Studies On Effective Parts And Mechanism Of Chaihu Jia Longgu Muli Decoction For Improving Sleep Disorders Of Menopausal Transition

Objective:Perimenopause is an inevitable physiological process for women. Sleep disorders are common symptoms in perimenopausal women. Low quality sleep significantly affects the quality of life of women. Chaihu Jia Longgu Muli Decoction, a Chinese herbal recipe composed of Longgu Muli Decoction with Radix Bupleuri, has obtained good clinical curative clinical effects in improving sleep disorders of menopausal transition. But the effective parts and machenism of it were not clear. Therefore, this study screened the effective parts and analyzed its machenism of improving sleep in perimenopausal women by extraction, separation technology and pharmacodynamics experiment.Method:1. The study on effective parts of Chaihu Jia Longgu Muli Decoction for improving sleep of ovariectomyized combined electrical stimulation mice.We used the technology of foramen-magnum absorbent resins, ovariectomy and electrical stimulation mice as the model of menopausal sleep disorders and pentobarbital-induced sleep experiment, to screen the effective parts and clarify main components of Chaihu Jia Longgu Muli Decoction.2. The chemical composition analiysis of 70% ethanol effective parts of Chaihu Jia Longgu Muli Decoction.A Diamonsil C18 column (250×4.6 mm,5μm) was used as the stationary phase and the mobile phase was consisted of acetonitrile and water, Gradient elution was carried out at the flow rate of 1.0 ml/min. The column temperature was 30 ℃. The ELSD detector was operated at 105 ℃ with the optimum flow rate of the nebulizing gas at 2.5 L/min.3. The study on the dose and time correlation of 70% ethanol effective parts of Chaihu Jia Longgu Muli Decoction for improving sleep of ovariectomyized combined electrical stimulation mice.Ovariectomy and electrical stimulation were given to the female mice to the model. And then, we observed different concentrations of 70% ethanol effective parts on pentobarbital-induced sleep time of the model mice, to define the optimal dose. On the basis of the optimal dose, we used the technology of foramen-magnum absorbent resins, ovariectomy and electrical stimulation mice as the model of menopausal sleep disorders and pentobarbital-induced sleep experiment, to observe different days on pentobarbital-induced sleep time of the model mice and define the optimal time of effective parts.4. The study on mechanism of 70% ethanol effective parts of Chaihu Jia Longgu Muli Decoction for improving sleep of ovariectomyized combined electrical stimulation mice.Ovariectomy and electrical stimulation were given to the female mice to the model. We analyzed its machenisms through organ coefficients of uterus, kidneys and the hormone level of Estradiol, Follicle-Stimulating Hormone, Luteinizing Hormone in serum and the hormone level of Gonadotropin-Releasing Hormone in hypothalamus during the day and night by separating the uterus and adrenal and glandenzyme-linked immunoassay (ELISA).Results:1. The study on effective parts of Chaihu Jia Longgu Muli Decoction for improving sleep of ovariectomyized combined electrical stimulation mice.Compared to the model group,70% ethanol elution of Chaihu Jia Longgu Muli Decoction had significant effect on prolonging the pentobarbital-induced sleep time (p<0.01).2. The chemical composition analiysis of 70% ethanol effective parts of Chaihu Jia Longgu Muli Decoction.The five ginsensides in effective parts of Chaihu Jia Longgu Muli Decoction were ginsenoside Rg1, ginsenoside Rb1, saikosaponin a, saikosaponin d, saikosaponin B2. They were linear in the ranges of 1.820-9.100μg,1.810-9.050μg,1.130-10.170μg,0.420-2.100μg, 3.125-15.625μg、respectively. The RSD of precision, reproducibility and recovery were all less than 3.0%. The method is rapid, simple, reliable and accurate.3. The study on the dose and time correlation of 70% ethanol effective parts of Chaihu Jia Longgu Muli Decoction for improving sleep of ovariectomyized combined electrical stimulation mice.Compared to the model group,70% ethanol elution of Chaihu Jia Longgu Muli Decoction(48 mg/kg) had significant effect on prolonging the pentobarbital-induced sleep time (p< 0.05); Compared with other groups,70% ethanol elution of Chaihu Jia Longgu Muli Decoction (48 mg/kg, for seven consecutive days) had significant effect on prolonging the pentobarbital-induced sleep time (p< 0.01, or p< 0.05).4. The study on mechanism of 70% ethanol effective parts of Chaihu Jia Longgu Muli Decoction for improving sleep of ovariectomyized combined electrical stimulation mice.Compared to the sham-operated group, the uterus coefficient (p<0.05) and the level of Estradiol in model group induced significantly during the day (p<0.01), the level of Gonadotropin-Releasing Hormone of model group was higher significantly during the day (p<0.05). Compared to the model group,70% ethanol elution (48 mg/kg per day, for seven consecutive days) of Chaihu Jia Longgu Muli Decoction had significant effect on increasing the uterus coefficient of the model mice (p<0.05) and the level of E2 in model group during the day (p<0.01), and induced the level of Gonadotropin-Releasing Hormone of model group during the day (p<0.05). Compared to the model group, estradiol valerate also had significant effect on increasing the uterus coefficient of the model mice (p<0.05), the level of E2 in estrogen group during the night was below sham-operated group and Chaihu Jia Longgu Muli Decoction group (p<0.01). The level of E2 in model group during the day was below the night.Conclusion:1. The part of Chaihu Jia Longgu Muli Decoction eluted by 70% ethanol is the effective part for improving sleep of ovariectomyized combined electrical stimulation mice.2. The five ginsensides in effective parts of Chaihu Jia Longgu Muli Decoction by HPLC-ELSD were ginsenoside Rg1, ginsenoside Rb1, saikosaponin a, saikosaponin d, saikosaponin B2. The average content of them were 0.474%,1.297%,1.031%,0.309% and 2.004% respectively. They were linear in the ranges of 1.820-9.100μg, 1.810-9.050μg,1.130-10.170μg,0.420-2.100μg,3.125-15.625μg respectively.3. The best doseage scheme of 70% ethanol effective parts of Chaihu Jia Longgu Muli Decoction is 48 mg/kg per day, and for seven consecutive days.4. The machenism of 70% ethanol effective parts of Chaihu Jia Longgu Muli Decoction in improving sleep of perimenopausal women may be stable Estradiol secretion of circadian rhythm and regulating Hypothalamus-Pituitary-Ovary axis.