Comparison Of The Some Chemical Compositions In Bark,leaves And Male Flowers Of Eucommia Ulmoides And Study On Their Anti-RA Activity In Vitro

Objective:Establish an analytical method based on HPLC multi-wavelength detection of the chemical compositions in the bark,leaves,and male flowers of E.ulmoides,and compare the content differences and distribution characteristics of the chemical compositions in the three medicinal parts of E.ulmoides;Establish an analysis method based on liquid-mass spectrometry UPLC-MS/MS to detect the changes in blood concentration of the chemical compositions in the three medicinal parts of E.ulmoides after entering the blood,and describe its pharmacokinetic characteristics and possible differences;Identify and characterize the chemical compositions of E.ulmoides and explore its metabolic pathways;The effect of E.ulmoides chemical composition on anti-Rheumatoid arthritis（RA）activity in vitro was discussed preliminarily.Methods:After establishing the methodology by investigating linearity,precision,stability,repeatability,sample extraction recovery rate,etc.,Aucubin（AN）,Geniposidic acid（GA）,Geniposide（GE）,Genipin（Ge）,Pinoresinol Glucopyranoside（PDG）,Chlorogenic acid（CA）,Quercetin（Qu）and Betulinic acid（BA）,which the contents of the 8 index chemical compositions were determined by HPLC multi-wavelength detection method.The liquid phase conditions are:Agilent 1260 instrument system and Agilent Zorbax Eclipse Plus C18（4.6 mm×250 mm,5μm）chromatographic column is used,acetonitrile（A）-0.1%formic acid aqueous solution（B）is used as the mobile phase,and the gradient elution program is:0-40 min,5-15%A;40-50 min,15-25%A;50-60 min,25-30%A;60-65 min,30-5%A;65-80 min,5%-5%A;isocratic elution program:0-30 min,82%A.the flow rate is controlled to 1.0m L/min,the injection volume is 10μL,the column temperature is set to 25℃,and the sample room temperature is 4℃.The detection wavelengths were set at 194 nm for AN,202 nm for PDG,238 nm for GA,GE and Ge,320 nm for CA,368 nm for Qu,and 210 nm for BA.The characteristic maps of 8 chemical compositions in multiple batches are established based on the results of peak area detection.Then use cluster analysis,PCA analysis,PLS-DA analysis and clustering heat map to perform one test and multiple evaluation.AB SCIEX Analyst TF software is used to collect injection data and images,and then use Peak View software to perform matching analysis to obtain the mass spectrum data and secondary mass spectrum of the compound.Combine the HMDB database search and literature review and comparison to qualitative identification and deduction the metabolic cleavage law of the ingredients.After optimizing the mass spectrometry and liquid phase conditions through single standard and mixed standard,the specificity,linearity,sensitivity,precision,accuracy,spiked extraction recovery rate,stability,matrix effect and other methodological content are investigated to establish a good UPLC-MS/MS analysis method,and then quantitatively detect the blood concentration changes of various index chemical compositions in the three medicinal parts of E.ulmoides in SD rats.The liquid phase conditions are:Waters Acquity UPLC HSS T3（100×2.1 mm²,id 1.8μm）chromatographic column connected to Waters Van Guard^TM Pre-Column 3/PK 2.1×5mm guard column;using 0.1%formic acid water（A）—acetonitrile（B）as the mobile phase for gradient elution,the elution time is 7 min,the flow rate is set to 0.3 m L/min,the injection volume was 5μL,the constant column temperature was 40°C,and the injector temperature was set to 15℃.Mass spectrometry conditions:Waters Triple Quiad 5500 system instrument is used,ESI is used as electrospray ionization source,scanning mode is multi-reaction positive and negative ion monitoring mode（MRM）,capillary ionization voltage is±4.5 KV,cone voltage is 30 V;the extrusion voltage 7V.The temperature of ESI positive and negative ion source is 500℃and 450℃,and the desolvation auxiliary gases GS₁ and GS₂ are both nitrogen,and the flow rate is 50 L/h,and the collision gas argon flow rate is 35 L/h.The quantification was performed using multiple reaction monitoring（MRM）of precursor-product ion transitions at m/z364.2[M+NH4]⁺→148.8 for AN,m/z 406.2[M+Na]⁺→148.9 for GE,m/z227.2[M+H]⁺→149.1 for Ge,m/z 417.1[M+H]⁺→297.0 for PU（IS）;m/z373.1[M-H]^-→123.2 for GA,m/z 353.3[M-H]^-→153.0 for CA,m/z 301.0[M-H]^-→150.9 for Qu and m/z 681.3[M-H]^-→356.9 for PDG.Graphpad is used to draw the drug-time curve of each ingredients,and the winnonlin 6.0 system software is used to analyze the main pharmacokinetic parameters such as C_max,T_max,AUC,MRT,etc.And using SPSS26.0software one-way analysis of variance to compare the differences between groups,P?0.05 has a significant difference.In vitro experiments were performed with MH7A cells to observe the effects of PDG and Ge active ingredients in E.ulmoides on RA-FLS.The cell viability was detected by MTT method,the amount of NO released in the cell supernatant after LPS stimulation was detected by the Griese reagent method,and the expression levels of IL-1βand IL-6 pro-inflammatory factors in the cells after TNF-αstimulation were detected by rt-PCR.Bonferroni test and one-way analysis of variance were performed using SPSS 26.0 software,and P?0.05 was considered statistically significant.Results:A method for HPLC multi-wavelength detection and analysis was successfully established.The linear correlation coefficients of AN,PDG,GE,GE,Ge,CA,CA and BA in their respective linear ranges are r≥0.9996.The RSD value of the instrument’s intra-day and inter-day precision is≤2.53%,and the repeatability RSD is≤2.95%.The stability of the 8 index ingredients in the E.ulmoides sample within 36h is RSD≤3.0%,the average recovery rate of the three levels of low,medium and high is95.02%～104.33%,and the RSD value of each ingredients is≤1.96%.The minimum detected content of 8 index ingredients in E.ulmoides bark,leaves and male flowers was 0.0016 mg/g,and the maximum was 52.6030 mg/g.The HPLC characteristic map and content determination showed that there were significant differences in the content and distribution of 8 index chemical compositions in the three medicinal parts of E.ulmoides bark,leaves and male flowers.PDG and Ge are mainly contained in E.ulmoides bark;AN,GE,GE and BA are mainly contained in the male flowers of E.ulmoides;CA is mainly contained in the leaves of E.ulmoides;CA is extremely low in the three medicinal parts of E.ulmoides.Multivariate statistical analysis showed that the three medicinal parts of E.ulmoides bark,leaves and male flowers were clustered into one group,and the content and distribution of the ingredients in the leaves and male flowers were more significantly different;The four chemical compositions of CA,PDG,GA and BA have a greater contribution to distinguishing the three medicinal parts of E.ulmoides,and can be used as characteristic variables to distinguish the three medicinal parts.Chemical composition among them,BA also has significant geographic differences.A total of 54 compounds in E.ulmoides bark and its plasma samples were identified and characterized,and 20 compounds were identified as prototype metabolites of the chemical compositions of E.ulmoides bark.The metabolic cleavage laws of 8 index ingredients including AN,GA,GE,Ge,CA,CA,PDG and BA were deduced.The UPLC-MS/MS analysis method was successfully established.AN,GA,GE,Ge,PDG,CA and Qu have good specificity,linear coefficient r≥0.9956,LLOQ and LLOD are respectively less than 7.6 ng/m L and 3.8 ng/m L,the instrument’s intraday and intraday precision RSD≤8.0%.The accuracy range of the six ingredients of AN,GA,GE,Ge,PDG and CA is 95.1%～104.4%;Extraction recovery rate and matrix effect are 91.2%～111.5%and 89.8%～104.3%respectively,RSD≤10.5%;Placed at room temperature（about 24°C）for 4 hours,refrigerated at 4°C for 24 hours,and-80°C freeze-thaw cycles for three RSD are less than 13%.The drug-time curve diagram and the analysis of pharmacokinetic parameters showed that:GA,GE,Ge,and CA have similar shapes in the E.ulmoides bark and male flower;while AN and GA and Ge have a bimodal trend.E.ulmoides bark alcohol extracts of AN,GA,GE,GE,and PDG showed higher plasma exposures,which were 1308.00±654.28,5483.33±3098.78,2234.33±1170.34,695.00±519.61 and 1076.00±697.06 ng/m L,respectively.The plasma exposure of CA in ethanol extract of E.ulmoides leaves was significantly higher than that in bark and male flowers,which was 1400.33±679.07 ng/m L.In addition,there are significant differences in the main pharmacokinetic parameters of the same ingredients after oral administration into the blood in the three medicinal parts of E.ulmoides.The range of T_max of the six ingredients after oral administration of the alcohol extract of E.ulmoides bark,leaves and male flowers is 0.08±0.00 h～3.33±3.30 h;AUC_0²4:0.43±0.51 hr·ng/m L～24502.92±5689.21 hr·ng/m L;Vz/F:1.24±0.46 L/kg～6903.7±1244.8 L/kg;CLz/F:0.20±0.05 L/hr/kg～592.10±338.85L/hr/kg;MRT0～24:0.34±0.53 h～11.86±0.11 h;T1/2z:2.12±0.00 h.AN,GA,GE,Ge,CA and PDG can significantly reduce the release of NO in MH7A cells stimulated by LPS,and PDG-Ge compatibility better than monomer.Both the effects of PDG and Ge monomer and the pairwise compatibility based on PDG can significantly inhibit the proliferation of MH7A cells,and PDG-Ge has the best inhibitory effect;PDG and Ge alone or in combination can significantly reduce the expression levels of IL-1βand IL-6 in cells stimulated by TNF-α,but the reduction effect of PDG and Ge monomer administration is significantly better than the compatibility of PDG-Ge.Conclusion:This study established HPLC multi-wavelength detection analysis method and UPLC-MS/MS analysis method with high accuracy,good reproducibility,high efficiency,sensitivity,and simple operation.Both methods meet the methodological verification requirements.The former can be used to deter mine the content of 8 index chemical compositions in the three medicinal parts of E.ulmoides.The latter can be used to quantify and analyze the pharmacokinetic characteristics of the changes in blood concentration of various ingredients of E.ulmoides after entering the blood.There are significant differences in the chemical composition content and distribution of 8 indexes of E.ulmoides bark,leaves,and male flowers,and the three medicinal parts can be clearly distinguished by certain characteristic ingredients;The difference in the content and distribution of each ingredients in different medicinal parts may be a main reason for the difference in pharmacokinetics.The AN,GA,GE,Ge,CA and PDG in E.ulmoides can all enter the blood as a prototype,and have potential anti-RA effects.To some extend,the synergistic effect of PDG-Ge and other ingredients may be the pharmacological basis for the anti-RA effect of E.ulmoides.The role of inflammatory factors is not consistent,reflecting the complexity of the interaction of the active ingredients of E.ulmoides.