Study On Analgesic And Anti-Inflammatory Spectral Effect Relationship And Quality Analysis Of Dallergia Hancai Benth

Objective:1.To study the analgesic and anti-inflammatory effects and mechanism of water extract of Dallergia hancai Benth.2.To compare the analgesic and anti-inflammatory effects of water extracts of Dallergia hancai Benth from different areas.3.HPLC fingerprint was established for the aqueous extract of Dallergia hancai Benth.4.Analyze the analgesic and anti-inflammatory spectrum-effect relationships of the water extract of Dallergia hancai Benth.5.Determine the contents of protocatechuic acid and vitexin in the water extract of Dallergia hancai Benth.6.The contents of five kinds of heavy metals（Pb,Cd,As,Hg,Cu）in Dallergia hancai Benth were determined.7.Analyze and identify the chemical components in the water extract of Dallergia hancai Benth to provide reference for further development and research.Methods:1.Using the method of hot plate and acetic acid twisting,mouse ear swelling induced by xylene and carrageen glue induced foot swelling in mice experiment of water extract of Dallergia hancai Benth of analgesic anti-inflammatory effects,and serum levels of PGE₂（prostaglandin E₂）and cAMP（cyclic adenosine monophosphate）were determined by ELISA（enzyme-linked immunosorbent assay）to investigate the analgesic mechanism of glacial acetic acid-induced pain mice.2.The analgesic and anti-inflammatory effects of the water extracts of Dallergia hancai Benth from different producing areas were compared by acetic acid writhing and carragene-induced foot swelling experiments in mice.3.HPLC method was used to establish the fingerprints of ten batches of water extracts of Dallergia hancai Benth from different producing areas,and similarity analysis was carried out by the software of TCM chromatographic fingerprint similarity evaluation system.Meanwhile,cluster analysis and principal component analysis were carried out for the ten batches of HPLC fingerprints.4.Grey relational degree（GRA）and partial least squares regression analysis（PLS）were used to establish the analgesic and anti-inflammatory spectral effect relationships of the water extract of Dallergia hancai Benth,and then the analgesic and anti-inflammatory substance basis was discussed.5.HPLC was used to determine the contents of protocatechuic acid and vitexin in the water extract of Dallergia hancai Benth.6.The contents of five kinds of heavy metals（Pb,Cd,As,Hg,Cu）in 10 batches of Dallergia hancai Benth from different producing areas were determined by microwave digestion-ICP-MS method.7.UPLC-Q-Exactive-HRMS was used to analyze the chemical constituents in the water extract of Dallergia hancai Benth,so as to provide reference for the study of chemical constituents and promote the development and utilization of Dallergia hancai Benth.Results:1.The high,medium and low dose groups of water extract of Dallergia hancai Benth could significantly increase the hot plate pain threshold（P<0.01 or P<0.05）and reduce the times of acetic acid writhing（P<0.01 or P<0.05）;The swelling degree of ear swelling induced by xylenes and foot swelling induced by carrageenan were significantly reduced in the high and medium dose groups,but there was no significant difference in the low dose group（P>0.05）.PGE₂content in serum of mice induced by glacial acetic acid was decreased in water extract group of Dallergia hancai Benth（P<0.01）,but there was no significant difference in cAMP content in serum（P>0.05）.2.The number of acetic acid writhing could be reduced by water extract groups of Dallergia hancai Benth from different producing areas（P<0.01）,and the inhibition rate was higher from Nanning,Qinzhou,Guilin and Hezhou producing areas.The degree of toe swelling in the water extract groups of Dallergia hancai Benth from different producing areas was significantly decreased（P<0.01）,and the inhibition rate from Laibin and Hezhou producing areas was better.3.Fingerprints of the aqueous extracts of of Dallergia hancai Benth were established,12 common peaks were identified,and peak No.2（protocatechuic acid）and peak No.11（vitexin）were identified.The similarity of 10 batches of water extracts of Dallergia hancai Benth from different producing areas was above 0.85.The results of cluster analysis and principal component analysis were consistent,and the 10batches of medicinal materials from different producing areas were divided into three categories.4.In analgesic spectrum-effect relationship,according to the results of grey relational analysis（GRA）that the correlation degree of each chromatographic peak of the water extract of Dallergia hancai Benth on acetic acid writhing of mice analgesic effect of correlation order from large to small was peak 1>8>2>7>9>5>10>6>11>4>3>12,except for peaks 12 and 3,the correlation degrees of other peaks were all greater than 0.7.The results of partial least squares regression analysis（PLS）showed that 1,2,6,8,9,10,11,12 peaks were positively correlated with analgesia,and the order was X8>X9>X11>X10>X12>X6>X1>X2,and VIP values are all greater than 1.In anti-inflammatory spectrum-effect relationship,according to the results of grey relational analysis（GRA）that the correlation degree of each chromatographic peak of the water extract of Dallergia hancai Benth on toes swelling of mice anti-inflammatory effects of correlation order from large to small was peak 1>8>2>9>7>5>10>11>4>6>12>3,except for peaks 3 and 12,the correlation degrees of other peaks were all greater than 0.7.Partial least squares regression analysis（PLS）results showed that peak 2,6,8,9,10,11,12 were positively correlated with anti-inflammatory effect,and the order was X8>X2>X9>X10>X11>X12>X6,and VIP values are all greater than 1.5.The protocatechuic acid and vitexin of water extract of Dallergia hancai Benth in 0.0014 mg/m L～0.0332 mg/m L,0.0014 mg/m L～0.0326 mg/m L range had good linear relationship,the average sample recovery rate was 95.27%and 96.67%respectively,the experiment precision,repeatability and recovery rate test of relative standard deviation（RSD）<3%,meet the requirement analysis.6.The correlation coefficient（r）of the standard curve of Pb,Cd,As,Hg and Cu in Dallergia hancai Benth was about 0.999,the detection limit were 0.003 mg/kg,0.0002 mg/kg,0.0006 mg/kg,0.0005 mg/kg,0.002mg/kg.The recovery is 95.2%～103.2%.The detection limit,precision,reproducibility and recovery test results all met the requirements,and the relative standard deviations（RSDs）were all less than 3%（n=6）.7.The UPLC-Q-Exactive-HRMS preliminary infer the 15 chemical composition,which were（1）4-Hydroxybenzoic acid（2）Ononin（3）Choline（4）Kojic acid（5）Betaine（6）Citric acid（7）Isoliquiritigenin（8）Protocatechuic acid（9）Vitexin（10）Orientin（11）DL-Malic acid（12）Salicylic acid（13）Methylmalonic acid（14）Neochlorogenic acid（15）Gallic acid.Conclusion:1.The high,medium and low dose of water extract of Dallergia hancai Benth has good analgesic effect.2.The high and medium doses of water extract of Dallergia hancai Benth had good anti-inflammatory effect,but the low dose of water extract of Dallergia hancai Benth had no obvious anti-inflammatory effect.Its analgesic mechanism may be related to the decrease of PGE₂content in serum of mice,but not related to the content of cAMP in serum.2.The analgesic and anti-inflammatory effects of the water extracts of Dallergia hancai Benth from different origins were different.3.The fingerprints of ten batches of the water extracts of Dallergia hancai Benth from different origins showed good similarity,and the procatechuic acid and vitexin were identified.4.Based on the grey relational degree（GRA）and partial least squares regression analysis（PLS）,the peak 8,9,11,10,12,6,1 and 2 were selected as the most significant contribution to the analgesic effect from the analgesic spectrum,indicating that these peaks were the analgesic substance basis of the water extract of Dallergia hancai Benth.Peak8,2,9,10,11,12 and 6 were selected from the anti-inflammatory spectrum effect,which were the anti-inflammatory pharmacological material basis of the aqueous extract of Dallergia hancai Benth.Among them,peak No.2 was protocatechuic acid,peak No.11 was vitexin,and the rest of the characteristic peaks could not be identified,which needed further study.5.The method（HPLC）is accurate,stable and reproducible,and can be used for the determination of protocatechuic acid and vitexin in the water extract of Dallergia hancai Benth.6.The established method（microwave digestion-ICP-MS method）is rapid,simple,accurate and sensitive,and can be used for the determination of five kinds of heavy metals（Pb,Cd,As,Hg,Cu）in Dallergia hancai Benth.7.UPLC-Q-Exactive-HRMS can quickly predict the possible chemical components in the water extract of Dallergia hancai Benth,which can further provide experimental basis for the study of pharmacodynamic substances and chemical composition analysis of the water extract of Dallergia hancai Benth.