| Objective In order to solve the problem of indefinite material basis,unclear mechanism and dissociation of quality control index with effects in Jasminum elongatum,multiple methods and technologies in both pharmaceutical analysis and pharmacology were used to study the material basis,pathology and targets for its therapeutics under the guidance of traditional Chinese medicine theory.In addition,a comprehensive set of technology for separation and analysis technology was introduced for separation and purification of compounds found with anti-diarrhea effect in the active part of Jasminum elongatum.As a result,the obtained monomers were subsequently screened by their own effectiveness,and quality control standard were established using the selected active compounds.At the same time,the absorption,distribution,metabolism,excretion process in body of the active compounds would be investigated by pharmacokinetic research.In general,the proposed effect-guided evaluation method for Jasminum elongatum was critical for systematic investigation of its mechanism and material basis,which guaranteed Jasminum elongatum's further resource development and clinical use.Methods 1.Extraction of Jasminum elongatum effective parts The herb was first extracted by soaking using methanol,then,the dried extract was re-dissolved by water.The obtained solution was extracted by a series of solvents n with different polarity such as petroleum ether,chloroform,ethyl acetate,n-butanol for initial separation.2.Pharmacodynamics research on the parts extracted form Jasminum elongatum(1)Rhubarb was taken by mice to construct diarrhea model,and the loose stools rate within 6 hours was chosen as index to observe the anti-diarrheaeffect of Jasminum elongatum extracted parts.(2)Neostigmine-induced bowel movement hyperfunction mice model with advance at the end of the small intestine length and carbon length of small intestine propulsion rate as index was used to observe the influence of Jasminum elongatum extracted parts on small intestine propulsion hyperfunction caused.(3)The analgesic effect of Jasminum elongatum extracted parts were tested by using of ache model caused by acetic acid in mice,with body torsion frequency in mice as evaluation indicator.(4)The anti-inflammatory effects of Jasminum elongatum extracted parts were tested using the auricle swelling method induced by xylene and heightened capillary permeability caused by acetic acid in mice,and evaluation carried out by measuring the mice auricle swelling degree and celiac lotion OD value.3.The research of anti-diarrhea mechanism on effective parts(1)Four target proteins associated with diarrhea and inflammation,namely BDNF,p38 MAPK,NF-?B p65,p-p38 MAPK were selected as measurement indexes,taking the ileum tissue in mice from rhubarb caused diarrhea experiment groups as sample,the amount of target protein expression in was determined using Western Blot method.(2)According to the results of the target protein expression determination,we used LPS stimulated RAW264.7 inflammatory cell model to screen the anti-inflammatory activity of methanol extracts,petroleum ether,chloroform,ethyl acetate,n-butanol extracts of Jasminum elongatum.First,MTT method was used to detect the influence on RAW264.7 cell growth with different concentration extract parts,in order to determine the safe concentration of extractions.Then luciferase reporter gene method was used to measure the fluorescence value in NF-?B +/+ RAW264.7 cell which containing p-NF-?B-luc plasmid after joining extractions.At the same time,detection of the release quantity of NO in supernatant of wild type RAW264.7(WT)cells was carried out by Griess method,RT-q PCR method to measure i NOS m RNA expression level in the cell.4.Separation and Purification of chemical components in effective parts To obtain active component,the effective parts of Jasminum elongatum was separated and purified by systematic and comprehensive method,which includedusing chromatographic column such as MCI Gel,Diaion HP-20,Sephadex LH-20,silica Gel with various solid phase,analytical methods and preparative HPLC,the obtained compounds was injected to ESI-MS,1 H NMR,13 C NMR instrument to investigate their chemical structures for identification.5.Screening active ingredients of anti-inflammatory activity Using LPS induced RAW 264.7 inflammatory cells model for examination of the impact on the generation of NO and the NF-?B transcriptional activity of component separated from Jasminum elongatum effective extraction parts.MTT method was used to detect the influence on RAW264.7 cell growth with different concentration chemical compounds.The release quantity of NO in the cell supernatant and fluorescence value in LPS induced NF-?B+/+ RAW 264.7 cell were detected by Griess and luciferase reporter gene method under the non-toxic concentration of compounds.6.Research of pharmacokinetic With two main active components,methylcaffeate and isochlorogenic acid B as the index,ultra high performance liquid chromatography(UPLC)method was adopted to establish UPLC fingerprint and content determination method of active ingredients for Jasminum elongatum,as well as method validation.7.Research of quality evaluation The concentration of isochlorogenic acid B and methyl caffeate in plasma samples were determined by ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS/MS),and the pharmacokinetic parameters were obtained by the software of BAPP2.0,then the degree to which the compounds exploitation by the body were clarified.Results 1.Total methanol extract and parts with different polarity like petroleum ether,chloroform,ethyl acetate,n-butanol were obtained from Jasminum elongatum.2.The experimental results of rhubarb cause diarrhea in mice showed that chloroform and ethyl acetate extraction from Jasminum elongatum can significantly decrease the mice stool rate,which had certain anti-diarrhea effect.The influence on Neostigmine-induced bowel movement hyperfunction in mice by showed that chloroform and ethyl acetate extract of high and low dose group,petroleum ether extract parts of the high dose group could significantly reduce the small intestine propulsion rate in intestinal propulsionhyperfunction mice caused by neostigmine.Results of analgesic experiments showed that ethyl acetate and n-butanoln part showed significantly reduction in the number of mice body torsion.Anti-inflammatory experiments showed that chloroform,ethyl acetate,n-butanol extract and total methanol extractions could reduce the mice auricle swelling caused by xylene,the results indicated a certain inhibitory effect on acute inflammatory swelling;also,the capillary permeability in mice induced by acetic acid can be obviously inhibited by Petroleum ether,chloroform and n-butanol extraction parts in high doses,which showed good anti-inflammatory activity.3.The chloroform extraction parts of Jasminum elongatum significantly reduced the BDNF,p38 MAPK,p-p38 MAPK,NF-?B p65,expression in the mice ileum tissue from rhubarb cause diarrhea experiment groups.4.The extractive parts of Jasminum elongatum have no obvious effect on the growth of RAW264.7 cells on the concentration below 100?g/m L,and the survival rate is above 85%.Chloroform extraction part within the scope of non-toxic concentration could increase the inhibition rate of LPS induced RAW264.7 macrophages NF-?B transcription activity and NO generation,along whti reduction of NO content and the expression quantity of i NOS m RNA levels in the cell.5.From the effective parts of Jasminum elongatum,17 compounds were isolated and identified as protocatechuic acid,protocatechuic acid methyl ester,caffeic acid,methyl caffeate,salicylic acid,isovanillic acid,ferulic acid,caffeine,3,6-diisopropylpiperazin-2,5-dione,scopoletin,4-hydroxy-3,5-dimethoxy benzoic acid,4-hydroxy-4-(3-hydroxy-1-butenyl)-3,5,5-trimethyl-2-Cyclohexen-1-one,3-indole carboxaldehyde,isochlorogenic acid B,isochlorogenic acid A,3,4-di-O-caffeoylquinic acid methyl ester,3,5-di-O-caffeoylquinic acid methyl ester.6.Activity of NF-?B and the generation of NO can be inhibited by methyl caffeate,when the concentration was set at 12.5,25,50?M,the inhibition rates of NF-?B activity was 34.65%,33.44% and 36.12%,,the inhibition rates of NO generated were 39.28%,45.48% and 47.89%,respectively,with no obvious effect on the growth of RAW264.7 cells.7.The thesis established an UPLC fingerprint of Jasminum elongatum,9 batches of Jasminum elongatum among the samples were found higher than 0.9 insimilarities of correlation coefficients.The components of isochlorogenic acid B and methyl caffeate had a good linearity relationship between 9.67~38.35?g/m L and 9.60~76.8?g/m L.The average recoveries were 98.61%(RSD=0.84)and 99.09%(RSD=1.25),respectively.8.After successful establishment validation of a UPLC-MS/MS method,contents the target compounds were accurately determined and pharmacokinetic parameters were calculated according to pharmacokinetic research.The maximum concentration(Cmax)was 262.03ng/m L for isochlorogenic acid B and 575.35ng/m L for Methyl caffeate;the time reach maximum concentration(Tmax)were 0.25 h,0.50h;elimination half-life(T1/2)were 0.75 h,1.32h;and AUC0~? were 290.12 hng/m L and 879.85 hng/m L respectively.Conclusion The results of in vivo pharmacodynamics experiment indicated that chloroform and ethyl acetate parts of Jasminum elongatum had obvious effect on anti-diarrhea,and certain anti-inflammatory effect as well.Ethyl acetate and n-butanol parts showed obvious analgesia effect.Cells in vitro experiments combined with in vivo pharmacodynamics experiment further verified that chloroform extractive part of Jasminum elongatum is the most important effective parts for anti-diarrhea and anti-inflammatory,followed by the ethyl acetate extractive part.The effective parts play a role in anti-diarrhea by regulating the NF-?B pathway of inflammation,restraining the transcription factors activation of NF-?B,inhibiting some proinflammatory factors' expression such as i NOS at the transcription level,then reducinig the release of NO which reduce inflammation reaction.The activity of chemical compounds isolated from effective parts of Jasminum elongatum were screened by in vitro inflammatory cell model,the results indicated that Methyl caffeate has best activity of anti-inflammatory,followed by isochlorogenic acid B,which can be regarded as the main chemical constituents of anti-inflammatory and anti-diarrhea effect in Jasminum elongatum.As for its mechanism,the experiments found related suppression to the transcriptional activity of NF-?B and reduction the generation of NO.The methods for fingerprint establishment and quantity determination based on the active ingredients of Jasminum elongatum are simple,accurate and reproducible,and they can effectively control the quality of Jasminum elongatum,ensure the effectiveness of clinical application.Thepharmacokinetic research showed that isochlorogenic acid B and methyl caffeate could be rapidly absorbed and utilized by the body in rats and then eliminated.This study clarify the anti-diarrhea mechanism and the active chemical constituents of Jasminum elongatum,and provide a scientific proof for its further development and exploitation. |
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