Studies On Anti-osteoporosis Activity Of Tanshinone ?A And Its Extraction And Separation Technology

Objective:To study the effect and mechanism of tanshinone ?A on peak bone mass and bone mass of ovariectomized rats in growing period,to lay a foundation for the development of new anti-osteoporosis Chinese medicine monomer,and to investigate the extraction and separation process of tanshinone ?A suitable for industrial production.Method: Study on the effect of Tanshinone ?A on peak bone mass in growing rats,using the30 female Wistar rats one month year old,randomly divided into Tanshinone ?A group,Icariin Group and normal control group,10 mices in each group.Stuay on effect of Tanshinone ?A on bone mineral density and bone mass in ovariectomized rats,using 40 wistar female rats three months year old,randomly divided into Sham group,OVX group,estrogen treatment group and tanshinone ?A treatment group,10 mices in each group.Rats were weighed once two weeks in the two animal experiments,use DEXA to detect the overall bone mineral density of rats monthly,if the overall bone density between groups is statistically significant,to kill all experimental animals and to determine the vertebrae and femur in vitro bone mineral density and biomechanical parameters,and some indexes such as bone biomechanical analysis,VG staining of undecalcified proximal tibia,bone morphometry and Micro-CT scanning,and the levels of serum bone resorption index TRACP 5b and bone formation index OC detected by Elisa method,and to analyze the changes of body weight,pathological changes of main organs,etc.To extract Tanshinone ?A from 5 different habitats by heating reflux extraction,the content of Tanshinone ?A in samples was determined by HPLC,to study the effect of 4factors of solvent concentration,solvent dosage,extraction time and extraction times on the extraction result by the orthogonal design experiment and the content of Tanshinone IIA as the evaluation index,and the optimum extraction process was optimized.Compared with direct precipitation method,water precipitation method and sodium carbonate washing method,the optimum separation technology of tanshinone ?A in industrial production of tanshinone ?A was selected.Result:1.Oral administration of Tanshinone ?A after two months,BMD in the whole body,BMD of vertebrae and femur,trabeculae numbers,trabecular thickness,trabecular area and serum OC levels in the growing rats were significantly higher than those of the control group.The differences were statistically significant(P<0.05),the separation degree of trabecular bone and serum TRACP 5b were significantly lower than those of control group(P<0.05).There was no significant difference between Tanshinone ?A group and Icariin group(P>0.05).And there was no difference in body weight between the administration group and the control group.Pathological changes were not found in the main organs.2.Oral administration of Tanshinone ?A can inhibit bone loss in ovariectomized rats.Compared with OVX group,the BMD of whole body and in vitro,the maximal load offemur and vertebrae of Tanshinone ?A group were significantly increased.The difference was statistically significant(P<0.05).The trabecular bone of rats in the administration group became variable and dense obviously.The results of micro-CT scanning and analysis of bone trabeculae were consistent with those of non-decalcification section,and the body mass of estrogen group was significantly lower than that of OVX group(P<0.01).The endometrium atrophy in OVX group was thinner,the difference between Tanshinone ?A group and Sham group was not significant,and the endometrium thickening obviously in estrogen group.The uterine coefficient of Tanshinone ?A group was slightly higher than that of OVX group,but significantly lower than that of estrogen group(P<0.01).3.The contents of Tanshinone IIA in Salvia miltiorrhiza in Longnan,Hebei,Min County,Inner Mongolia and Sage gansi were 0.1%,0.25%,0.16%,0.1% and 0.23% respectively.The optimum extraction technology of Tanshinone ? A from Salvia sage was selected: 6 times the amount of 95% ethanol,reflux extraction 2 times,1h each time;The optimum separation method of tanshinone ?A in the extract was as follows: recovery of ethanol to small volume from extraction solution,the ochre red powder was obtained,and the crude extract of tanshinone ?A was obtained by recrystallization with 50% ethanol,the content of which reached 51.12%.It is proved that the extraction and separation process are reliable and reproducible,and suitable for industrial production.Conclusion:1.Oral Tanshinone ?A could improve bone mineral density and microstructure of bone tissue,and can increase peak bone mass in growing rats;2.Oral Tanshinone IIA can inhibit bone resorption activity and improve bone formation activity in ovariectomized rats,thereby significantly reducing bone loss and preventing osteoporosis.3.Using the orthogonal design experiment method to optimize the extraction technology of Tanshinone ?A from Salvia sage by heating reflux;The method to improve the content of tanshinone ?A in the extract was selected,and the extraction and separation process was simple and effective.