Analysis Of Main Primary And Secondary Metabolites And Screening Of Anti-inflammatory Active Components In Shuanghuanglian Injection

Shuanghuanglian injection,a water injection prepared by Lonicerae japonicae Flos,Forsythiae Fructus and Scutellariae Radix,through modern pharmaceutical technology,has the functions of clearing heat and detoxifying toxin,as well as soothing the wind and relieving exterior symptoms.Modern pharmacological research shows that it possesses antibacterial,anti-inflammatory,antiviral,and enhanced body immune function,and mainly used to clinical treatment of headaches,fever and other symptoms caused by upper respiratory infections cliniclly.It's play an important role for the quality control of Shuanghuanglian injection to clarify the chemical composition.Shuanghuanglian injection is composed of multiple drugs with complex chemistry,and its material basis still needs to be further identified;Shuanghuanglian injection has significant Anti-inflammatory activity,but the anti-inflammatory active substances of Shuanghuanglian injection need to be further studied.In this study,the primary and secondary metabolites in Shuanghuanglian injection were analyzed by using nuclear magnetic resonance spectroscopy?NMR?and full two-dimensional gas chromatography-mass spectrometry?GC×GC-MS?.The chemical components were determined,which laid the foundation for the quality control of Shuanghuanglian injection.Finally,focusing on the secondary metabolites in Shuanghuanglian injection,the anti-inflammatory components among the secondary metabolites were screened.1.Through nuclear magnetic resonance spectroscopy technology,the structures of 17compounds were identified relying on the chemical shift values and coupling splitting conditions of proton signal peaks in the ¹H-NMR spectrum,and further combined with the¹³C-NMR,the ¹H-¹H COSY,HSQC and HMBC spectra information and comparison with the standard,including eight primary metabolites and nine secondary metabolites in the Shuanghuanglian injection.Eight primary metabolites included one amino acid?valine?,three monosaccharides?glucose,fructose and mannose?,one disaccharide sugar?sucrose?,two organic acids?formic acid and succinic acid?,one cycloalkane?myo-inositol?,and nine secondary metabolites were four phenylpropionic acids?chlorogenic acid,neochlorogenic acid,cryptochlorogenic acid and caffeic acid?,three phenylethanol glycosides?forsythoside A,isoforsythiaside and forsythoside E?,one flavone?baicalin?and one lridoid?secoxyloganin?.The chemical compositions of Shuanghuanglian injection were further clarified.2.After systematic methodological verification,a quantitative nuclear magnetic analysis method established by ¹H-NMR technique was applied to measure the contents of the primary metabolites in 20 batches of Shuanghuanglian injections.The average contents of valine,glucose,fructose,mannose,sucrose,succinic acid and myo-inositol in 20 batches of Shuanghuanglian injection were 0.0338,1.5585,3.1405,0.1652,0.5127,0.0356 and 1.3999mg/m L separately.The total contents of the primary metabolites in different batches of Shuanghuanglian injection ranged from 5.5780 to 8.1840 mg/m L,and the average total content was 6.8461 mg/m L.The average total solid content of 20 batches of Shuanghuanglian injection was determined as 27.504 mg/m L,while the contents of measurable components accounts for 24.84%of the total solid.The primary metabolites of Shuanghuanglian injection were mainly monosaccharides,among which the contents of fructose and glucose were higher.3.Applying full two-dimensional gas chromatography-mass spectrometry?GC×GC-MS?method,the volatile components in Shuanghuanglian injection were two-dimensionally analyzed by optimizing the preparation method of test samples,and the conditions of chromatography and mass spectrometry.Through the search of computer database,29compounds with forward and reverse match values greater than 800 were detected.The main compounds were organic acids and aromatic compounds,among which the content of acetophenone was the highest.4.Nine secondary metabolites in Shuanghuanglian injection were identified based on nuclear magnetic resonance technology,combined with the 22 secondary metabolites identified using liquid chromatography-mass spectrometry?nine secondary metabolites were consistent with nuclear magnetic identification results?,the anti-inflammatory activities of Shuanghuanglian injection and 22 secondary metabolites were researched systematically through an LPS-induced inflammation model in RAW264.7 macrophages.MTT and Griess method were employed to evaluate the cell viability and anti-inflammatory activity of the compounds respectively.Preliminary results showed that Shuanghuanglian injection had anti-inflammatory effects.In addition,the chlorogenic acid,neochlorogenic acid,cryptochlorogenic acid,isochlorogenic acid A,isochlorogenic acid B and isochlorogenic acid C in Shuanghuanglian injection,caffeic acid,secologanic acid,secoxyloganin,,forsythoside A,Isoforsythiaside,baicalein,baicalin,hyperin,and phillyrin were anti-inflammatory components of Shuanghuanglian injection,among which theand phenylpropionic acid components behaved better anti-inflammatory activities.In this study,the primary and secondary metabolites of Shuanghuanglian injection were qualitatively analyzed by NMR and GC×GC-MS techniques respectively,and the chemical substance basis were further clarified.the quantitative analysis method of Shuanghuanglian injection was constructed based on ¹H-NMR technology,24.84%of the components of Shuanghuanglian injection were quantified by using ¹H-NMR technology.Based on the results of the anti-inflammatory activities research on Shuanghuanglian injection and 22secondary metabolites,16 chemical components from Shuanghuanglian injection were screened out to be anti-inflammatory activities.The results would provide a scientific basis for the quality control of Shuanghuanglian injection.