Study On Anti-Inflammatory Activity Of Index Components Of Forsythia

Forsythia suspensa is the main ingredient of various compound medicines such as Lianhua Qingwen Capsules and Vitamin C Yinqiao Tablets.Forsythia suspensa contains abundant active ingredients,among which forsythiaside A（FSA）and phillyrin（PHR）are specified as the indicator components of Forsythia suspensa in the 2015 and 2020editions of the Pharmacopoeia of the People’s Republic of China.Forsythia suspensa has significant anti-inflammatory activity,and finding new anti-inflammatory drugs from Forsythia suspensa is an effective way.In this paper,the index components and fingerprint components of Forsythia suspensa are taken as the research objects.Firstly,an in vitro enzyme target screening model is established,and the anti-inflammatory activity of the index components of Forsythia suspensa is evaluated with elastase（PPE）as the anti-inflammatory target enzyme.Secondly,multi-spectral method is used to study the interaction of the components and PPE.Finally,the molecular docking simulation is used to clarify the action site and structure-activity relationship.The main results are as follows:Forsythiaside A（FSA）,the index component of Forsythia,has anti-inflammatory activity,and its inhibitory type on PPE is competitive inhibition,IC₅₀=1.5 mmol/L,K_i=8.1×10^-4mol/L.According to the fluorescence spectrum,the quenching type of PPE by FSA is static quenching,the binding ratio is 1:1,the binding distance is 2.181 nm,and occur non-radiative energy transfer.Fluorescence and UV-Vis show that FSA act on the Trp and enhance the hydrophobicity of PPE.FT-IR shows that FSA made PPE more stable through H-bond.CD spectra further demonstrate that FSA increase the proportion ofα-helix and random coil of PPE,and decrease the proportion ofβ-sheet,increase the disorder and extend the peptide chain of PPE.H-bond and hydrophobic interaction between FSA and PPE are consistent with the spectral results.The polyhydroxy structure of FSA can produce H-bond with PPE.The longerπ-πconjugate structure makes Van der Waals force more extensive.In addition,hydrophobic interaction andπ-cation interaction can’t be ignored.In conclusion,there are a variety of interaction forces between FSA and PPE,which reduce the energy of the system to form a stable complex and inhibit the activity of PPE,thus achieving anti-inflammatory effect.Phillyrin（PHR）,the index component of Forsythia,has anti-inflammatory activity,the inhibition type of PPE is competitive inhibition,IC₅₀=1.5 mmol/L,K_i=8.9×10^-4mol/L.UV-vis show that PHR can combine with PPE;and FT-IR show that PHR can change the secondary structure of PPE.CD spectra showed that PHR can increase the proportion ofα-helix and random coil of PPE,while decreaseβ-sheet.Molecular docking results show that there areπ-cation,H-bond,Van der Waals,hydrophobic and electrostatic interactions between PHR and PPE.Phillygenin（PHG）,the fingerprint component of Forsythia suspensa,and its anti-inflammatory activity is higher than that of PHR.The conclusions were as follows:the inhibition type of PPE is competitive inhibition,IC₅₀=0.5 mmol/L,K_i=3.8×10^-4mol/L.UV-vis show that PHG can combine with PPE;and FT-IR show that PHG can change the secondary structure of PPE.CD spectra shows that PHG can increase the proportion ofα-helix and random coil structure of PPE,while decrease the proportion ofβ-sheet structure.Molecular docking results show that there areπ-cation,H-bond,Van der Waals,hydrophobic and electrostatic interactions between PHG and PPE.Arctigenin（ARC）,the main ingredient of Forsythia,has anti-inflammatory activity,and the inhibition type of PPE is competitive inhibition,IC₅₀=1.25 mmol/L,K_i=6.2×10^-4mol/L.UV-vis show that ARC can combine with PPE;show that ARC combined with PPE;and FT-IR show that Arc interacts with PPE and changes PPE secondary structure.CD spectra shows that Arc increases the proportion ofα-helix and random coil in PPE.Molecular docking shows that there areπ-cation,H-bond,Van der Waals,hydrophobic and electrostatic interaction between Arc and PPE.The four monomeric antibodies in forsythia suspensa can combine well with PPE,interfere with the decomposition of PPE on the substrate,reduce the activity of PPE,and play an anti-inflammatory role.The order of their activity is PHG,Arc,FSA,and PHR.From the structural analysis,phenylethyl alcohol glycosides as the mother nucleus,3,4,3’bits can provide protons and H-bonds formed between PPE,4’hydroxyl and caffeic acid esterification,ester base does not provide a locus,but longπ-πconjugate structure,wide range of Van der Waals forces,it is beneficial for anti-inflammatory activity.The parent nucleus of PHR and PHG is ditetrahydrofuran.The 7’-benzene ring doesn’t provide action site,the 7-benzene ring provides action site,and the 3 glucoside and 3 hydroxy group can provide action site;The 3-position hydroxyl group increases theπ-Sigma interaction between PHG and PPE.Although the 3-position glycoside of PHR provides more H-bond sites,it also increases the steric hindrance,making PHG have stronger anti-inflammatory activity than PHR,that is,the introduction of glycosides at the 3-position of tetrahydrofuran is not as active as the hydroxyl group.ARC uses diphenylbutyrolactones as the mother nucleus,and the carbonyl oxygen,benzene ring,and methoxy groups of pentamembered lactones are all good action sites.The index component and fingerprint component of Forsythia suspensa possess the basic structural units of phenylethanoside and ditetrahydrofuran,including the dibenzyl butyrolactone structure of the main component ARC,which provides a potential parent structure for inhibiting PPE.On this basis,appropriate action sites and effective forces can enhance its inhibitory activity.The above research provides new ideas for the design,modification,and synthesis of novel anti-inflammatory drugs.