Study On Preparation Of Total Alkaloids Of Peganum Nigellastrum Bunge And Their Therapeutic Effects On Rheumatoid Arthritis

Objective:In this paper,by optimizing the extraction and separation method of total alkaloids of Artemisia camelaris and its treatment of rheumatoid arthritis,the unclear problem of total alkaloids of Artemisia camelaris will be solved,and the mechanism of action of the treatment of rheumatoid arthritis will be preliminarily discussed,which has certain guiding significance for the clinical application of this drug.Method:Through the percolation extraction method,alkali-soluble acid precipitation method and organic solvent extraction method were compared to determine the extraction method of total alkaloids of Artemisia argyi.On this basis,the extraction rate of total alkaloids of Artemisia argyi was taken as the objective.Single factor experiment and response surface method were used to optimize the extraction process with powder size,soaking time,concentration of hydrochloric acid and dosage of hydrochloric acid as the factors.On this basis,by determining the two-phase solvent system based on the reference reports and measuring the distribution coefficient K,HPCCC separation method of luotonin B,harmaline and3-phenylquinoline was established.In addition,by establishing the SD rat model of adjuvant arthritis,observing the degree of joint and foot swelling before and after the model,the serum levels of LCK,Fyn PTKs(Fyn),Zap70,CD45,and the pathological changes of ankle synovium between each group,the effect of total alkaloids of Artemisopsis camelaris on the treatment of rheumatoid arthritis rat model was preliminarily investigated.Results:The average extraction rate of total alkaloids was 21.048 ± 0.145%,which was the highest among the three extraction methods.The main influencing factors such as powder size,soaking time,hydrochloric acid concentration and dosage were investigated in the single factor experiment.When each factor is a variable,the other factors are invariants.Results The optimum conditions for powder size,soaking time,hydrochloric acid concentration and dosage were 60 mesh,48 h,5 % and 50 m L,respectively.On the basis of single factor experiment,the extraction process of total alkaloids from A.camelis was optimized by acid-soluble alkali precipitation method combined with response surface Box-Behnken analysis method.The optimal extraction process was determined as follows: powder size 60 mesh,soaking time 49 h,hydrochloric acid concentration 7 %,hydrochloric acid dosage 40 m L,and the yield was 31.913%.A two-phase solvent system of n-hexane,ethyl acetate,methanol-water 7:3:3:5(v/v/v/v/V)was established and HPCCC separation method for luotonin B,harmaline and 3-phenylquinoline was developed to obtain a yellowish powder solid with three absorption peaks.HPLC analysis of three absorption peaks of light yellow powder solid confirmed that the purity of 98.6 %,99.1 % and 99.6 %,respectively.Its structural formulae were determined by NMR data for luotonin B(1),harmaline(2)and 3-phenylquinoline(3),respectively.After the establishment of the rat model of adjuvant arthritis,the swelling degree of Artemisia argyi low-dose total alkaloids group decreased significantly after continuous administration for 28 days(P< 0.05).Serum LCK,Fyn PTKs(Fyn),z AP70 and CD45 contents of total alkaloids in low,medium and high dose groups were decreased in different degrees compared with model group.In low,medium and high dose groups,inflammatory cell infiltration was rare in synovium,and the changes of fibrous tissue hyperplasia were slight.The changes of inflammatory cell infiltration and fibroplasia in low and medium dose groups were the most slight,almost close to the normal group.Conclusion : By comparing different extraction methods,single factor experiment and response surface method,the extraction process of total alkaloids of A.camphoris was optimized.The optimal extraction process was determined as follows: powder size 60 mesh,soaking time 49 h,concentration of hydrochloric acid7 %,dosage of hydrochloric acid 40 m L.These alkaloids,such as luotonin B,harmaline and 3-phenylquinoline,were isolated by HPCCC,and their formulae were determined by NMR.In addition,according to the experimental results of the treatment of adjuvant arthritis rat model by total alkaloids of A.camelaris,total alkaloids of A.camelaris can improve the degree of joint swelling,serum LCK,Fyn PTKs(Fyn),z AP70,CD45 and the pathological changes of ankle synovium in rats to a certain extent.