| Herba houttuyniae(H.cordata)injection is the extract from H.cordata by steam distillation,redistillation,solubilization with Twain 80,filtration,subpackage and sterilizing.With pharmacological effects on anti-microbial,anti-virus,enhance immunity,anti-inflammatory,anti-allergic,antitussive,hypoglycemic,etc.H.cordata injection is used to treat respiratory tract infection,viral enteritis,eczema and malignant tumor in clinics.From 1988 to 2006,there were 222 reports ahout serious adverse reaction of H.cordata injection in clinics.The SFDA issued a announcement about moratorium using and examination of seven injections,which have H.cordata in 2006.This implies,at present,Chinese medicine injection safety detection method cannot accurately and effectively eliminate sensibilisins from H.cordata injection.So this test method cannot guarantee its clinical medication safety.In order to further ensure the medication safety of H.cordata injection in clinical,this study is devoted to preliminary explore the sensibilisins of H.cordata injection according to the differences between its different preparation technology in animal allergy test and chemical constituents which were detected by GC/MS and LC/MS.And establish a relative safety preparation technology of H.cordata injection to improve its safety in clinical.The details are as follows:(1)Optimizing the animal models of type ? hypersensitivity ahout H.cordata injection.Through sensitive animals screening test and enhancing sensitivity test by adjuvant to optimize the animal models of type ? hypersensitivity.This study screened four kinds of animals,which were ICR mice,SD rats,guinea pigs and BN rats.And it also compared the sensitization on the BN rats between H.cordata injection and H.cordata injection which uesd adjuvant.The results showed that the optimal animal models of type ? hypersensitivity ahout H.cordata injection was as follows:with the rate of change of IgE,histamine,tryptase and beta aminocaproic glycosidase in plasma for the targets,ovenalbumin for positive drug,BN rats for sensitive animals and adjuvant to enhance sensitivity.There are other allergic substance in H.cordata injection except Twain 80.(2)A preliminary study ahout animal models of anaphylactoid reaction of H.cordata injection.With the rate of change of IgE,histamine,tryptase,beta aminocaproic glycosidase,anti-C4b and C5b-9 in plasma for the targets and sensitive animal screening test to preliminary study animal models of anaphylactoid reaction of H.cordata injection.The experimental results showed that 1 percent of ovenalbumin can be used as a positive drug in type I allergic reaction,not in anaphylactoid reaction.So positive drug screening tests must be increased afer this study.Analysis from the angle of anaphylactoid reaction,four kinds of animals,which were ICR mice,SD rats,guinea pigs and BN rats,had low sensitivity to H.cordata injection and twain 80.So the scope of animal screening test must be expanded.(3)Establishment a relatively safe preparation technology of H.cordata injection.Using the optimization model of allergic to discuss the security of two kinds of volatile oil extraction technology ahout H.cordata.Using the 2-hydroxypropyl-beta-cyclodextrins as solubilizer,because of the relatively high security in clinical,to eliminate the influence of auxiliary materials for the allergy test to make the results more reliable.The results demonstrated that some chemical components in H.cordata forerunning liquid,extracted by water vapor distillation method,can cause BN rats to produce type I allergic reaction.But this chemical components were not in H.cordata volatile oil which made by supercritical fluid extraction.Due to its low allergenic,the carbon dioxide supercritical fluid extraction is superior to steam distillation to extract H.cordata volatile oil.With the content of H.cordata volatile oil and 2-undecanone for the targets,single factorexperiment and orthogonal experiment to optimize the supercritical fluid extraction,distillation and inclusion process.The best preparation technology conditions for H.cordata injection were as follows:takeing Heartleaf houttuynia herb to carbon dioxide supercritical fluid extraction.The optimum condition process was established as following:25 MPa as extractor pressure,45 ? as extractor temperature and 1.5 h as extractor time.Then adding the right amount of distilled water to distillation 2 h with 140 ? as distillation temperature.And adding about 7.5g/kg 2-hydroxypropyl-beta-cyclodextrin to 0.5 L/kg distillate liquid to magnetically stirre 3 h with 60 ?as magnetic stirring temperature.Finally,joining the right amount of sodium chloride,keeping the volume to 0.5 L/kg with injected water,0.45?m filtration and sterilizing.There are four kinds of differences between the new preparation process and the original process.(1)The difference of technology:Compared with the original process of water vapor distillation,using supercritical carbon dioxide extraction method to extract houttuynia cordata have a high extraction rate and safty.(2)The difference from excipient:Compared with the original process,new techniques adopted in 2-hydroxypropyl-beta-cyclodextrin as solubilizer to replace polysorbate 80 in order to achieve a great safety in clinical.(3)Using ultrafiltration to instead of microfiltration to remove the macromolecular impurities from H.cordata injection,the new preparation process had been enhance the clarity of injection and the security.(4)The difference of formulation:Original formulation of H.cordata injection is the water-soluble injection,which have a poor stability.The new technology will be made a freeze-dried injection products to improve the stability of the injection.At the same time,using the optimize allergy model to discuss the security of H.cordata injection which extracted by the new preparation technology.It turned out that H.cordata injection extracted by the new preparation technology cannot cause BN rats to produce type I allergic reaction.Namely,sensitizations of H.cordata were not generated in supercritical fluid extraction,distillation and inclusion process.The irritability of new H.cordata injection to BN rats was far lower than the injection that extracting by original technology.(3)Finding 15 kinds of suspected allergic substance and establishing a ELISA detection method for H.cordata protein.Using GC/MS and LC/MS to analysis the chemical compositions of H.cordata volatile oil,that respectively extract by water vapor distillation and supercritical fluid extraction.24 same components consisted of erpenes,aldehydes,ketones,alcohols,lower fatty acids and esters were identified in H.cordata volatile oil prepared by two methods by gas chromatography-mass spectrometry(GC-MS)analysis.31 different components were identified in two kinds of volatile oil,and there are 17 compounds only in H.cordata volatile oil prepared by water vapor distillation.But only 11 components consisted of 2-hexene aldehyde,2,3-dimethyl pyridine,benzaldehyde,linalool,isoborneol,bomyl acetate,kwai acyl methyl,lauryl aldehyde,2-13 ketones and caryophyllene oxide have been confirmed according to the Similarity in more than 85%.4 components,only in oil prepared by water vapor distillation,were identified by liquid chromatography-mass spectrometry(LC-MS)analysis.Two components,the retention time were 0.52 and 20.12 min,were identified in the positive ion mode.And two components,the retention time were 21.78 and 24.36 min,were identified in the negative ion mode.While,due to the carbonyl and hydroxyl active group,this 11 kinds of components are more easy coupling with serum protein into complete antigen than others,that increasing the possibility of the sensitization to BN rats.A strong specificity,high sensitivity ELISA detection method of H.cordata protein was set up,according to the principle of the specificity combining between antigen ang antibody.The method are as follows:H.cordata antigen is diluted in proportion with carbonate buffer solution.The diluent was added to the 96 hole polyethylene elisa plate,then 4 ? wet box for the night,wash the plate with phosphate buffer for twice,and the specific binding between H.cordata protein and the elisa plat was stoped by the 2%calf serum albumin solution for 10 minutes,which dissolved with PBS solution of pH 7.4.Add 100 ?L of the rabbit anti H.cordata serum,which dissolved with PBS solution in a ratio of 1:1000,to the wells.Incubation the residuai liquid after 2 hours and wash the elisa plate with phosphate buffer for 5 times.Add 100?L of the goat anti-rabbit IgG solution,which labeled by Horseradish peroxidase and dissolved with PBS solution in a ratio of 1:10000,to the wells.Incubation the residuai liquid after 2 hours and wash the elisa plate with phosphate buffer for 5 times.Add 100 ?L of the substrate phosphate buffer which made by OPD and H2O2.Finally,add 100 ?L of the 2 mol/L sulfuric acid to stop the reaction and read the O.D value at 490 nm by the microplate reader.The linear range of the method from 0.038 to 3.8 ?g/mL,the detection limit of 9.88 ng/mL and the quantificationlimit of 14 ng/mL. |
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