| The gynecological illness,which was about irregularly bled by abortion and the inflammation relate to the infection,frightened the women's healthy seriously at recently.Till now,about one hundred million women need the fast gynecologic hemostatic and antiphlogistic medicine.However,unfortunately,no such gynecological medicine has been found now.Therefore,the clinic calls an efficient,low toxicity,high selectivity of novel hemostatic blood,removing blood stasis and anti-fungal drugs urgently.Blumeari Paria(BL.)DC,a Chinese medicinal herb,also as the main ingredient of the medicine Fuxuekang in Guangxi,playing an important role in gynecological diseases.Till now most of the researches about the Blumeari Paria(BL.)DC.are focus on the separation and the verification,few researches are focus on the blood activities about the compounds extract from the herb.Our preliminary research shows that the phenolic acids are some of the active components of Blumeari Paria(BL.)DC,which have certain hemostatic physiological,but the activities not good enough.P-hydroxycinnamic acid,is one of the component in Blumeari Paria(BL.)DC,has good hemostatic physiological,and also can excited the mice womb in vitro.This thesis based on the result of the former research,selecting p-hydroxycinnamic acid and the similar compounds as origination materials,using the simple aromatic amine,fatty amine and also sulfanilamides as the synthon,synthetizing several series amines compounds,the detail as following:(1)Using p-hydroxycinnamic acid,m-hydroxycinnamic acid,p-methoxy cinnamic acid,m-methoxy cinnamic acid,caffeic acid and chlorogenic acid as raw material,enlightening by the prior hemostasis drug structure characteristics,we selected the simple aromatic amines,fatty amines and sulfa drug as the amine part of the raw material,70 new compounds have been synthetized,these compounds identified by IR,1H-NMR,13C-NMR,MS and X-Ray.(2)Through the blood activity screening by four coagulation indexes on the compounds,compounds 1,2,3,4,5,6,27,28,31,32,41,42 and 43 showed certain procoagulant activity in a test of concentration range(0.0625,0.125 and 0.25 mg/mL),but the strength is not significant;35 and 36 have significant anticoagulant activity.Compounds 33,34,54,55,56,57 can be procoagulant at low concentrations,and show anticoagulant activity in high concentration.Compounds 35,57,54,58,55,56,49,60,68,36,48,39,62 and 63 in 0.125mg/mL concentration can be significantly extend the TT,compared with blank group increased by 120%,123%,44.9%,50.67%,36.03%,33.33%,19.08%,19.46%,15.15%,15.99%,separately,compare with the positive control of compound 70(Yunnan Baiyao,extension 14.77%)extended a longer time,implying that these compounds have strong inhibition on thrombin and fibrin polymerization.At low concentrations,the compounds can affect on FIB at some extent.(3)The compounds with better blood physiological active were selected for plasma recalcification test,the test result showed that,for cinnamic acid compounds,35 and 28,in the tested concentration range mainly showed anticoagulant activity,compounds 31 and 58 exhibited procoagulant activity only,compound 57 showed that at low concentrations present procoagulant,and at high concentration was anticoagulant.The rest of the compounds in different concentration range showed different activity.Compounds 1,2,4,5,6 and 43 mainly showed of procoagulant activity,3,35,27,28,33,41,42,54,55 and 59 exhibit anticoagulant activity.Compounds of caffeic acid series,showing a good two-way activity,i.e.,at low concentrations showed procoagulant,at high concentrations showed anticoagulant,the compound of chlorogenic acid benzene toluene amide(68),when at a high concentration,showing good anticoagulant activity,the anticoagulation inhibition rate can reach to 73%.(4)After screened by blood activity experiment,the compounds with good activity were selected for the antibacterial experiment,and the bacteria contain Salmonella bacteria,Candida albicans,pneumococcal and golden glucose coccus.The experimental results show that,36 and 37 can inhibit to four kinds of bacteria,compound 37 can inhibit to Candida albicans,golden glucose coccus and pneumococcal,the minimum inhibitory concentrations was up to 0.078mg/mL,on the Salmonella,the minimum inhibitory concentration is 0.156 mg/mL.The MIC of compound 36 for these four bacteria are range from 0.3125mg/mL?1.25 mg/mL.Compound 57 inhibit the Salmonella,Candida albicans,golden glucose coccus,the MICs are 2.5 mg/mL,to the pneumococcal,the MIC was higher than 10 mg/mL.compound 32,has a of very good inhibitory effect on Candida albicans and golden glucose,the MIC were 0.3125 and 2.5 mg/mL.(5)In order to understand how the compounds effect the organization in the process of hemostasis,we did the experiment of mice uterus in vitro,which were screened by the former steps in the process of hemostasis on tissue factor(the tension and of frequency).The study found that the compound 37and 39,at a concentration of 2.49x 10-5mol/mL and 4.98 x10-smol/mL,separately,the frequency can be reduced greatly(p<0.05),which implying the anticoagulant activity.Compounds 1 and 4 can increased the frequency of mice uterus in vitro and shown the procoagulant activity,but the strength is not significant.Compound 36,when adding at a concentration of 19.61x 10-5mol/L,can reduced intensity of the tension of the mice uterus in vitro(p<0.01).For compounds 4,35 and 57,when at low concentrations,can increased the average tension slightly,other compounds at a different concentration,all can make the tension reduced,which implying the compounds had anticoagulant effect.(6)In order to a better understanding of the binding and transport situation in vivo for the good physiological activity and antibacterial activity compounds,compounds 1(procoagulant and antibacterial),35(anticoagulant),36(anticoagulation and inhibition)and 57(bidirectional regulation and antibacterial)were selected to interact with HSA and investigated by fluorescence spectrometry.The Stern-Volmer quenching constants and corresponding thermodynamic parameters were calculated.The result shown that the binding model was static quenching,the binding sites for these four compounds with HSA all were 1,at the same time.The distances between the donor(HSA)and the acceptors(these four compounds)were obtained according to fluorescence resonance energy transfer,all were less than 8nm.The primary forces stabilizing the compound-HSA complexes were mainly hydrogen bonds,hydrophobic interactions and van der Waals force.Synchronous fluorescence spectra shown that,the addition of compound,both effected on the tryptophan and tyrosine residues. |
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