Study On Anti-Inflammatory Spectrum Effect And Quality Evaluation Of Green Forsythia

Forsythiae Fructus has the effect of clearing heat and removing toxicity,detumescence and resolving hard mass.It is widely used in the treatment of respiratory inflammation,fever and other related diseases.Anti-inflammatory through detumescence and resolving hard mass is one of the main pharmacological effects of Forsythiae Fructus.According to different harvesting periods,Forsythiae Fructus is divided into Green Forsythia fructus(GF)and Ripe Forsythia fructus(RF).Current researches showed that the ethyl acetate part is the anti-inflammatory active part of Green Forsythia fructus whose main activity material basis may be forsythin,phillygenin and other medium polar components.The efficacy of traditional Chinese medicine collabotates by multi-component,multi-target.Spectral-effect relationship can associates fingerprint of chemical compositions in traditional Chinese medicines with pharma-codynamic effect to screen the effective constituents.Therefore,this study utilized the Green Forsythia medicinal materials from different producing areas as the target to establish the fingerprint.We conducted the pharmacodynamic research in vitro and construct the anti-inflammatory spectrum effect relationship in order to screen the potential active ingredients and establish the multi-indicator content determination method.It provided the basis for improving the quality evaluation of GF medicinal materials.Objective:The fingerprint of GF was studied by HPLC.The anti-inflammatory-spectrum-effect relationship in vitro was established and potential antiinflammatory active ingredients was analyzed.On the basis of that,an approach for determining the content of multi-indicator components was established.Methods:GF medicinal materials from different producing areas were collected and the fingerprint conditions were optimized.The fingerprint of GF was then established by HPLC method.HPLC-Q-TOF/MS method was adopted to identify ingredients of GF.LPS(Lipopolysaccharides)was used to induce RAW264.7 cells to establish an inflammatory model.The effects of extracts from sixteen batches of medicinal materials on TNF-α,IL-1β,IL-6 inflammatory factors were studied by treating RAW264.7 inflammatory cells.Spectrum-effect relationship was established by multiple linear regression analysis and gray correlation analysis.On the basis of that,the quality evaluation method of multi-index components of GF was established by choosing four kinds of index components with better anti-inflammatory activity.Results:1.Fingerprint study on distinct GF samples The HPLC fingerprint of GF was optimized,and the precision,repeatability and stability RSD values were all less than 3%.The method was practical and reliable,and could be used for fingerprint research of GF.The similarity evaluation results showed that the similarity for 16 batches of GF medicines from different producing areas was above 89%.The RSD values of the common peak area in 16 groups were between 25% and 160%.HPLC-Q-TOF/MS identified 21 compounds,namely Methyl4-hydroxyphenylacetate,Quince acid,Cornoside,3,4-dihydroxyphenylglycoside,Ado-xosidic acid,Forsythiaside E,p-coumarin-(6-O-caffeoyl)-glucoside,β-hydroxyforsyl Forsythoside A,Forsythiaside B,Forsythiaside J,Forsythiaside F,Forsythiaside A,Rutin,Forsythiaside H,(+)-epipinoresinol-4-O-β-D-glucoside,(+)-pinoresinol-4-O-β-D-glucoside,Arctigenin,Phillyrin,phillygenin,Isoprenoid,Matairesinol.2.Spectrum-effect relationship study and screening of anti-inflammatory active ingredients Cell inflammatory model in vitro was established by RWA264.7 invigorated with LPS.The study of anti-inflammatory activity in vitro for sixteen batches of GF medicinal materials from different areas used inflammatory factors of NO,IL-1β,IL-6 and TNF-α as detection indicators.Eleven anti-inflammatory active common peaks were screened by multiple linear regression analysis and gray correlation analysis.Four anti-inflammatory active ingredients including Forsythiaside E,Forsythiaside B,Forsythiaside A and Phillyrin were identified by standard substances.Five anti-inflammatory active ingredients including Cornoside,p-coumarin-(6-O-caffeoyl)-glucoside,Forsythiaside H,(+)-epipi-noresinol-4-O-β-D-glucoside,(+)-pinoresinol-4-O-β-D-glucoside were identified by comparing the retention time of HPLC-Q-TOF/MS and HPLC.3.Establishment and evaluation of determination methods for multiple indicator components Method of determination according to four anti-inflammatory components was established.The linear range was good and the method had high sensitivity,good repeatability and high reliability.The method can be used for quality control of GF.Conclusions:1.The HPLC fingerprint of GF is accurate and reliable and it can reflect the whole quality of GF.2.Nine anti-inflammatory active ingredients of GF were selected and identified by the study of spectral-effect relationship in vitro.3.The method for determination of multi-indicator contents which were selected from main anti-inflammatory components of GF was stable and reliable and it had good reproducibility.It provided a basis for quality control and evaluation of GF.