Study On The Material Foundation Of Vinegar-baked Curcuma Kwangsiensis On Promoting Blood Circulation And Removing Blood Stasis

Objective:The Chinese medicine curcuma is the dry rootstocks of Curcuma phaeocaulisValeton,Curcuma kwangsiensisS.G.Lee et C.F.Liang,CurcumawenyujinY.H.Chen et C.Ling,and has the effect of removing blood stasis and eliminating accumulation of pain,often used for the treatment of amenorrhea congestion,dyspeptic pain and other symptoms.Modern research showed that it has obvious anti-tumor and anti-angiogensis effect,and is a commonly used medicine for promoting blood circulation and removing blood stasis.Although three kinds of curcuma had similar effects,the components in it were different.The relationship between active components and pharmacological effecs of curcuma kwangsiensis remains unlear.Therefore,in this paper,effective parts of the blood circulation in vinegar-baked curcuma kwangsiensis（VBCK）were studied first and then the components in it were isolated.Thereafter effects of isolated component on growth of Human Umbilical Vein Endothelial Cell（HUVEC）and A549 cell were investigated.The study will provide experimental basis for its further development and utilization.Methods:1.Optimal dose screen of VBCK on promoting blood circulation and removing blood stasis.40 SD rats were divided into control,model,VBCK high dose(3.72 g·kg^-1),medium dose(1.86 g·kg^-1),low dose(0.93 g·kg^-1)group.Besieds control group,rats in other groups had ice water swimming daily stimulation and bounded stimulation for 15 days to establish blood stasis model.After that,VBCK with different doses were continuous administration to treated groups for three days respectively.The coagulation time,blood rheology index were measured on the fourth days to investigate the effective dose.2.Active parts screen of vinegar-baked curcuma.50 SD rats were divided into control group,model group,ethyl acetate group,alcohol group,water extracted group.After establish the blood stasis model,different parts VBCK were given to the corresponding rats for 3days.After that,the coagulation time,blood rheology,prothrombin time,partial thromboplastin time and thrombin time were determined to evaluate its effects.3.Isolation and identification of the components in active parts of VBCK.Sequentially partitioned the active extract by silica gel column with cyclohexane: ethyl acetate（15: 1）,cyclohexane: ethyl acetate（10: 1）,cyclohexane: ethyl acetate（10: 3）,cyclohexane: ethyl acetate（2: 1）,dichloromethane: methanol（15: 1）,dichloromethane: methanol（1: 1）.The structure of the isolated compounds was identified by UV,ESI-MS,¹H-NMR,¹³C-NMR and DEPT.4.Activity of the isolated compounds on HUVEC and A549.The isolated compounds with different concentration were given to HUVEC and A549 cell respectively,and the cell viability was detected by MTT method in 24 h,48 h to investigate the anti angiogenesis and anti-tumor activity.Results:1.The water decoction of VBCK dose dependently shortened the coagulation time,decreasing the viscosity of plasma,and whole blood,as well as the shear index of whole blood,suggested that it has anticoagulant effect.All parts of VBCK showed anti-coagulation effect,and the effect of ethyl acetate extraction was the strongest.2.Seven compounds were isolated and purified from the ethyl acetate extraction.Among them,the zederone（1）,germacrone（2）,cuezerene（5）,furanodienone（6）were sesquiterpenes,1-（3,4-dihydroxy-phenyl）-7-（4-hydroxyphenyl）-（6E）-6-hepten-3-ol（3）,（3R,5R）-3,5-di-acetoxy-1-（3,4-dihydroxyphenyl）-7-（4-hydroxyphenyl）heptane（4）,1,7-bis（4-hydr oxyphenyl）hepta-4E-6E-dien-3-one（7）were diphenylheptane class.3.Compounds 3,4,6,7 inhibited the growth of HUVEC,and compound 3,5,6,7 inhibited the growth of A549,shown the effect of anti-angiogenesis and anti-cancer effect.Among them,compound 7showed stronger effect than that of curcumin.Conclusions:The ethyl acetate extraction of VBCK has a good effect on promoting blood circulation and removing blood stasis,compound 7 had the effect of anti-angiogenesis and anti-cancer,may be the active components of VBCK.