Study On Chemical Constituents And Bio-activities Of Apostichopus Japonicus Selenka

Apostichopus japonicus Selenka is a marine animal of Echinodermata,Holothuroidea,Aspidochirotida,Stichopodidae and Apostichopus.Apostichopus japonicus Selenka is the most valuable species of sea cucumber in China with extremely high economic value.As a traditional medicinal and edible variety,Apostichopus japonicus Selenka not only has high nutritional value and dietary supplement,but also has high medicinal value.Modern medical research shows that Apostichopus japonicus Selenka contains sea cucumber saponins,cerebrosides,gangliosides,polysaccharides,polypeptides,sterols,lipids and other biologically active substances,which have the effects of anti-coagulation,anti-tumor,anti-bacterial,anti-radiation,anti-virus and enhancement of immunity and so on.The research on Apostichopus japonicus Selenka mainly focuses on the extraction,isolation,purification and structural identification of chemical components,screening of biological activities,processing technology and quality standards and so on.The in-depth study of the chemical components in Apostichopus japonicus Selenka is the premise and foundation for other related researches.Continuously searching for new chemical components in Apostichopus japonicus Selenka and screening their physiological activities will not only help to develop the medicinal use,but also expand the health care use of Apostichopus japonicus Selenka.In addition,the establishment of new methods for the determination of the active or index components in Apostichopus japonicus Selenka will make the evaluation system of its quality more scientific and reasonable,which will help to improve the competitiveness in the market.In conclusion,an in-depth study on the chemical composition and biological activity of imitation ginseng can provide more scientific and technological support for the further development of Apostichopus japonicus Selenka culture industry and the development and utilization of its resources.Therefore,on the basis of summarizing the research results of the previous researchers,this thesis conducted a further in-depth study on the chemical composition and biological activity of Apostichopus japonicus Selenka.This thesis mainly includes the following aspects:1.Firstly,this paper used fresh Apostichopus japonicus Selenka as raw material,extracted by 95%ethanol refluxed,the extract was recovered from ethanol and extracted with ethyl acetate and n-butanol,respectively,and the extract was separated and purified by silica gel column chromatography coupled with semi-preparative high-performance liquid chromatography,and a total of 15 monomeric compounds were isolated,which included ten sea cucumber saponins and five cerebrosides.The chemical structures of seven sea cucumber saponins were identified by 1D-NMR,2D-NMR,HR-ESI-MS,and other spectroscopic methods,and the structures of the other compounds are still under identification.The seven sea cucumber saponins included two new sea cucumber saponins and five known ones.Two new sea cucumber saponins were 3β-O-{2-O-[3-O-methyl-β-D-glucopyranosyl-（1→3）-β-D-xylopyranosyl-（1→4）-β-D-quinovopyranosyl]-4-O-[β-D-glucopyranosyl]-β-D-xylopyranosyl}-holosta-7（8）,25（26）-dien-16-one（compound HS-1,named Holotoxin E₁）and 3β-O-{2-O-[3-O-methyl-β-D-glucopyranosyl-（1→3）-β-D-xylopyranosyl-（1→4）-β-D-glucopyranosyl]-4-O-[β-D-glucopyranosyl-（1→3）-β-D-glucopyranosyl]-β-D-xylopyranosyl}-holosta-7（8）,25（26）-dien-16-one（compound HS-2,named Holotoxin E₂）.The five known sea cucumber saponins are Cladoloside B,Holotoxin A₁,Holotoxin B₁,Holotoxin E and Holotoxin D.In addition,this paper summarizes some regular characteristics of the NMR spectral data of sea cucumber saponins by analyzing the NMR spectral data of sea cucumber saponins,which provides a reference basis for the identification of the chemical structure of sea cucumber saponins.2.On the basis of the above research on chemical composition,this paper established a new method for the simultaneous determination of three sea cucumber holostane-type saponins Holotoxin B₁,Holotoxin E and Holotoxin A₁in fresh Apostichopus japonicus Selenka by MSPD-HPLC-UV method,and the conditions of MSPD were as follows:the dispersant was silica gel and the ratio of the mass of sample to the dispersant was 1:1;the cleaning agent was ethyl acetate（40 m L）,and the eluent was methanol（60 m L）.The HPLC-UV conditions were as follows:the chromatographic column was a Tnature C18 column（5μm,4.6×250 mm,Waters,Ireland）at 37℃;the mobile phase was acetonitrile-water（35.5:64.5）at a flow rate of1 m L/min;the detection wavelength was 203 nm.Methodological investigations showed that the linear ranges of the three sea cucumber holostane-type saponins were Holotoxin B₁0.24-9.55μg,Holotoxin E0.25-9.85μg and Holotoxin A₁0.21-8.31μg,respectively;the detection limits were Holotoxin B₁5.05 ng,Holotoxin E 4.56 ng and Holotoxin A₁7.58 ng,respectively.The analytical method developed in this paper has the advantages of wide linear range,high sensitivity,high accuracy,good precision as well as rapid,convenient,environmentally friendly and economical.The method was used to determine the contents of three sea cucumber saponins in fresh Apostichopus japonicus Selenka from Dalian and the results showed that the contents of the three sea cucumber holostane-type saponins Holotoxin B₁,Holotoxin E and Holotoxin A₁,were 6.24μg/g,9.58μg/g and 96.2μg/g,respectively.3.In this paper,the cytotoxic activities of the six fractionated sea cucumber saponins were firstly investigated.The results showed that,except for Holotoxin E₂,Holotoxin A₁,Holotoxin B₁,Holotoxin E,Cladoloside B and Holotoxin E₁exhibited high cytotoxic activities against two types of cancer cells,MDA-MB-231 and Hep G2,with IC₅₀values ranging from 2.386-18.86μmol/L.Then,using sea cucumber saponins and their secondary saponins as research objects,this paper analyzed the intersecting targets,PPI protein interactions,KEGG and GO enrichment results by using network pharmacology research methods and inferred the core targets related to liver and breast cancers that sea cucumber saponins and their secondary saponins might act on.The interaction between sea cucumber saponins and their secondary saponins and SRC was investigated by molecular docking technique,and the affinity of the interaction between sea cucumber saponins and their secondary saponins and SRC was systematically explored,and the conformational relationship between them was also discussed in depth.The results showed that the affinity of the interaction between sea cucumber saponins and their secondary saponins with SRC had a certain correlation with the number of sugars attached to the 3-position of the aglycone and the mode of attachment,and it was deduced that sea cucumber saponins and their secondary saponins containing fewer sugar groups might have better anticancer activities.Finally,in this paper,combined with the above results,in order to obtain the secondary saponins,acid hydrolysis and alkaline hydrolysis of sea cucumber saponin Holotoxin A₁and total sea cucumber saponin from Apostichopus japonicus Selenka were carried out,and the hydrolyzed products were isolated and purified.As a result,one Apostichopus japonicus Selenka secondary saponin monomer was isolated from the acid hydrolysis of Holotoxin A₁,which were 3β-O-{4-O-[3-O-methyl-β-D-glucopyranosyl-（1→3）-β-D-glucopyranosyl]-β-D-xylopyranosyl}-holosta-9（11）-en-16-one(compound HSS-1,named Holotoxin A_1-1),a secondary saponin was fractionated in the alkaline hydrolysis product,and ¹³C NMR showed that its parent structure was also changed and the structure is being characterized.The experimental results showed that acid hydrolysis and alkali hydrolysis can hydrolyze the sugar group of saponin,but the structure of the aglycone will be changed at the same time.It is suggested that the secondary saponins whose aglycone structure is not changed cannot be obtained by acid hydrolysis or alkali hydrolysis and the subsequent studies should be carried out by using more mild hydrolysis methods（such as enzyme hydrolysis）to obtain the secondary saponins whoseaglycones are unchanged.In summary,this dissertation has achieved innovative research results in the study of chemical composition of Apostichopus japonicus Selenka,determination of the content of active ingredients and the study of anti-tumor constitutive relationship of sea cucumber saponins.The results of this thesis have enriched the number of sea cucumber saponins in Apostichopus japonicus Selenka,established a new content determination method with guiding significance for the quality evaluation of Apostichopus japonicus Selenka as well as the research and development of related health foods and new drugs,provided a new research idea and research direction to find secondary sea cucumber saponins with stronger anti-tumor activity from the hydrolysis products of natural sea cucumber saponins,and provided new scientific bases for the promotion of the development and utilization of imitation ginseng.The new research idea and research direction provide a new scientific basis for promoting the development and utilization of Apostichopus japonicus Selenka.