| Menopause is a special transition period that ovarian function get weaker and weaker,until disappeared. And Menopausal Syndrome which is due to the disorder of menstrual cycle and the descend of hormone level that most women should go through, usually with a series of autonomic nervous system and a set of syndrome with neuropsytoms. During this period, with most of the changes in estrogen levels are associated with varying degrees of disease, such as:menstrual disorders, depression, mood swings, breast cancer, irritability, insomnia, the atrophy of sexual organs, the decreased of immune function and the like. With the social development and the ascent of the old people,more and more people are affected by it.The traditional Chinese medicine formula Yining Gengnian Granules include Oviductus Ranae, Fructus Ligustri lucidi, Herba Ecliptae, Radix Angelica sinensis and Rhizoma chuanxiong. It is good at MPS which have been proved well clinical treatment for activate blood and the kidney. However, its initial preparation exists some disadvantages,we decide to improve its use,carry,dosege etc to be better for clinical application. Therefore, according to relevant provision,this study will establish the research of preparation, quality standard as well as primary pharmacodynamic studies.Objective:To optimize a extraction technology establish quality standards,observe the initial stability and investigate the immunomodulatory effects of Yining Gengnian Granules on immunosuppression mice for providing a reliable theoretical and clinical basis.Method:1. preparation procedureThe content of specnuezhenide is tested by HPLC, with composite score of the content of specnuezhenide and dry extract rate as indexes, orthogonal experiment is adopted to optimize extraction method of Yining Gengnian Granules with the soak time, solvent content and extraction time as factors;the content of volatile oil is evaluated the extraction process of Radix Angelica sinensis and Rhizoma chuanxiong to choose the suitable particle size,water amount and extraction time. The yield of volatile oil from β-CD inclusion compound is investigated the proportion of P-CD to volatile oil,temperature and water amount. Finally, choosing the appropriate auxiliary materials to make particles and observing bulk densty, angle of repose,the critical relative humidity and so on. The initial pharmacodynamics of new technology is evaluated by immunosuppressive murine model.2. quality standardIn times of requirements of Chinese Pharmacopoeia 2010, Fructus Ligustri lucidi, Herba Ecliptae and Radix Angelica sinensis are identified by the Thin layer chromatographic method(TLC).The Oviductus Ranae is indentified by HPLC. Column is Agilenet column(4.6x250 mm,5μm); the mobile is water; the flow rate is lml ·min-1 and detective wavelength was set at 224 nm. Specnuezhenide is also analyzed by HPLC. Methanol-water(35:65) was the mobile phase; the flow rate is 0.7 ml-min-1,time is thirty minutes and detective wavelength was set at 215 nm.Initial stability:the sample is choosed in 0,1,2,3 months to evaluate the content of specnuezhenide at room temperature. And to test the properties,particle,size, moisture,arsenic salts and so on.3. primary pharmacodynamicsThe immunosuppressive murine model was induced by intraperitoneally injecting female Kunming mice with cyclophamide(Cy). The Cy(80mg/kg) is injected for initial three days,then once a week the injection is strengthen again. The experimental animals’ groups and the dosages of drug are as follows:model group(equal volume solvent),the high dose group(9.6g/kg), the middle dose group(6.4g/kg), the low dose group(3.2g/kg), the original process group(6.4g/kg), the positive control group(3.3g/kg); the drug is administrated by Ig once a day for 15 days. After that they were treated with different kinds of drugs by intragastric fifteen days, then the carbon clearance function, the weight of immune organs,the proliferationactivities of splenic lymphocyte,the level of serum hemolysin and the activities of natural killer (NK) cells were measured. The method of carbon clearance function is as follows:after the last administration 1h, the diluted India ink is injected by intravenous and the blood is got by inner canthus in 2 min,10 min, then placed 0.1% sodium carbonate solution the absorbance is measured at 600 nm. The index of immune organs is got by weighing the weight of dead mice. The transformation test of splenic lymphocyte:after the last administration, the mice are killed and get the sterile spleen to be adjusted the concentration of cell,then put them in 37℃ incubator,after 68 hours added ConA to continue incubate 4 hours,finally to be measured at 490nm. The method of serum hemolysin:chicken red blood cell are injected by ip after administration 7 days and separate serum to measure the OD540. the experiment of activities of natural killer (NK) cells:after the last administration, the all mice are killed and get the sterile spleen to be adjusted the concentration of cell,then the MTT is added and put them in 37℃ incubator to be measured at 490nm.Result:1. preparation procedureThe optimized method of Fructus Ligustri lucidi and Herba Ecliptae is as follows:soak for half an hour later,extracting twice with water, the first time is 10 folds water for 2 h, the second time is 8 folds water for 1 h. The extraction of volatile oil from Radix Angelica sinensis and Rhizoma chuanxiong is 5 folds water for 5 h.The best processing conditions for β-CD inclusion compound are as follows: the proportion of volatile oil to β-CD is 1:8, the temperature is 40℃, the time is 3 h, then make all of those to concentrate,dry and mix with auxiliary material.2. quality standardThe identification of Radix Angelica sinensis, Ligustrum lucidum Ait, Eclipta prostrata L shows clear spots,good specificity. The result of Oviductus Ranae shows the same peak of 1-methyl hydantoin and the sample, this method is simple,accurate. The content of specnuezhenide is tested by HPLC,and less than 25 mg per bag. Method validation results showed that:The method is accurate, stable, reproducible, and can effectively separate each component, also has a special properties.Results of stability display that each indexes are good and the content of specnuezhenide keep stable mainly during three months.Granules routine examination results:Angle of repose less than 30°, bulk density is 0.49, excellent mobility; moisture was less than 6%,other indexes such as particlessize,arsenic salt,dissolubility,content uniformity etc are all qualified.3. primary pharmacodynamicsThe immunosuppressive female Kunming murine model is established by intraperitoneally injecting with cyclophamide(Cy),then some different kinds of drugs are treated them by intragastric administration 15 days; finally,observing those factors as followings:carbon clearance, the weight of immune organs,the proliferation activities of splenic lymphocyte,the level of serum hemolysin and the activities of natural killer (NK) cells. Compare with original process, different dosages of YNGN proved better immunity adjustment function, and the increased index of different dosages YNGN have a significant difference(P<0.05) compared with the model group, but it is lower than the positive groups and has no significant difference(P> 0.05) compared with the positive group.Conclusion:Specnuezhenide measured by HPLC can be more effectively extracted by the optimized process and pharmacodynamic tests further validate the preferred process, the method of anaysis is accurate,stabilized,good reproducibility and specificity,the identifition of TLC is also. Primary pharmacodynamics show that:the immunosuppressive murine model is established successfully,the immunological indicators contain that:the weight of immune organs,the proliferation activities of splenic lymphocyte,the level of serum hemolysin, carbon clearance and the activities of natural killer (NK) cells, the results of these indicate that optimized method is better than initial program, and own well regulation in the specific immune or non-specific immunity. |
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