| Objective:Prepared the main ingredients with anti-tumor active of Phellinus igniarius by anti-tumor screening in vitro and high-speed countercurrent chromatography combined with semi-preparative liquid chromatography platform,and used the main ingredient as the guiding component to optimize the artificial cultivation conditions of Phellinus igniarius.Based on this,analyzed the chemical composition of alcohol extract of the cultivar of Phellinus igniarius by UPLC-HDMS technology in vivo and in vitro to clear the absorbed components in blood,and evaluated the influence of metabolic pathways of the cultivar of Phellinus igniarius by metabolomics investigation of rat serum and urine following intragastric administration of decoction of the cultivar of Phellinus igniarius after 28 days.The results will provide the reliable guidance for scaling artificial cultivation of Phellinus igniarius and lay the foundation for further research on the effective material basis and pharmacological effects of the cultivar of Phellinus igniarius.MethodsSeparated and prepared the main anti-tumor ingredients of Phellinus igniarius by anti-tumor screening in vitro and high-speed countercurrent chromatography combined with semi-preparative liquid chromatography platform,and used it as the guiding component to optimize the artificial cultivation conditions of Phellinus igniarius by response surface methodology.Based on this,applied the theory and method of Serum Medicinal Chemistry of Chinese medicine,utilized ultra-performance liquid chromatography high-resolution mass spectrometry(UPLC-HDMS)with MetaboLynx and Markerlynx software,to characterize and identify the chemical composition of alcohol extract of the cultivar of Phellinus igniarius and the transitional components in blood.Applied the theory and method of metabolomics,used Markerlynx software and metabolic databases of HMDB and KEGG,to investigate the metabolomics of rat serum and urine following intragastric administration of decoction of the cultivar of Phellinus igniarius for 28 days.Results1.The optimization of scale artificial cultivation conditions of Phellinus igniarius based on anti-tumor components guidingUtilized MTT method,to screen out the anti-tumor portion between alcohol extract,dichloromethane site of alcohol extract,ethyl acetate extract site of alcohol extract and crude polysaccharide extract of Phellinus igniarius in vitro,to observe the inhibition for human gastric carcinoma cells,human liver cancer cells,human lung cancer cells and human colon cancer cells.The results showed that dichloromethane site of alcohol extract of Phellinus igniarius has the better inhibition for digestive tumors,and has the strongest inhibition for the human colon cancer cell HT-29.The IC50 value was 13.08 ?g/mL.There were two major compounds prepared by high-speed countercurrent chromatography combined with semi-preparative liquid chromatography platform,which were identified as Hypholomine B and Inoscavin A.The anti-tumor activity of the two compounds was evaluated by cell chip technology.The results showed that Hypholomine B and Inoscavin A had a strong anti-tumor effect,IC50 values were 17.4 ?M and 11.3?M respectively.Establishing the UPLC method to simultaneous determine the content of Hypholomine B and Inoscavin A of Phellinus igniarius,and to analyze the contents of 14 batches of Phellinus igniarius collected from different national origins.On this basis,Hypholomine B as a higher content and stronger anti-tumor effect index component was used to optimize the scale artificial cultivation conditions of Phellinus igniarius.The optimized cultivation conditions were that cultivation temperature is 35?,cultivation humidity is 95%and the substrate is wheat.Under the conditions the average content of Hypholomine B is 1.046%2.Chemical analysis of alcohol extract of the cultivar of Phellinus igniarius in vitro and in vivo based on UPLC-HDMSThe analysis method for the chemical composition of alcohol extract of the cultivar of Phellinus igniarius and the transitional components in blood after intragastric administration of alcohol extract of the cultivar of Phellinus igniarius was established.20 chemical components in vitro and 27 absorbed components in blood were characterized,including 9 prototype components and 18 metabolites.20 chemical components in vitro included 2-Methyl-4-oxo-4H-pyran-3-yl-6-O-(3,4,5-trihydroxybenzoyl)-?-D-glucopyranoside,Protocatechuic aldehyde,Protocatechuic acid,Caffeic acid,4-Hydroxy-6-(4-hydroxystyryl)-2-pyrone,Osmundacetone,Hispidin,Interfungin B,Inotilone,Sternbin,Phelligridimer A,Davallialactone,Phellinstatin,Hypholomine B,Interfungin A,Inoscavin A,Phelligridin D,3-[4,5-Dihydroxy-2-(6-methyl-4-oxo-4H-pyran-2-yl)phenyl]-6-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-4-hydroxy-2H-pyran-2-one,Inonoblin A and(4Z)-3-(3,4-Dihydroxyphenyl)-6-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-1-hydroxy-4-(2-oxopro pylidene)-3a,4-dihydro-3H,8H-furo[3,4-c]oxepin-8-one.9 prototype components included Protocatechuic acid,Protocatechuic aldehyde,Caffeic acid,Osmundacetone,Hispidin,Interfungin B,Davallialactone,Hypholomine B and Interfungin A.18 metabolites are all the metabolite produces of prototype components in blood and their transformation pathways are mainly based on the glucuronidation.3.Influence of metabolic pathways by the cultivar of Phellinus igniariusThrough metabolomics study on serum and urine of rat following intragastric administration of decoction of the cultivar of Phellinus igniarius after 28 days,to investigate the influence of metabolism pathways by the cultivar of Phellinus igniarius.Serum metabolomics analysis showed that Phellinus igniarius could increase the levels of Cholic acid,Allocholic acid,Taurocholic acid,7-ketodeoxycholic acid,PC(20:3(5Z,8Z,11Z)/18:1(9Z)),LysoPC(20:0),Cer(d18:0/12:0),SM(d18:1/14:0)and 7a-Hydroxy-3-oxo-5b-cholanoic acid,and reduce the levels of LysoPE(22:4(7Z,10Z,13Z,16Z)/0:0,Palmitic amid,4-Hydroxy-L-proline,2,4-Diaminobutyric acid,PC(16:1(9Z)/18:1(11Z)),Oleamide and Corticosterone.Urine metabolomics analysis showed that Phellinus igniarius could increase the levels of D-Fructose 1-phosphate,S-Adenosylmethioninamine,Carboxytolbutamide and Hypoxanthine,and reduce the levels of Octanoylglucuronide,Estrone 3-sulfate,2-Hydroxydecanedioic acid,Neuraminic acid,D-(+)-Xylose,Methylguanine and 4-Hydroxy-L-threonine.After analysis of metabolic pathways the decoction of the cultivar of Phellinus igniarius colud affect some metabolic pathways included Glycerophospholipid metabolism,Primary bile acid biosynthesis,Steroid hormone biosynthesis,Cysteine and methionine metabolism,Arginine and proline metabolism and Purine metabolism,and could lower cholesterol levels and regulate the body's lipid metabolism.Through the in-depth analysis of differentiated metabolites we speculated that the cultivar of Phellinus igniarius may have the lipid regulation,hypoglycemic and antitumor functions.ConclusionIn this study,firstly we separated and prepared the anti-tumor components of Phellinus igniarius,and used Hypholomine B as component guiding to optimize the scale artificial cultivation conditions of Phellinus igniarius.The optimized cultivation conditions were that cultivation temperature is 35?,cultivation humidity is 95%and the substrate is wheat.Then we had systematic study on the chemical composition of the cultivar of Phellinus igniarius by UPLC-HDMS.20 compounds were identified in vitro and 27 absorbed components in blood included 9 prototype components and 18 metabolites were identified in vivo.The identified metabolites were mainly the glucuronide conjugates of the prototype components which were absorbed into blood.At last,we investigated the metabolomics of rat serum and urine following intragastric administration of decoction of the cultivar of Phellinus igniarius after 28 days.The results showed that the cultivar of Phellinus igniarius could influence some metabolic pathways included glycerophospholipid metabolism,primary bile acid biosynthesis,steroid hormone biosynthesis,cysteine and methionine metabolism,arginine and proline metabolism and purine metabolism.After the in-depth analysis of differentiated metabolites we speculated that the cultivar of Phellinus igniarius may have the lipid regulation,hypoglycemic and antitumor functions.These results would provide the guidance for large-scale artificial cultivation of Phellinus igniarius and lay the foundation for further research on the effective material basis and pharmacological effects of the cultivar of Phellinus igniarius. |
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