| According to the 2015 edition of Chinese pharmacopoeia,Coriolus versicolor has the functions and indications of invigorating the spleen,removing heat and detoxification,and it is used for jaundiced dampness,side pain,poor appetite and fatigue.The second class national drug,the intracellular glycoseptide,is mainly used in the treatment of liver protection and cancer.However,due to the traditional method of water extraction and alcohol precipitation for the preparation of intracellular glycoseptide,the crude extract was obtained with complex components,so the active components and structural characteristics of the intracellular glycoseptide were not clear.This paper will study the material basis of the effect of glycopeptide on neovascularization,and make efforts to develop glycopeptide drugs with clear active ingredients and clear therapeutic targets.The main research contents of this paper are as follows:1 Molecular pharmacognosy identification of Coriolus versicolorIn order to ensure the scientific production,research and drug safety.Therefore,it is necessary to adopt accurate and rapid modern molecular biological methods to identify the strains after successful culture.A genealogical tree was established to establish the relationship between the five strains.CHANGBAISHAN and LIGONGDA wild strains were isolated and cultured by tissue separation method.The culture of S1090,S1037 and S1340 mycelium slant surface was directly propagated for culture.The mycelia of five strains were cultured and identified by ITS sequence analysis.Sequencing results showed by BLAST: All five strains belong Eukaryota Fungi Dikarya Basidiomycota Agaricomycotina Agaricomycetes Polyporales Polyporaceae Trametes versicolor.Genealogical tree results show that: S1034 and LIGONGDA strains,S1090 and CHANGBAISHAN strains are closely related.2 Research on fermentation technology of Trametes versicolorTo meet a lot of people's needs.Both rhododendron and its intracellular glycoseptide were obtained by deep liquid fermentation.So it is necessary to screen the good strain and study the fermentation technology.In this chapter,the yield of wet mycelium,dry mycelium and glycoseptide were taken as indexes.Five strains were screened.Finally,three strains with the highest yield of S1090 were selected as production strains.According to the national standard of WS-XG-021-2002,the preliminary quality analysis of four batches of glycopeptide was carried out.The results show that: The color reaction of ninhydrin and naphthol were positive;The total sugar content was above 35%;The peptide content was above 25%;The monosaccharide content was all less than 10%.All the indexes were in line with the existing national standards for intracellular glycoseptide.Then the fermentation of industrial fermenters was studied,monitoring fermentation process,after 72 h fermentation.Monitoring results show that: the concentration of bacteria,pH and total sugar all decreased,dissolved oxygen did not pick up,microscopic examination of the thick mycelium,dyeing shallow,to meet the requirements of the can,so carry out the pot,the fermentation broth was obtained.3 Chemical property study of PSPThe chemical constituents of glucoseptide prepared by different strains under different fermentation conditions were different,therefore,we will analyze the chemical composition of the intracellular glycoseptide obtained by S1090 fermentation in an industrial fermentation tank,to provide a basis for the identification of active ingredients.This chapter refers to the national standard WS-XG-021-2002,Color identification,molecular weight distribution identification,total sugar content,peptide content,monosaccharide content,acidity determination,water determination were analyzed.The experimental results show that: the color reactions of ninhydrin and naphthol were positive;the retention time of the main peak of the molecular weight distribution was 17.930 min,the retention time of the main peak of the glucoseptide was 17.842 min,the retention time of the two main peaks is basically the same.;the total sugar content was 43%;The peptide content was 42%;the content of monosaccharide was 8.6%;the pH value of aqueous solution was 4.81;the water content of glucoseptide was 5.8%.4 Preliminary study of chemical composition of glycopeptide of Trametes versicolorOnly its mycelium was precipitated by water extraction and alcohol precipitation,there is no further research and development on other sites,in order to improve the utilization rate of the fermentation broth,it also provides the possibility for the development of new drugs for ganoderma.In this paper,the fermentation broth of ganoderma lucidum was systematically prepared and its chemical composition was preliminarily studied.After deep fermentation,the fermentation broth was obtained,fermentation metabolites are divided into intracellular and extracellular,the precipitation and supernatant were obtained after water extraction and alcohol precipitation treatment respectively.After drying,four products were obtained: CPV-a(extracellular supernatant),CPV-b(extracellular precipitate),CPV-c(intracellular supernatant),and intracellular glucoseptide(intracellular precipitate).The total sugar content,peptide content,monosaccharide composition and amino acid composition of the products were studied.The experimental results show that: the total sugar content of CPV-a is 51%,the peptide content is 29%,the monosaccharide composition is mainly glucose and mannose,the amino acid composition is mainly tyrosine,aspartic acid,threonine,proline and valine;Cpv-b total sugar content is 35%,peptide content is 11%,monosaccharide composition is mainly glucose and mannose,amino acid composition is mainly glutamic acid,alanine,threonine,semi-fatty acid and valine;Cpv-c total sugar content 56%,peptide content 20%,monosaccharide composition mainly for glucose and mannose,amino acid composition mainly for threonine,aspartic acid,alanine,glutamate and valine;The content of total sugar and peptide in rhododendron was 43% and 42% respectively.The monosaccharide was mainly composed of glucose,mannose and galactose,and the amino acid was mainly composed of aspartic acid,threonine and tyrosine.5 Structural feature analysis of glycopeptide of Trametes versicolorThe intracellular glycoseptide was purified,analyze its structural characteristics,provide reference for discussing material basis.In this chapter,DEAE-52 and CL-6B were used to purify the intracellular glycoseptide of Trametes versicolor,five purified intracellular glycoseptide components CPV-1-1,CPV-1-2,CPV-2-1,CPV-2-2 and CPV-2-3 were obtained.The structural characteristics of the five components were analyzed in terms of molecular weight,ultraviolet,infrared,nuclear magnetism,amino acid composition and monosaccharide composition.The molecular weights of the five components were 22486 Da,16982 Da,8813 Da,5228 Da and 6748 Da,respectively;Ultraviolet full-wavelength scanning results showed that the maximum absorption wavelength of the five components was about 210 nm,and there was an acromion around 280 nm;The infrared results showed that there were characteristic absorption peaks of polysaccharides and carboxyl protonation;The results of NMR are due to the larger molecular weight and the larger viscosity of aqueous solution,most of them have a uniform field and cannot get effective information,only CPV-1-2 and CPV-2-3 can be analyzed,There are characteristic peak information of benzene ring,glucose,pyranose and-glucoside bond;The five components of monosaccharide are identical,contain glucose,mannose and galactose;To establish a TLC identification method for intracellular glycoseptide of Trametes versicolor,N-butanol: acetone: water =5:1:1 expansion,P-aminobenzoic acid-glacial acetic acid-phosphoric acid as a chromogenic agent,sunlight and UV lamp at 365 nm,under this condition,the separation of monosaccharides was significant,the results were consistent with that of HPLC monosaccharide analysis;The amino acid composition of the five components is basically the same,main contents: aspartic acid,threonine and tyrosine.6 Study on the neovascularization of glycoseptide of Trametes versicolorTo identify the active components of intracellular glycoseptide and its therapeutic target,therefore,it is necessary to study the neovascularization activity of rhododendrin.This chapter selects the experimental model of chicken embryo allantoic membrane.In this paper,the activities of various glucopeptides and purified components of intracellular glucopeptides were compared and statistically analyzed,combined with the previous composition and structure analysis,a preliminary discussion of the material basis.The results of the activity study showed that all of the glucoseptide had inhibitory effect on the angiogenesis.The activity results showed that: The inhibition rates of CPV-a,CPV-b,CPV-c and intracellular glycoseptide were 37%,30%,36% and 28%,respectively.The results of statistical analysis showed that there was a significant difference in inhibition between supernatant products and precipitation products,the inhibition rates of intracellular glycoseptide were 28% and 30%,respectively.Statistical analysis results showed that there was no significant difference between the two: The inhibition rates of CPV-1-1,CPV-1-2,CPV-2-1,CPV-2-2 and CPV-2-3 were 27.4%,26.2%,27.9%,29.8% and 28.3%,respectively.The results of statistical analysis showed that there was no significant difference in the inhibitory effect among the five components. |
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