Characterization Of Chemical Constituents Of Poria Cocos And Primary Study Of Their Protective Effects Against Cisplatin-induced Intestinal Injury

Poria is the dried sclerotia of Poria cocos(Schw.)Wolf.It is one of the most important traditional Chinese medicines which is considered to be diuretic,sedative and tranquilizing.The herb also has the effect of tonifying spleen and strengthening the body.Many prescriptions containing Poria,such as Shen-Ling-Bai-Shu-San and Qiong-Yu-Gao,are also famous for their effect of replenishing qi to invigorate the spleen.The chemical constituents in Poria can be divided into 3 groups according to their different solubility which are triterpene acids,water-soluble and alkali-soluble polysaccharides.In those prescriptions famous for their effect of invigorating the spleen,Poria often exists in the form of powder.When it is eaten,all 3 groups of constituents enter the body's digestive system and are absorbed and metabolized.However,no systematic research has been done to determine the constituents contributing to protective efficacy of Poria.In order to better develop and utilize this herb resource,it is necessary to systematically study the constituents contributing to its efficacy.Based on the extraction and characterization of 3 groups of constituents in Poria,mice model of intestinal injury induced by cisplatin was established,to evaluate the role of 3 groups of constituents in the intestinal protective activity of Poria from the perspective of anti-inflammatory effects,intestinal microbial diversity and metabolic regulation.The research can be divided into 3 parts:1.Preparation and quality control of triterpene acids in Poria.Huge datasets are acquired when characterizing triterpene acids in Poria using ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight tandem mass spectrometry(UPLC-ESI-QTOF-MS/MS).Data interpretation can be laborious and time-consuming.Furthermore,it is often a challenge to discriminate positional isomers that share identical molecular weights and similar fragmentation behaviour in MS-based analysis.In order to facilitate data interpretation,an in-silico predicted analogue database was established based on the skeletons and modifying groups of known triterpene acids previously reported in Poria.UPLC-QTOF-MS/MS data of triterpene acids was used to rapid screen the interested triterpene acids in Poria.Then,combining the fragment patterns and chromatographic behavior of reference compounds and triterpene acids reported,the screened compounds were identified.As a result,a total of 62 triterpene acids were unequivocally or tentatively identified,among which 17 were identified for the first time in Poria.In this study,when extracting the triterpene acids from Poria,ethanol was firstly used,and ethanol extracts were further extracted with acetic ether to enrich the active ingredients,and the final extraction yield was 0.30%.However,the study on the chemical consistency of triterpene acids in ethanol extracts and acetic ether extracts thereof has not been reported,it is necessary to scientifically evaluate the value of further extraction with acetic ether.Based on the result mentioned above,a mimic multiple reaction monitoring(mMRM)mode was established using UPLC-QTOF-MS/MS to quantify the triterpene acids in ethanol extracts and acetic ether extracts thereof.The calculation of transfer and enrichment rate proved that the ethanol extracts and acetic ether extracts have good chemical consistency,and triterpene acids can be effectively enriched by the extraction process.The present study provides scientific evidence for further extraction of triterpene acids in Poria by acetic ether.2.Preparation and quality control of polysaccharides in Poria.The water-soluble and alkali-soluble polysaccharides in Poria were extracted with water and alkali,and the final extraction yields were 0.38%and 63.7%,respectively.A HPGPC method was developed to characterize the molecular weight distribution of water-soluble polysaccharides.The molecular weight ranges from 3.0×103 to 2.3×107 Da as detected by HPGPC.To characterize the monosaccharide composition of water-soluble and alkali-soluble polysaccharides,a HPLC-DAD method was developed and validated.The results showed that the water-soluble polysaccharides in Poria were mainly composed of mannose,glucose and galactose,with the molar ratio of 1.00:8.37:2.23,and the alkali-soluble polysaccharides were mainly composed of glucose.3.Primary study on constituents contributing to protective efficacy of Poria against cisplatin-induced intestinal inj uryMice model of intestinal inj ury induced by cisplatin was established,to evaluate the role of 3 groups of constituents in the intestinal protective activity of Poria from the perspective of anti-inflammatory effects,intestinal microbial diversity and metabolic regulation.Cisplatin could cause structural disorder of intestinal mucosa in mice,abnormal crypt structure and inflammatory cell infiltration,and can also induce the rise of inflammatory factors IL-2 and TNF-a in peripheral blood,water-soluble polysaccharides had a good regulation effect on the abnormal change of colonic morphology and inflammatory reaction induced by cisplatin;from the perspective of intestinal microbial diversity,water-soluble polysaccharides can regulate the composition of intestinal flora changed by cisplatin,increasing the relative contents of probiotics(such as Prevotellaceae)while reducing the relative contents of pathogenic bacteria(such as Ruminococcaceae and Helicobacteraceae);from the perspective of metabolic regulation,water-soluble polysaccharides had relatively better effects on the regulation of the metabolic profile of mouse serum,urine and feces changed by cisplatin.Analysis of metabolic pathway indicated that water-soluble polysaccharides can restore the energy,amino acid and nucleotide metabolism balance by regulating the disorder of metabolic pathways such as lipid metabolism,amino acid metabolism,and purine metabolism to alleviate intestinal damage caused by cisplatin.Therefore,combined with anti-inflammatory effects,intestinal microbial diversity and metabolic regulation,it is speculated that water-soluble polysaccharides play a major role in the intestinal protective activity and have a similar mechanism of action to Poria.