Study On Material Basic Of ’Benglouting’ And Molecular Cloning And Effect Evaluation Of A Bioactive Peptide From The Red-eyed Leaf Frog

Metrorrhagia,also known as functional uterine bleeding,has a total incidence of about 20%to 22%in women.Clinical manifestations include loss of normal menstrual cycles,excessive menstruation,prolonged periods,and irregular vaginal bleeding.The disease has severe effects on patients’ daily lives.Benglouting is a clinical experience.It has shown good curative effects in many years of use,and the small amount of medication makes patients better compliance.But its effective ingredients are not clear,which hinders its further development and application.Therefore,this thesis aims to study its material foundation for the treatment of functional uterine bleeding.The thesis is divided into five chapters.The first chapter systematically introduce the Functional uterine bleeding,the chemical components of the constituent herbs in Benglouting（Typhae Pollen,Trogopterori Faeces,Notoginseng Radix et Rhizoma,Angelicae Sinensis Radix,Cyperi Rhizoma）,and the research progress on the material basis of traditional Chinese medicine compounds.Chapters 2 to 5 are experimental parts.The specific research contents and corresponding conclusions are as follows:Study on the preparation process of Benglouting intermediates:The composition of Benglouting is Typhae Pollen（Puhuang,PH）,Trogopterori Faeces（Wulingzhi,WLZ）,Notoginseng Radix et Rhizoma（Sanqi,SQ）,Angelicae Sinensis Radix（Danggui,DG）and Cyperi Rhizoma with vinegar（Cuxiangfu,CXF）.It is necessary to prepare intermediates to provide test products for subsequent experiments,so this section first examines the process route of intermediate preparation.First of all,the relative extraction rates of index components in each medicinal material,mouse coagulation and bleeding test results are used as indicators for the inspection of three process routes（Route one:total water extraction and alcohol precipitation;Route two:alcohol extraction of DG,CXF and SQ,water extraction of the combination of drug extraction and PH and WLZ,and then alcohol precipitation;Route three:alcohol extraction of PH,DG,CXF and SQ,water extraction of the combination of drug extraction and WLZ,and then alcohol precipitation）.The inspection results showed that route three is the best.After the general process route was determined,the number of times of alcohol reflux extraction and water extraction was optimized.The results showed it was more reasonable that PH,SQ,DG and CXF were extracted by alcohol once,and the combination of dregs and WLZ was extracted by water twice.After the superior process route has been screened,and the process conditions were optimized then,including different alcohol concentrations,alcohol addition amounts,alcohol extraction time,water extraction time,water volumes,the concentration of alcohol solution for alcohol precipitation,alcohol concentration and alcohol precipitation time.Finally,the optimized intermediate preparation process is to take PH,DG5 CXF and SQ,add 9 times of 60%ethanol,heat and reflux for 1.5 hours,filter,recover the ethanol from the filtrate,and concentrate under reduced pressure to obtain the paste one;combine dregs with WLZ,add 8+6 times of water,extract for 2+1 hours,filter,concentrate to the paste with a drug concentration of 3 g/ml add ethanol to the alcohol content to 60%,and let stand for 12 hours,filter,recovere ethanol,and concentrate under reduced pressure to obtain the paste two;combine the past one and paste two,mix well,and concentrate under reduced pressure to obtain an intermediate.Research on Chemical Composition of Benglouting:The research on the material basis of the traditional Chinese medicine compound is based on a foll understanding of its chemical composition.Therefore,in this part,the combination of fingerprints and serum medicinal chemistry was used to find the intersection of the chemical constituents represented by the characteristic peaks and the blood-introducing constituents,thereby narrowing the basic range of pharmacodynamic substances.（1）Study of the fringerprint of Benglouting:Due to the complex chemical composition of the compound,HPLC-UV-ELSD was used to establish the fingerprint of Benglouting.The conditions such as elution conditions,mobile phase,UV detection wavelength,evaporative light scattering detector drift tube temperature,and atomization volume flow were optimized.The final chromatographic conditions were Eclipse XDB-C18 column（4.6mm×4.6mm 5μm）,mobile phase acetonitrile（B）-0.1%formic acid（C）,gradient elution conditions,0～10 min:5%B)～10%（B）,10～12 min:10%（B）～13%（B）,12～16 min:13%（B）～15%（B）,16～27 min:15%（B）～16%（B）,27～37 min:16%（B）～25%（B）,37～52 min:25%（B）～38%（B）,52～57 min:38%（B）～45%（B）,57～69 min:45%（B）～95%（B）,69～77 min:95%（B）～97%（B）,77～80 min:97%（B）～100%（B）.The flow rate was 1 ml·min-1,the UV detection wavelength was 265 nm,the column temperature was 30℃,and the injection volume was 10 μl.The temperature of the evaporative photodetector drift tube was 110℃,the carrier gas flow rate was 2.0 L·min-1,and the recording time was 80 min.The results showed that 27 common peaks were calibrated under the UV detector,and 5 common peaks were ELSD detector.The retention time and peak area RSD in the precision,repeatability,and stability experimental results were all less than 3%,and the similarities of the ten batches of Benglouting test solutions were all greater than 0.9.Then,the spectra of each medicinal material were compared with the Benglouting fingerprint to assign characteristic peaks to medicinal materials.The results showed that under UV detector,8 peaks were from PH,3 peaks were from WLZ,6 peaks were from DG,6 peaks were from CXF,1 peak was from PH and WLZ,2 peaks were from WLZ and CXF,and 1 peak was from WLZ,DG and CXF.Under ELSD detector,4 peaks were from SQ,and 1 peak was from PH and SQ.In addition,by comparison with the chromatogram of the mixed standard solution,it was determined that under UV detector,the chromatographic peaks of No.2,4,13,14,16,and 26 were gallic acid,protoeatechuic acid,typhaneoside,ferulic acid,isorhamnetin-3-O-neohesperidin,α-cyperone,while under ELSD detector,the chromatographic peaks No.1’,2’and 3’were notoginsenoside R1,ginsenoside Rgl,and ginsenoside Rb1.（2）Analysis of transitional components of Benglouting in blood:The composition of traditional Chinese medicine compound is complex,but for oral compound,the ingredients can only exert their effects in the body when they enter the blood.Therefore,in this section,the blood-injection components of Benglouting was studied through serum pharmacological methods.LC-MS was used to analyze the chemical composition of the Benglouting extraction and the drug-containing serum,and the intersection of the characteristic peaks of the fingerprint and the blood components was found.The results showed that a total of 19 components were detected in the Benglouting extraction in negative ion mode and 52 components were detected in positive ion mode,of which 9 components could be detected in both modes.And the retention time was used to determine the 25 characteristic peaks in the fingerprint.On this basis,these components were searched in the LC-MS map of medicated serum,and 17 cross-linked components were found,which were typhaneoside,neohesperidin,hesperidin,quercitrin,naringetol,gallic acid,protocatechuic acid,caffeic acid,ferulic acid,ligustilide,notoginsenoside Rl,ginsenoside Rgl,ginsenoside Re,ginsenoside Rbl,ginsenoside Rd,n-butylidenephthalide,α-cyperone.Therefore,the scope of the medicinal substances of Benglouting was narrowed down to these 17 chemical components.However,the efficacy of each component needed to be proved by subsequent experiments.Study of the Medicinal Effect of Benglouting inn Vitro:In the previous part,we studied the whole formula of Benglouting and its blood-inducing components,and finally identified 17 components that may be effective components.In this part,by administering the deeomposed formula and the monomer eomponents to rat plasma in vitro,the parts and components that have bidirectional effects,hemostatic and activating blood,were initially determined from two aspects.（1）Study on the in vitro pharmacodynamic effect of each part of Benglouting:The whole Benglouting was divided into PH and WLZ part,and SQ,DG and CXF part that were used to administered to rat plasma in vitro respectively.Using thrombin time（TT）,prothrombin time（PT）,and activated partial thromboplastin time（APTT）as indicators,the hemostatic and blood activating effects of the whole prescription and each decomposed prescription in vitro were measured.The results showed that the whole BLT and SQ,DG and CXF part had a significant effect on prolonging the coagulation time,while the PH and WLZ part had a significant effect on shortening the coagulation time.（2）In vitro pharmacodynamic research of various chemical components in Benglouting:The above-mentioned 17 kinds of chemical components were administered to rat plasma in vitro,and PT,TT,and APTT were used as indicators to determine the hemostatic and activating blood effects of each chemical component in vitro.The results showed that flavonoids and their aglycones,most organic acids could shorten the coagulation time,saponins,volatile oils in DG and ferulic acid could extend the coagulation time,while a-cyperone in CXF had no signifieant impact.Network Pharmacology Research of Benglouting:In the last part,17 chemical components were found to play an important role for Benglouting in the process of hemostatic and activating blood through in vitro screening.Therefore,in this part,taking these components as the research object,we used network pharmacology to explore the mechanism of Benglouting treatment for fiunctional uterine bleeding.The analysis results showed that the mechanism may be related to promoting angiogenesis,participating in the estrogen signaling pathway,and interfering with the cell cycle.Natural products,as rich and varied resources for the discovery of novel drags,have been essential for human healthcare systeim throughout history.A variety of bioactive components with unique structures and functions isolated from the skin secretion of amphibians,especially peptides,have enormous potential for medical purposes.Here,a peptide from the phylloseptin femily was identified from the Red-eyed Leaf Frog,Agalychnis callidryas,by molecular cloning.This peptide was named as QUB-1541 based on its molecule weight.Then the mature peptide was successfully synthesised by Tribute? 2_channel peptide synthesiser.Reversed-phase high-performance liquid chromatography（RP-HPLC）was used for purification and matrix-assisted laser desorption ionisation time-of-flight（MALDI-TOF）MS was used to confirm its molecular mass.QUB-1541 has better inhibitory activity against gram-positive bacteria than gram-negative bacteria.The MIC for S.aureus is 8μM while for E,coli is 512μM Also,QUB-1541 inhibits the growth of yeast,C.albicans and the MIC is 128μM pM.The results of MBC show that QUB-1541 has no bactericidal effect on E.coli and C.albicans at the highest concentration.However,QUB-1541 kills S.aureus at a lower level of 16μM,while the haemolysis is weak.QUB-1541 was only effective against cancer cells,PC-3,U251 MG and HI57 at the highest concentration of 10^-4μM.Thus,QUB-1541 has the value of subsequent research.