Pharmaceutical, Pharmacological And Toxicological Research Of Chinese Formula Li AnKang On Treating The Perimenopausal Osteoporosis

ObjectLi An Kang Formula (LAK), which is based on Danggui Buxue Tang (DBT), is testified to be active as estrogen-like effect and stimulating the osteoblast in the MCF-7 cell line and MG-63 cell line respectively. Therefore, the biological result provides the evident for curing the Perimenopausal Osteoporosis (POP) in vitro. However, its effective component is not under control and lack of monitor, neither is its experiment in vivo. Therefore, the research aims to offer the macro evidence for the effect and reveal the mechanism in vivo, which is carried on further through pharmaceutical, pharmacological and toxicological aspects.Method and Result1. Research on Extraction Process of LAKAccording to the biogical result and the feature of the medical material, the optimization of the extraction process is implemented by orthogonal test, seting 4 factors and 3 levels each factor and selecting the relative effective components as index. Specifically, in term of the aqueous extracting of RA and RAS, the AstragalosideⅣand total solids are choosen as index, and amount of water, hour of extraction, times of extraction and drying temperature are considered as 4 factors. As the result shows, Radix Angelicae Sinensis (RAS) and Radix Astragalus (RA) should be extracted with 8 times water for 1.5 hour each time, and repeat for 3 times. Afterward, the extract is filtered and dried under 70℃. In term of extraction process of Aceranthus sagittatus (AS), the icariin and total solids are choosen as index, and ethanol concentration, amount of ethanol, hour of extraction and times of extraction are considered as 4 factors. As the result shows, AS should be extracted with 14 times 70% enthanol for 1 hour, and repeat for 2 times.After the optimization of extraction process is completed, the tablet-form research of LAK is the next step to guarantee the effect. Due to the existence of abundance polysaccharide in the extract, the tablet is prone to absorb the moisture and be sticky. Therefore, disintegration time, percentage of absorbing the moisture, angle of repose and critical relative moisture are tested respectively to improve the quality. As the result demonstrates, the dried extract should be mixed with some avicel and CMC-Na, and be added with 4.2% crospovidone (as disintegrant).Then, the mixture is dried under 55℃and become particle. After that,the particle is added with 2.3% avicel (as lubricant) and tabletting and coating. Additionally, the tablet's disintegration time is limited within 26 minutes and the critical relative moisture is 64%.2. Research on Determination of Effective Component in LAKChemical derivative is applied to determine the AstragalosideⅣin LAK, and the AstragalosideⅣis benzoylated by using the pyridine and benzoyl chloride as derivatization reagent. After the derivatization, the AstragalosideⅣis determined by HPLC, which is separated through the Phenomenex Gemini C18 (50 mm×4.6 mm,5μm) column by using the Acetonitrile-0.1% triethylamine solution for 30 minutes and detected under 230 nm. Compared with HPLC-ELSD, the chemical derivative method has advantage in procedure, specificity, accuracy, sensitivity and repeatability. Other two components in LAK, including ferulic acid and icariin, are simultaneously determined by HPLC, and they are separated through Phenomenex Luna C18 column (250 mm X 4.6 mm,5μm) by using Acetonitrile-0.1% acetic acid as mobile phase, and are detected under 230nm UV. The mothod is superior in the accuracy, celerity, sensitivity and repeatability. As the results demonstrate, there are 0.38mg of AstragalosideⅣ,2.02mg of icariin and 0.017mg of ferulic acid per tablet.3. Research on Pharmacology of LAKThe pharmacological research is carried out in vivo, and index, including bone situation, is analyzed with camparison between spontaneous-aging-mouse model and emasculated-mouse model, secretions in peripheral blood, hypothalamic neurotransmitters, lymphocyte subsets and relative organ. As the results show, in term of bone, LAK eases osteoporosis and the deterioration of trabecula; in term of secrections, LAK mediates theβ-EP, E2, SOD and MDA in both aniamal model, but it only dramatically decreases the ratio of FSH/LH in the spontaneous-aging model; in term of hypothalamic neurotransmitters, LAK increases the concentration of NE and 5-HT in the the spontaneous-aging model and decreases the concentration of NE, DA and 5-HT in the emasculated-mouse model; in term of lymphocyte subsets, LAK eases the decline of CD4/CD8 in the the spontaneous-aging model, and it indicates that LAK enhance the immunity in this animal model.In term of relative organ, LAK improves the situation of endometrium and vagina mucous membrane in both two models.4. Research on Assistant Pharmacology of LAKPerimenopausal women often suffer from the deterioration of immunity and memory and hectic fever, which weakens the quality of life and the therapeutic effect. Therefore, the immunity-inhabited model, memory-inhabited model and pilocarpine-induced sweating model are set to confirm the assistant effect. As result demonstrates, Firstly, LAK improves the humoral immune function, specific immune response, macrophage phagocytic function and thymus index and splenic index which are inhabited by prednisone acetate. Secondly, LAK improves the memory in the forming-depressing, consolidate-depressing, and reappear-depressing models. Lastly, LAK eases pilocarpine-induced sweating.5. Research on Toxicology of LAKDue to the therapeutic effect of LAK, the toxicology focuses on relative organ including ovary and uterus. As the result shows, in all administration groups and control group, all the animal survive and behave normally; compared with control group, other groups do not show difference in food-intake, water comsuption, weight increase and excrement and urine. Additionally, there is no difference on uterus and ovary between LAK high-dose group and other groups.ConclusionThe result above shows, LAK is perfect in the preparation technology, and all the process parameters indicates that transport rate of the index in the extraction and form process meets the requirement of the experiment and all the factors are clear and controllable in rule and quantification. The test method of the index is accurate and repeatable, consequently it can guarantee the quality of the product, which means reserve the effective component in the biological experiment and lay the foundation for completing its pharmacological research. Then, the pharmacological result shows that LAK has the experiment evidence in curing POP and is verified to work as estrongen-like effect and stimulation of bone growth in vivo. Integrated analysis of result in vivo and in vitro, LAK owns two different ways in curing POP:Firstly, LAK works directly in bone cell and ease the decrease of bone mineral density (BMD). Secondly, LAK improves the ovarian structure and function and consequently enhances the secretion of estrogen, then improve circumstance which causes POP through hypothalamic-pituitary-ovarian axis (HPO). Lastly, the toxicological results shows that LAK is safe to the relative. In sum, the research comprise optimization of extraction process, determination of index component, pharmacological and toxicological research, and demonstrates the superiority of Chinese Medicine in curing the primary disease and reveal the pharmacological mechanism, then set up the creative model for researching and applying LAK.