| Chinese medicines have been widely studied because they contain a variety of active ingredients and have a variety of pharmacological effects.The efficient extraction and separation of the active ingredients with high purity in Chinese medicines and the investigation of their related pharmacological effects are the hot research topics of interest in the field of Chinese medicine research.In this thesis,Chinese medicines with origin in Jilin Province: Inonotus obliquus,Phellinus igniarius and Trifolium repens were studied,the extraction,screening,isolation and pharmacological effects of α-glucosidase and lactate dehydrogenase inhibitors associated with diabetes and lactate dehydrogenase inhibitors associated with heart failure in Inonotus obliquus and Phellinus igniarius,and lipoxygenase inhibitors associated with inflammation in Trifolium repens were systematically investigated.The specific work of the thesis is as follows:1.Study of α-glucosidase and lactate dehydrogenase inhibitors in Inonotus obliquus.A method for the extraction of triterpenoids from Inonotus obliquus was established by combining the ionic liquid 1-butyl-3-methylimidazole bromide salt with heat assisted extraction,and the extraction rate was increased by 58.0% at the same extraction time.Using ultrafiltration technology,the α-glucosidase and lactate dehydrogenase related to diabetes and heart failure were selected as the targets,and the aromatic compounds of Inonotus obliquus:(E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one as α-glucosidase inhibitor and the triterpenoids:betulin and lanosterol as lactate dehydrogenase inhibitor.The mechanism of action between(E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one and α-glucosidase,betulin and lactate dehydrogenase,as well as lanosterol and lactate dehydrogenase was investigated by molecular docking technique.The separation method of(E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one in Inonotus obliquus was established by combining the UNIFAC mathematical model with high speed countercurrent chromatography.10.3 mg of(E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one with 95.9% purity was obtained from 900 mg of Inonotus obliquus extract.The method improved the separation efficiency,reduced the amount of solvent and shortened the separation time.The separation of lactate dehydrogenase inhibitors from Inonotus obliquus was performed by high speed countercurrent chromatography.After optimizing the separation conditions,a total of 95.9% and 97.8% purity of betulin and lanosterol were obtained,respectively.The effects of the extracts of Inonotus obliquus,(E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one,betulin and lanosterol on the central nervous system were investigated by gavage administration in ICR mice,and the high safety of Inonotus obliquus in clinical use was verified.The experimental results provide a basis for the application of the active ingredients in Inonotus obliquus and guarantee the safety of Inonotus obliquus.2.Study of α-glucosidase and lactate dehydrogenase inhibitors in Phellinus igniarius.Using ultrafiltration technology,the α-glucosidase and lactate dehydrogenase,which are related to diabetes and heart failure,were selected as targets for the screening of active ingredients in Phellinus igniarius,respectively.The key residues of active ingredients in Phellinus igniarius interacting with α-glucosidase were analyzed by molecular docking technique as TRP1355,PHE1559 and HIS1584,interacting with the A and B chains ofα-glucosidase through hydrophobic interactions,hydrogen bonding,π-π stacking and salt bridges.The key residues in Phellinus igniarius that interact with lactate dehydrogenase are LEU108,ASN137,GLU191,ASP194,ALA29,TYR246,VAL30 and THR247,which interact with lactate dehydrogenase through hydrophobic interactions,hydrogen bonds and salt bridges.The active components in Phellinus igniarius were separated using semi-preparative high performance liquid chromatography technique,and three active components were obtained: Davallialactone,Inoscavin A and Hypholomine B,with purity of 97.6%,96.1% and97.1%,respectively.The effects of Phellinus igniarius extract,Davallialactone,Inoscavin A and Hypholomine B on the central nervous system were investigated by gavage administration experiments in ICR mice,which verified the high safety of Phellinus igniarius when used in clinical practice.The experimental results provide a new method for the isolation of the active ingredients of Phellinus igniarius and guarantee the safety of Phellinus igniarius.3.Study of lipoxygenase inhibitors in Trifolium repens.An ultrasound-assisted enzymatic extraction method for isoflavones in Trifolium repens was established,the extraction conditions were optimized by response surface methodology,and the extraction rate was significantly improved.Using ultrafiltration technology,the active ingredients in Trifolium repens were screened as the targets of lipoxygenase related to anti-inflammatory effect.The key residues of the active ingredients in Trifolium repens interacting with lipoxygenase were analyzed by molecular docking technique as ARG99,ARG166,PRO110,LYS388,PHE167,ASP164 and TRP100,interacting with the A and B chains of lipoxygenase mainly through hydrophobic interaction and hydrogen bonding.The separation of lipoxygenase inhibitors from Trifolium repens was carried out by high speed countercurrent chromatography with gradient flow rate combined with semi-preparative liquid chromatography.Five lipoxygenase inhibitors were obtained: ononin,daidzein,genistein,formononetin and biochanin A.The purity was 98.7%,96.5%,96.2%,97.9% and 98.1% in order.The effects of Trifolium repens extract,ononin,daidzein,genistein,formononetin and biochanin A on the central nervous system were investigated by gavage administration experiments in ICR mice,and the high safety of Trifolium repens in clinical use was verified.The experimental results provide a new approach for the development of Trifolium repens in anti-inflammatory drugs and guarantee the safety of Trifolium repens. |
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