Chemical Composition Analysis,Antipyretic And Anti-inflammatory Substances Research And Product Development Of Polygoni Chinensis Herba

Polygoni Chinensis Herba have antipyretic and anti-inflammatory effects,but the specific chemical components are not clear,and the mechanism of action is not clear enough.in this paper,the antipyretic and anti-inflammatory mechanism of the active substances in Polygoni Chinensis Herba was taken as the starting point.Focusing on the five factors of“differential metabolites-pyrogenic factors-metabolic pathways-pharmacological effects-product development”,to carry out the separation and identification of chemical constituents,screening of active substances,research on the mechanism of antipyretic and anti-inflammatory effects and product development.The chemical components of Polygoni Chinensis Herba were detec-ted by UPLC-MS,a total of 359 chemical components in 20catego-ries were identified,including 146 phenolic acids,78 flavono ids,49 alkaloids,44 tannins,16 lignans and coumarins,2 terpenoids,1 qui-none,5 stilbenes and 18 others.The chemical constituents of Polygoni Chinensis Herba were extracted and separated,17 compone-nts were obtained and 10 components were identified,which wereellagic acid（309.0 mg）>hyperoside（15.0 mg）>gallic acid（14.4 mg）>rutin（14.2 mg）>β-sitosterol（12.0 mg）>quercetin（9.6 mg）>corilagin（6.1mg）>brocchlin carboxylic acid（5.3 mg）>brevifolin（5.0 mg）>quercetin-3-α-O-rhamnoside（4.8 mg）.The activity of LPS induced RAW264.7 cell inflammation mo-del was screened.Based on the inhibition rate of cell viability,9com-pounds had different degrees of inhibition rate,from strong to weak:gallic acid（99.22%）>quercetin（88.42%）>corilagin（75.18%）>Ellagic acid（4.77%）>brocchlin carboxylic acid（65.90%）>hyperoside（57.91%）>brevifolin（54.99%）>rutin（29.88%）>quercetin-3-α-O-rhamnoside（2.09%）.The inhibition rate of quercetin,gallic acid,corilagin and ella-gic acidmore than 70%.LPS-induced rabbit fever model was used to screen the antipyretic effect of ethanol extract and three compounds.On the basis of reducing the maximum change rate of rectal temp-erature in rabbits,the intensity of antipyretic effect was ellagic acid（139.1%）>ethanol extract（81.1%）>gallic acid（80.0%）>quercetin（75.7%）.Combined with the results of anti-inflammatory effect of RAW264.7 cells,ellagic acid was determined to be the active substance of ant-ipyretic and anti-inflammatory effect of Polygoni Chinensis Her-ba.The rabbit fever model induced by LPS was established to investigate the anti-inflammatory mechanism of ellagic acid（EA）.The rectal temperature of rabbits in each dose group of EA decreased significantly until 7h.The contents of IL-1β,IL-6,TNF-αand MDA in serum were significantly decreased,and the activities of SOD and GSH were increased.The levels of c AMP and PGE₂in cerebrospinal fluid were significantly decreased.Significantly reduced c AMP and 5-HT levels in the hypothalamus;except that there was no significant difference in the expression of COX-2 protein in EALG,the expression of p-NF-κB p65and COX-2 protein in the hypothalamus of the other dose groups was significantly down-regulated.EA can inhibit the secretion of inflammatory factors,alleviate oxidative stress,and exert anti-inflammatory and antipyretic effects by blocking the NF-κB/COX-2 signaling pathway.GC-MS technology was used to analyze the metabolomics of fever rabbit serum at 4h and 7h.the results showed that 12 differential metabolites were screened at 4h,and 7 of them were reversed to normal levels by EA.Combined with the correlation coefficient values with pharmacodynamic indicators,2 key differential metabolites of propionic-acid and pyridine were obtained.At 7h,10 differential metabolites were screened out,and 6 of them were reversed to normal levels.Combined with the correlation coefficient values,4 key differential metabolites of L-valine,inositol,urea and 2-Monopalmitin were obtained.When the levels of propionic acid,pyridine and L-valine were up-regulated by EA,the expression of MDA,IL-1βand TNF-αwas inhibited,and the activity of GSH was increased.When the levels of inositol,urea and2-Monopalmitin were down-regulated by EA,the expression of MDA,IL-1βand TNF-αwas inhibited,the activity of SOD and GSH was increased,the inflammation was reduced,and the oxidative stress was alleviated.The metabolic pathways of 4h and 7h differential metabolites involved galactose metabolism,synthesis and degradation of ketone bodies,ascorbate and aldarate metabolism,D-galactose and inositol hit pathways.In addition,pyruvate metabolism,glycerolipid metabolism,inositol phosphate metabolism,valine,leucine and isoleucine biosynthesis pathways involve sugar,fat,amino acids and other energy substances,indicating that EA exerts antipyretic and anti-inflammatory effects by regulating metabolic pathways and metabolites on the pathways,which is closely related to energy metabolism and fat metabolism.The fever model of rats induced by yeast was established.The results showed that the body surface temperature and rectal temperature of rats in each dose group of EA decreased significantly until the 19h.The levels of TNF-α,IL-6,IL-1β,PGE₂and MDA in serum were significantly decreased,and the activities of SOD and GSH were increased.The levels of c AMP and PGE₂in spinal fluid were decreased.Except that there was no significant difference in the expression of IκB-αprotein of rats in EALG,the expression of p-NF-κB p65 and IκB-αprotein in the hypothalamus of the other dose groups was significantly down-regulated.Based on the NF-κB signaling pathway,EA can inhibit the expression of IκB-αprotein,block the translocation of NF-κB p65 from cytoplasm to nucleus,reduce the production of TNF-αand IL-6 downstream pro-inflammatory factors,and achieve antipyretic and anti-inflammatory effects.UPLC-MS/MS was used to analyze the metabolomics of the br ain of fever rats,and 11 differential metabolites were screened.Incl uding 2-hydroxyphenylacetic acid,SM（d34:1）,PC（18:2（9Z,12Z）/P-18:1（11Z））,PC（20:4（8Z,11Z,14Z,17Z）/P-18:0）,PC（O-16:0/20:5（5Z,8Z,11Z,14Z,17Z））,PC（16:0/16:0）,PC（18:2（9Z,11Z）/15:0）,SM（d18:1/14:0）,PC（16:0/22:6（4Z,7Z,10Z,13Z,16Z,19Z））,PE（16:0/18:2（9Z,12Z））,PC（18:1（9Z）/18:1（9Z））.All of them belong to glycerophospholipids and sphingomy elins.the levels of 8 glycerophospholipid metabolites increased,which were converted into arachidonic acid,and PGE₂was generated under the action of COX-2.The levels of 2 sphingolipid metabolites incre-ased,which increased the expression of S1P and promoted the relea-se of IL-1β.EA can significantly down-regulate the levels of glyce-rophospholipids and sphingolipids,inhibit the expression of S1P and COX-2,block the inflammatory pathway to produce heat-causingfactors,and achieve antipyretic and anti-inflammatory effects.GC-MS technology was used to analyze the serum of febrilerats by metabolomics,and 15 differential metabolites were screened.Among them,9 metabolites could be reversed by EA,and the P value of 5 metabolites was statistically significant.Four metabolites were negatively correlated with TNF-α,except for L-tyrosine,pvalues were statistically significant.After fever,the levels of D-（-）-l actic acid,glycerin,phosphoric acid and 5-oxo-L-proline decreased,and the expression of TNF-α,IL-6,PGE2 and MDA increased.Afte r administration of EA,the contents of four metabolites increased,and the levels of TNF-α,IL-6,PGE₂and MDA were inhibited.Low levels of L-tyrosine can increase SOD activity.The metabolic path ways of 15 potential biomarkers involved Linoleic acid metabolism,Phenylalanine,tyrosine and tryptophan biosynthesis,Galactose metabol-ism,Biosynthesis of unsaturated fatty acids,Glycerolipid metabolism,and 8 potential biomarkers hit pathways.When the heat source acts on the temperature regulation center,fat accelerates decomposition and heat production increases.The significant increases in glycerin,hexadecanoic acid and oleic acid were associated with lipolysis,in-dicating that eight metabolites were potential biomarkers of fever.EA first regulates the level of differential metabolites,inhibits therelease of inflammatory factors,and then regulates the energy meta-bolism pathway.The differential metabolites mainly belong to amino acids,carbohydrates and lipids,which are related to amino acidmetabolism,carbohydrate metabolism and lipid metabolism,showing target polymorphism.The moisture absorption percentage,angle of repose,critical relative humidity and other micropowder data were used as the indexes to investigate the preparation process of Huotanmu capsules,and the stability of Huotanmu capsules was examined.The optimum molding process of Huotanmu capsules was determined as follows:the main drug and microcrystalline cellulose were mixed evenly at a ratio of 2.5:1,and80%ethanol was used as a wetting agent to granulate.After the whole grain was filled into No.0 capsule.The water content was 8.6%,the average loading difference was 2.36%,and the disintegration time was13.3 min.The results of pilot test,stability test and quality test showed that the process was reasonable and feasible.In summary,the chemical constituents of Polygoni Chinensis Herba were extracted,isolated and identified.EA was identified as the an-tipyretic and anti-inflammatory active substance of Polygoni Chinensis Herba by in vitro experiments and in vivo screening.Based on the LPS induced fever model of rabbits and the yeast induc-ed fever model of rats,the relationship between EA and TNF-α,IL-6,IL-1β,PGE₂and c AMP was systematically established.The mechanis-m of COX-2/IκB-α/NF-κB signaling pathway was elucidated.Combi-ned with the overall efficacy evaluation of metabolomics,the antip-yretic and anti-inflammatory effects of EA and the mechanism of endogenous biomarkers were revealed.The research model and theor-etical viewpoint of“differentialmetabolites-pyrogenicfactors-metabo-lic pathways-pharmacodynamic effects-product development”（five in one）were put forward,which reflected the characteristics of multi-component,multi-target and multi-pathway,and provided scientific basisfor the chemical composition,antipyretic and anti-inflammatory mechan-ism and product development of Polygoni Chinensis Herba,which was beneficial to promote the high-quality development of characteristic medicinal materials in Guangxi of Polygoni Chinensis Herba.