| Object:Taking Tetrastigma hemsleyanum Diels et Gilg as the research object,it’s antiviral pedigree,virus species and inhibitory effect of three different extraction products and their extraction parts were inspected.On this basis,the author characterizes material base by UPLC-Q-Exactive/MS and mass defect filtering(MDF).At the same time,the author systematically separated the antiviral substances of T.hemsleyanum,explored the most valuable antiviral sites(components),and discussed the mechanism of anti-Ebola pseudovirus action of its corresponding parts(components)in vitro,at the last,animal models were used to verify the anti-influenza A(H1N1)virus effect in vivo and to explore the mechanism of action.This paper provides experimental data for the comprehensive discovery of the substance basis of the antiviral effect of T.hemsleyanum,provides reference for further clarifying its mechanism of action,provides ideas for the systematic development of T.hemsleyanum products.Methods:(1)By literature method,the historical evolution,plant primordia,natural distribution and modern research of T.hemsleyanum were reviewed.Access to nearly 150 ancient and modern documents,among them,more than 60 books and 80 papers,this paper provide support for the research on the pharmacodynamic material basis,mechanism of action and clinical development and application of T.hemsleyanum.(2)In this paper,traditional laboratory water extraction,70% ethanol extraction and 70% ethanol cold extraction are used,and petroleum ether,dichloromethane,ethyl acetate and n-butanol are used for extraction respectively.For each extraction part,the cytopathic effect method(CPE method)was used to carry out in vitro antiviral experiments,including influenza virus A(H1N1),herpes simplex virus type 1(HSV-1),enterovirus type 71(EV71),coxsackie virus B3,B5(COX-B3,COX-B5)and respiratory syncytial virus(RSV),to calculate the poison suppression index(TI)by daily observating of cytopathology.(3)Cationic liposome method was used to transfect and package HIV/Ebola and HIV/Lassa pseudoviruses.the pseudoviruses were used to screen 6 different extraction sites of T.hemsleyanum alcohol cold soaking and to track the antiviral effect of 13 fractions in vitro.At the same time,the antiviral effect of drugs at 100μg·m L-1,50μg·m L-1,25μg·m L-1,12.5μg·m L-1 was evaluated by luciferase activity detection method.Cell Titer-Glo? Luminescence Method was used to detect the cell viability to evaluate the toxic effect of the drug to be detected on cells at different concentrations.(4)Under isoflurane respiratory anesthesia,we established a Balb/c mouse model of H1N1 virus nasal drop infection,with weight change and death protection rate as indicators,to verify the antiviral effect of ethanol cold-soaked extract of T.hemsleyanum in vivo,setting the drug concentration to 200mg·kg-1,100 mg·kg-1 and 50 mg·kg-1.After the model is established,daily observating and recording of mice weight and death to 14 days.If the weight of the mouse is reduced by more than 20%,it will be regarded as death,then,calculate the LD50 according to Reed-Muench method.(5)The pneumonia mouse model prepared by influenza A(H1N1)virus was selected as the research object,through MTT,ELISA,real-time fluorescence quantitative PCR technical methods,to investigate the effects of T.hemsleyanum extract on lung index,lung index inhibition rate,spleen T cell proliferation and NK cell activity,concentration of three inflammatory factors such as IL-2/IFN-γ/TNF-α in serum and m RNA expression of three cytokines such as TNF-α in lung tissue of model mice.In-depth research is carried out from different levels of tissues,cells,molecules,genes.To explore the mechanism of antiviral activity of T.hemsleyanum in vivo.(6)Using classical extraction and separation methods,the 70% ethanol room temperature cold-soaked extract from root tuber of T.hemsleyanum was separated.Methods such as macroporous resin gel column chromatography,silica gel column chromatography,Sephadex LH-20,normal(reverse)phase column chromatography,and preparation of high performance liquid chromatography were adopted to separating and purifying all extraction parts and fractions.At the same time,the structure of the obtained substance was identified by nuclear magnetic resonance spectroscopy.Using UPLC-Q-Exactive LC/MS-mass defect filtering MDF-OTCML database retrieval identification strategy,we analysis qualitatively the chemical constituents of T.hemsleyanum,especially flavonoids.The main parameters for MDF analysis are set to: in positive ion mode,flavonoid aglycone takes 5,7,4’-trihydroxyflavone as a template,the mass range is 271-391 amu,the range of mass defect is 28-93 mmu;Flavonoid glycoside uses 5,7,4’-trihydroxyflavone plus monosubstituted hexose as template,the mass range is 432-770 amu,the range of mass defect is 79-180 mmu.In negative ion mode,flavonoid aglycone takes 5,7,4’-trihydroxyflavone as a template,the mass range is 269-389 amu,the range of mass defect is 28-93 mmu;Flavonoid glycoside uses 5,7,4’-trihydroxyflavone plus monosubstituted hexose as template,the mass range is 430-768 amu,the range of mass defect is 79-180 mmu.Results:(1)Literature research found that T.hemsleyanum was first recorded in Wu qijun,a botanist in Qing dynasty,who wrote a study of plant names and real maps,and it has not been included in the Chinese Pharmacopoeia so far.Modern scholars seldom carry out systematic research and textual research on it,and it has many other names,thus,the phenomenon of homonymy and homonymy is frequent.According to the literature,the author confirms its plant primordial: Tetrastigma hemsleyanum Diels et Gilg.,a plant of the subgenus Periploca of the family Vitaceae in the round umbilicus group,and firstly summarizes the natural distribution of its resources: in our country,all the provinces south of the Yangtze River and most of the provinces through which the Yangtze River flows have natural distribution,but they are rare or not seen everywhere north of the Yangtze River.This is the first time to systematically analyze the causes of its synonym and homonymy.(2)Experimental data on extraction of pharmacodynamic substances show that,yield of Ethyl Acetate part: cold alcohol extraction > alcohol extraction > > water extraction,yield of n-butanol part: water extraction > cold alcohol extraction > alcohol extraction.Count by TI > 16,comprehensive results of virus inhibition: H1N1> EV71> HSV-1>COX-B3>RSV> COX-B5;In the H1N1 experiment,4 parts of alcohol cold extraction mother liquor,ethyl acetate,n-butanol and water,and 3 parts of alcohol extraction mother liquor,ethyl acetate and n-butanol have the best toxicity inhibition effect;In HSV-1 and EV71 experiments,alcohol extraction of n-butanol,water,mother liquor and alcohol cold extraction water part have strong inhibitory effect,but the inhibitory effect of water extraction of n-butanol and water parts is slightly weak;At the same time,alcohol cold extraction of ethyl acetate,n-butanol and water also inhibited HSV-1,COX-B3 and COX-B5 to some extent.(3)The experimental results of anti-pseudovirus particles in vitro show that,for the Ebola pseudovirus,no significant inhibitory effect was observed at 4 concentrations at part 1#,and only at 100μg·m L-1 and 50μg·m L-1,it showed a certain inhibitory effect at part 2#,at the same time,when the parts of 3 # 4 # 5 # 6 # is 12.5μg·m L-1,the inhibition rate is above 70%,the inhibition rate also increases as the drug concentration increases,so this indicates a dose dependence.for the Lassa pseudovirus,there was no obvious inhibitory effect on 6 parts at 4 concentrations.Drug toxicity test results show that,the CC50 of 3 # 4 # 5 # 6 # parts is more than 100μg·m L-1 when the highest concentration is 100μg·m L-1,thus they have no cytotoxicity in vitro.Further experiments show that,the portions E7 at 3 # and N1 and N2 at 4 # have good inhibitory effects on Ebola pseudovirus.(4)In vivo pharmacodynamic experimental data show that,the weight of mice in the three dose groups of T.hemsleyanum(200mg·kg-1,100mg·kg-1,50mg·kg-1)showed a downward trend in the first 8 days,and they gradually gain weight after 8 days,and the weight recovery of the small dose group was the closest to that of the normal group,at the same time,the death rate of model group was 80% by the 8th day.From the perspective of mortality protection,by the 15 th day,the survival rate of small dose group was 100%,however,the survival rate of medium dose and large dose is 80%,but it was only 20% in the model group.The experimental results show a certain aging and dose-effect relationship at the same time.(5)In vivo pharmacodynamic mechanism experimental data show that large and medium doses(200mg·kg-1,100mg·kg-1)of T.hemsleyanum extract can reduce lung index and improve lung index inhibition rate of H1N1 influenza virus pneumonia model mice.Both large and medium doses of T.hemsleyanum extract can enhance spleen T cell proliferation and NK cell killing activity in model mice,and there are significant differences.Further experimental data show that,the large and medium doses of T.hemsleyanum extract can increase the serum cytokines IL-2 and IFN-γ and reduce the content of TNF-α in model mice.At the same time,the drug can increase the m RNA expression of three inflammatory factors IFN-γ,IL-2 and TNF-α in the lungs of model mice with time prolonging.In addition,IFN-γ and IL-2 expressed faster and more than TNF-α m RNA on days 3-7 and 3-5 respectively.(6)In classical extraction and separation experiments,Through further separation and purification of ethyl acetate and n-butanol parts of T.hemsleyanum,we get 21 compounds.We used nuclear magnetic resonance spectroscopy to identify its structure,among them,12 flavonoids,1 anthraquinone,2 steroids and 6 phenolic acids.In the experiment of modern technology to characterize the material basis of T.hemsleyanum,before being processed by MDF,we identified 50 compounds(30 flavonoids),among them,36 species in negative ion mode(22 flavonoids)and 32 species in positive ion mode(23 flavonoids),and there are 19 components that can be detected in both ion modes.After MDF technology treatment,4 more components were identified in negative ion mode,and 19 more components were identified in positive ion mode.Finally,73 compounds were identified from methanol ultrasonic extraction of T.hemsleyanum.Among them,53 compounds are flavonoids.Conclusion:(1)Study on virus spectrum screening.It is found that,the extraction rate of effective components from root tuber of T.hemsleyanum is the highest by cold soaking with 70% ethanol at room temperature;T.hemsleyanum repens extract has broad spectrum antiviral characteristics,and the inhibitory effect of petroleum ether and dichloromethane extracts is weak(TI < 8),all other parts have different degrees of inhibition on 6 kinds of test viruses such as H1N1(TI > 12),and the comprehensive antiviral effect is obviously characterized by H1N1> EV71> HSV-1>COX-B3>RSV> COX-B5(counted by the part of TI > 16),In the H1N1 experiment,4 parts of alcohol cold extraction mother liquor,ethyl acetate,n-butanol and water,and 3 parts of alcohol extraction mother liquor,ethyl acetate and n-butanol have the best toxicity inhibition effect;at the same time explain,the anti-H1N1 drugs of T.hemsleyanum are likely to be distributed in the middle and high polarity parts,extremely unlikely to be located in the low polarity part.(2)Basic material research.Using UPLC-Q-Exactive LC/MS-mass defect Filtering MDF-OTCML database retrieval identification strategy,we analysis qualitatively the chemical constituents of T.hemsleyanum,a relatively comprehensive,rapid and efficient chemical composition identification method has been established.We have identified a total of 73 chemical constituents,mainly flavonoids,together with other undiscovered ingredients,they form the pharmacodynamic material basis of T.hemsleyanum extract.(3)Study on anti-Ebola pseudovirus in vitro.Through in vitro anti-Ebola pseudovirus experiment of T.hemsleyanum extract,it is found that,the toxicity inhibition effects of the four alcohol cold extraction parts show the characteristics of n-butanol > ethyl acetate > water > mother liquor,and the CC50 of the four parts is more than 100μg·m L-1 when the highest concentration is 100μg·m L-1,thus they have no cytotoxicity in vitro for normal cells;Further experiments show that,when the concentration of N1 and N2 from n-butanol part and E7 from ethyl acetate part is as low as 12.5μg·m L-1,the inhibition rate of Ebola pseudovirus is still more than 90%,indicating that it has the best inhibition effect on Ebola pseudovirus,and the pharmacodynamic substances in the three polar ranges are most sensitive to Ebola pseudovirus.According to the structural characteristics of pseudovirus particles,it can be inferred that the effective part(component)of T.hemsleyanum exerts antiviral effect by blocking Ebola pseudovirus from invading normal cells and has protective effect on host cells.The mechanism of action may be blocking the haemagglutinin glycoprotein(HA)on the outer surface of the virus outer membrane from binding to host cells,at the same time,the neuraminidase(NA)of another neuraminidase glycoprotein cannot continue to diffuse and reproduce,so that newly generated virus particles cannot be released,and jointly ensure the toxic inhibiting effect of the active substances.(4)Study on drug effect in vivo.Experimental study on the mechanism of drug effect in vivo found that,Large and medium doses(200mg·kg-1,100mg·kg-1)of T.hemsleyanum extract can improve the survival rate of mice infected with influenza A H1N1 virus,decrease the lung index of influenza virus pneumonia model mice and increase the inhibition rate of lung index.The pharmacodynamic effect of anti-H1N1 virus in vivo was preliminarily determined.(5)Study on the mechanism of drug effect.Large and medium dose of alcohol extract from root tuber of T.hemsleyanum can enhance spleen T cell proliferation and NK cell killing activity,improve the cellular immune function of mice,reduce the damage of exogenous viruses to cells.Further experimental data show that,the large and medium doses of T.hemsleyanum extract can increase the serum cytokines IL-2 and IFN-γ and reduce the content of TNF-α in model mice,prove its role in the process of inflammatory reaction in the body.At the same time found that,the drug can increase the m RNA expression of three inflammatory factors IFN-γ,IL-2 and TNF-α in the lungs of model mice with time prolonging.In addition,IFN-γ and IL-2 expressed faster and more than TNF-α m RNA on days 3-7 and 3-5 respectively.This indicates that these DNA did undergo transcription,thus releasing different amounts of m RNA.This process is also a process in which inflammatory factors exert physiological activities.It also shows that the pharmacodynamic effect of Trifolium repens extract occurs before the formation of inflammatory cytokines or in the process of inflammatory reaction,and has the characteristics of bidirectional regulation and dose dependence to different degrees.The mechanism of action may be related to the up-regulation of the concentrations of pro-inflammatory factors IFN-γ and IL-2 in serum and lung tissue,downregulating the concentration of inflammatory factor TNF-α in serum or inhibiting the up-regulation of the concentration of inflammatory factor TNF-α in lung tissue. |
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