raising the level of progestin, reducing the level of E2/P and increasing the synthesis of5-HT. |
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