Study On Senile Osteoporosis Inhibitory Effect Of Bajitian Pill And Its Preparation Technology

Objective:Bajitianwan（BJTW）,first recorded in Gu Jin Yi Tong Da Quan,is a classic Chinese formulation consisting of seven herbal drugs:the root of Morinda officinalis F.C.How.,root and rhizome of Acorus tatarinowii Schott,the root bark of Lycium chinense Mill.,the sclerotium of Poria cocos（Schw.）Wolf,the root of Polygala tenuifolia Willd.,sclerotium with host wood of Poria cocos（Schw.）Wolf and root and rhizome of Panax ginseng C.A.Mey.BJTW is an ancient prescription with the ability of improving memories and the potential of anti-osteoporosis according to our pre-experiment.In this paper,we adopted D-galalactose-induced rats to observe the therapeutic effect of BJTW on age-related osteoporosis.We also adopted D-gal-induced osteoblast to evaluate BJTW’s therapeutic effect and its possible mechanism.On this basis,the preparation technology of Bajitian pill was optimized.Methods:In chapter 1,D-galalactose（D-gal）was injected subcutaneously into male Wistar rats to establish the model of age-related osteoporosis,and the aqueous extract of BJTW（6g/kg/d,12g/kg/d）were given for 16 weeks.Parameters of bone formation and oxidation were measured by kits;OPG,RANKL/OPG,Fox O1 and SOD2 were measured by WB and IHC staining;bone mass and microstructure were evaluated by micro-CT.The effects of water extract of BJTW on senile osteoporosis induced by D-galactose were investigated In chapter 2,osteoblasts were stimulated by 100 m M D-galactose to achieve a senile cell model.The effects on osteoblast’s proliferation,bone formation and antioxidant capacity of BJTW and 5 active ingredients were measured via Flow cytometry.The total protein of cells were extracted.The Nrf2,Akt,HO-1,NQO1 were measured by Western blot and immunofluorescence.In chapter 3,the content of monotropein and scopoletin in Bajitian Pill was determined by HPLC.Based on the results of content determination,an orthogonal test was applied to optimize the preparation process.Results:Chapter 1:BJTW（6g/kg/d,12g/kg/d）can significantly improve bone mass and the related indexes（BV/TV,BS/BV,Tb.Sp,Tb.N,Tb.Th and SMI）of bone microstructure;increased serum BALP,OCN,increase the expression of OPG in femur and decreased the ratio of RANKL/OPG;MDA was decreased with the increase of CAT,SOD in serum at the same time.The expression of Fox O1 and SOD2 was increased.Chapter 2:The osteoblasts were successfully extracted by the 2-time digestion method.D-gal-induced osteoblast model was successfully established at a dosage of 100 m M for 48 h.Cell senescence was proved byβ-Galactosidase staining.At the set concentration,BJTW（L,H）,MON(10^-2,10^-1μM),SCO(10^-2、10^-1μM),Rb1(10^-4,10^-3μM),Rg1(10^-1,1μM)and FER(10^-4,10^-3μM)could promote the proliferation and increase the activity of ALP.The ROS levels of osteoblasts in D-gal model were significantly reduced in HB（L,H）,MON(10^-1μM),SCO(10^-1μM),Rb1(10^-3μM).BJTW（L,H）,HB（L,H）,MON(10^-1μM)can significantly up-regulate the SOD activity;BJTW（L,H）,HB（L,H）,FER(10^-3μM)can significantly up-regulate the CAT activity.BJTW（L,H）and HB（L,H）can activate Nrf2/Akt pathway by increasing Akt phosphorylation,promoting Nrf2 protein into nucleus,and then increasing the expression of downstream antioxidant enzymes HO-1 and NQO1,thus playing an antioxidant role.Chapter 3:A stable and feasible HPLC method was established for the determination of the 2 compounds in Bajitian Pill.The optimized preparation process of Bajitian Pill was as follow:80 mesh herb powder added with 40%aleurone powder to make pills of 6 mm in diameter.The average content of monotropein and scopoletin in 3 batches under this method was 0.348 mg/g,1.430 mg/g,with the RSD 0.524%,0.400%.Conclusion:（1）BJTW can effectively reduce the level of oxidative stress,increase the activity of antioxidant enzymes,increase the bone mass density of the aged osteoporosis rats caused by D-gal.（2）Aqueous extract of BJTW can effectively enhance the proliferation activity and ALP activity of osteoblasts injured by D-gal,improve the activity of antioxidant enzymes,and reduce the level of ROS in the cells.BJTW’s effect is almost equal to the mixtures of 5 ingredients,and is better than that of single active ingredien.The antioxidant and osteogenic effects of BJTW may be related to the activation of Nrf2/Akt pathway in osteoblasts.（3）The preparation process was proved to be practical and stable.