CN110698532A - Method for extracting sea cucumber saponin Cladoloside A - Google Patents

Method for extracting sea cucumber saponin Cladoloside A Download PDF

Info

Publication number
CN110698532A
CN110698532A CN201911039830.4A CN201911039830A CN110698532A CN 110698532 A CN110698532 A CN 110698532A CN 201911039830 A CN201911039830 A CN 201911039830A CN 110698532 A CN110698532 A CN 110698532A
Authority
CN
China
Prior art keywords
extraction method
cladoloside
water
methanol
macroporous resin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201911039830.4A
Other languages
Chinese (zh)
Other versions
CN110698532B (en
Inventor
刘桂英
王旭达
周遵春
宋伦
吴金浩
王昆
杜静
葛坤
李爱
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
RESEARCH INSTITUTE OF OCEAN FISHERY SCIENCE LIAONING PROVINCE
Original Assignee
RESEARCH INSTITUTE OF OCEAN FISHERY SCIENCE LIAONING PROVINCE
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by RESEARCH INSTITUTE OF OCEAN FISHERY SCIENCE LIAONING PROVINCE filed Critical RESEARCH INSTITUTE OF OCEAN FISHERY SCIENCE LIAONING PROVINCE
Priority to CN201911039830.4A priority Critical patent/CN110698532B/en
Publication of CN110698532A publication Critical patent/CN110698532A/en
Application granted granted Critical
Publication of CN110698532B publication Critical patent/CN110698532B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring

Abstract

The invention discloses an extraction method of sea cucumber saponin Cladoloside A, belonging to the technical field of biological separation. The invention leaches the apostichopus japonicus body wall through ethanol water solution, concentrates and separates the concentrated solution by AB-8 macroporous resin, collects extractum, and obtains holothurin crude extract. Separating and purifying by using a forward and reverse silica gel column to obtain a pure product, and performing structural analysis and identification by using nuclear magnetic resonance spectroscopy to determine the compound Cladoloside A. The extraction method is simple, convenient, fast, efficient and green, the holothurin Cladoloside A with higher purity can be obtained, and the compound is extracted from the apostichopus japonicus for the first time, so that a foundation is laid for further exploring the pharmacological activity of the holothurin Cladoloside A, and the method has scientific significance for deeply researching the physiological synthesis of the holothurin in the apostichopus japonicus.

Description

Method for extracting sea cucumber saponin Cladoloside A
Technical Field
The invention relates to a method for extracting sea cucumber saponin Cladoloside A, belonging to the technical field of biological separation.
Background
Stichopus japonicus selenka saponin is the main active component of Apostichopus japonicus selenka, and is distributed in any part of body wall, muscle, intestinal tract, respiratory tree, body cavity fluid, etc., but the types and contents of Stichopus japonicus selenka saponin are different. The apostichopus japonicus wall has high content of holothurin, has activities of resisting fungi, tumors, blood coagulation and the like, and has more and more approved medicinal and health-care values. Therefore, the pharmacological activity of holothurin is receiving wide attention. At present, scholars at home and abroad find and determine more than 100 holothurin saponins with structures, most aglycones of the holothurin saponins have basic skeletons of lanosterol, but no related report about extracting holothurin Cladoloside A from apostichopus japonicus body walls is found.
At present, the extraction of total saponins of sea cucumber is more in China, the extraction of sea cucumber saponin monomers is less, the technical difficulty is higher, monomer compounds with higher purity are not easy to obtain, and the further pharmacological activity research is difficult to carry out. The invention provides a method for separating and obtaining holothurian saponin Cladoloside A from the wall of an apostichopus japonicus selenka body.
Disclosure of Invention
The invention provides an extraction method of sea cucumber saponin Cladoloside A, which can be used for simply, efficiently and quickly purifying the sea cucumber saponin Cladoloside A and comprises the following steps:
(1) selecting fresh Apostichopus japonicus, dissecting, removing viscera, pulverizing to obtain Apostichopus japonicus meat, adding 80% ethanol water solution, reflux-extracting for 3 times, filtering the extractive solution while it is hot, collecting filtrate, concentrating, and recovering to obtain concentrated extract as total extract;
(2) uniformly dispersing the total extract obtained in the step (1) in water, performing gradient elution through a macroporous resin column, performing thin-layer chromatography analysis on water and 80% ethanol in the order of elution, and collecting 80% ethanol eluent;
(3) recovering the 80% ethanol eluate obtained in step (2), and concentrating to obtain total saponins extract of Stichopus japonicus;
(4) and (3) passing the sea cucumber total saponin extract obtained in the step (3) through a forward silica gel column, performing gradient elution by using chloroform, methanol and water in different proportions as elution solvents, performing thin-layer chromatography on the eluent, further separating and purifying the part with large content by twice reverse silica gel column chromatography, and concentrating to finally obtain colorless crystalline powder, namely the sea cucumber saponin Cladoloside A.
Further, in the above technical scheme, the mass ratio of the stichopus japonicus meat and the 80% ethanol aqueous solution in the step (1) is 1: and 7, extracting for 2 hours, 1.5 hours and 0.5 hour respectively, and concentrating the filtrate in a vacuum rotary concentration mode, wherein the concentration temperature is 40 ℃.
Further, in the above technical scheme, the particle size of the macroporous resin filler in the macroporous resin chromatographic column in step (2) is 0.3-1.25 mm.
Further, in the above technical scheme, the mass ratio of the total extract to the macroporous resin in the step (2) is 1: 7-1: 8.
further, in the above technical scheme, the elution solvent in the elution of the macroporous resin in the step (2) is 6-7 times of the mass of the macroporous resin, and the elution flow rate is 1 bed volume per hour.
Further, in the above technical scheme, in the step (2), the thin layer chromatography developing solvent is chloroform, methanol and ethyl acetate, and the volume ratio is 9: 3: 2; in the step (4), the thin-layer chromatography developing solvent is chloroform, methanol and water, and the volume ratio is 4:1: 0.1.
further, in the above technical scheme, the concentration in step (3) and step (4) is performed by vacuum rotary concentration, and the concentration temperature is 40 ℃.
Further, in the above technical solution, in the step (4), chloroform: methanol: the volume ratio of water is 10:1:0.1, 9:1:0.1, 4.5:1:0.1, 4:1:0.1, 3:1:0.1, 2:1:0.1 and 1:1:0.1 in sequence.
Further, in the above technical scheme, the elution solvents for the two reverse silica gel column chromatographies in step (4) are methanol and water in a volume ratio of 5:1 and methanol and water in a volume ratio of 1:1, respectively.
Advantageous effects of the invention
The invention provides an extraction method of sea cucumber saponin Cladoloside A, which is characterized in that high-purity monomer sea cucumber saponin is obtained by refluxing 80% ethanol water solution, enriching macroporous resin and separating and purifying column chromatography, ethanol and water are used as solvents, organic solvents such as acetonitrile, acetone, ethyl acetate, n-butyl alcohol and the like which pollute the environment are avoided, and the ethanol is recycled in the production process, so that the production cost is greatly reduced. Therefore, the method provides a process method for obtaining the holothurin Cladoloside A with the purity of more than 90 percent through one-step enrichment and one-step purification, the extraction process is simplified, the operation steps are shortened, the holothurin Cladoloside A is ensured not to be greatly lost due to too long purification time, the cost is lower, the pollution is less, green extraction is realized, the method is suitable for large-scale industrial production, the characteristics of simple separation process, rapidness, economy, high efficiency and green are realized, and the foundation is laid for further exploring the pharmacological activity of the holothurin Cladoloside A. The compound is extracted from the apostichopus japonicus for the first time, and has scientific significance for further researching the physiological synthesis of the holothurian saponins in the apostichopus japonicus.
Drawings
FIG. 1 shows the liquid chromatography analysis of the compound holothurian saponin Cladoloside A extracted in example 1 of the present invention.
FIG. 2 shows the Cladoloside A, a compound extracted in example 1 of the present invention1HNMR spectra.
FIG. 3 shows the Cladoloside A, a compound extracted in example 1 of the present invention13CNMR spectra.
FIG. 4 is DEPT spectrum of the compound holothurian saponin Cladoloside A extracted in example 1 of the present invention.
FIG. 5 shows the structural formula of Cladoloside A, which is a compound extracted in example 1 of the present invention.
Detailed Description
The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.
Example 1
The sea cucumber saponin Cladoloside A is extracted by the following extraction method:
(1) raw material selection and processing
Selecting fresh apostichopus japonicus, dissecting and removing internal organs, crushing by a high-speed tissue triturator, adding 80% ethanol water solution which is 7 times of the mass of the apostichopus japonicus meat into the fresh apostichopus japonicus meat, performing reflux extraction for 3 times in batches, wherein the extraction time is 2 hours, 1.5 hours and 0.5 hour respectively, filtering while hot, collecting filtrate, performing vacuum rotary concentration and recovery at 40 ℃, and obtaining concentrated extract as a total extract.
(2) Treatment of the Total extract
Uniformly dispersing the total extract obtained in the step (1) in water, adding the uniformly dispersed total extract into a chromatographic column filled with AB-8 macroporous resin, wherein the chromatographic column is filled with AB-8 macroporous resin filler (the granularity is 0.3-1.25mm, shin-Etsu fine chemical research institute in Tianjin), the length of the chromatographic column is 120cm, the diameter of the chromatographic column is 10cm, the resin amount is 7-8 times of the mass of the extract, firstly, water is used as a solvent to wash off saccharides, inorganic salts and some impurities in the extract, then, 80% ethanol water solution is used as a solvent to wash and enrich the sea cucumber total saponin eluent, the elution solvent is 6-7 times of the mass of the macroporous resin, the elution flow rate is controlled to be 1 column bed volume per hour, the sea cucumber total saponin eluent is subjected to thin-layer chromatography analysis (the volume ratio of a developing solvent is chloroform: methanol: ethyl acetate: 9: 3: 2), collecting 80% ethanol eluent, performing vacuum rotary concentration by a rotary evaporator at 40 ℃, obtaining the total saponins extract of sea cucumber.
(3) Separation of
Separating the sea cucumber total saponin extract obtained in the step (2) by normal phase silica gel (granularity: 200-.
(4) Purification of
Purifying the extract obtained in step (3) by ODS reverse silica gel (particle size: 50 μm, Tianjin Borna Aijiel technology Co., Ltd.) column chromatography, eluting with methanol and water at a solvent volume ratio of 5:1, performing secondary purification with methanol and water at a solvent volume ratio of 1:1, and performing vacuum rotary concentration at 40 deg.C to obtain colorless crystalline powder.
1mg of the powder was dissolved in 1ml of methanol, and liquid chromatography analysis was performed using HPLC-DVD to determine the monomer compound having a purity of 90%. The following chromatographic conditions were used:
using a Zorbax SB-C18 column (250 mm. times.4.6 mm, 5 μm), column temperature: 30 ℃, ultraviolet detection wavelength: 205nm, flow rate: 1ml/min, sample size: 10ul, acetonitrile and water as mobile phase, gradient elution, and the elution order is shown in the table:
Figure BDA0002252533740000051
the liquid chromatogram of the monomeric compound is shown in FIG. 1.
(6) Structural identification
About 10mg of the powder was dissolved in deuterated dimethyl sulfoxide, and structural analysis was performed using a Bruker 400M nuclear magnetic resonance spectrometer (AVANCE III 400MHz),13the CNMR spectrum gives a 53-carbon signal, which is known from the DEPT spectrum and contains 8 methyl groups, 13 methylene groups, 25 methine groups, 7 quaternary carbons, deltaC213.6,175.97 suggests that the structural formula contains two C ═ O, deltaC150.73, 145.3, 110.75 and 129.89 show that the structural formula contains two double bonds, and the structural formula contains xylose, carbostyril and glucose according to signals given by an H spectrum. Combining the above analyses13The CNMR data is compared with Cladoloside A reported in the literature, and the Cladoloside A is consistent with the CNMR data, so that the sea cucumber saponin Cladoloside A is identified. The compound is extracted from Apostichopus japonicus for the first time, and has a molecular formula of C53H82O21Molecular weight of 1055, structural formula shown in FIG. 5, FIG. 2, and FIG. 23 and 4 are respectively1HNMR spectrum,13CNMR spectra and DEPT spectra.

Claims (10)

1. The extraction method of the holothurian saponin Cladoloside A is characterized by comprising the following steps:
(1) selecting fresh Apostichopus japonicus, dissecting, removing viscera, pulverizing to obtain Apostichopus japonicus meat, adding 80% ethanol water solution, reflux-extracting for 3 times, filtering the extractive solution while it is hot, collecting filtrate, concentrating, and recovering to obtain concentrated extract as total extract;
(2) uniformly dispersing the total extract obtained in the step (1) in water, performing gradient elution by using a macroporous resin column, performing thin-layer chromatography analysis on water and 80% ethanol in order, and collecting 80% ethanol eluent;
(3) recovering the 80% ethanol eluate obtained in step (2), and concentrating to obtain total saponins extract of Stichopus japonicus;
(4) and (3) passing the sea cucumber total saponin extract obtained in the step (3) through a forward silica gel column, performing gradient elution by using chloroform, methanol and water in different proportions as elution solvents, performing thin-layer chromatography analysis on the eluent, further separating and purifying the part with large content by twice reverse silica gel column chromatography, and concentrating to obtain the sea cucumber saponin Cladoloside A.
2. The extraction method as claimed in claim 1, wherein the mass ratio of the stichopus japonicus meat to the 80% ethanol water solution in the step (1) is 1: and 7, extracting for 2 hours, 1.5 hours and 0.5 hour respectively, and concentrating the filtrate in a vacuum rotary concentration mode, wherein the concentration temperature is 40 ℃.
3. The extraction method according to claim 1, wherein the particle size of the macroporous resin filler in the macroporous resin chromatographic column in the step (2) is 0.3-1.25 mm.
4. The extraction method according to claim 1, wherein the mass ratio of the total extract to the macroporous resin in the step (2) is 1: 7-1: 8.
5. the extraction method according to claim 1, wherein the elution solvent in the elution of the macroporous resin in the step (2) is 6-7 times of the mass of the macroporous resin, and the elution flow rate is 1 bed volume per hour.
6. The extraction method according to claim 1, wherein the thin layer chromatography developing solvent in the step (2) is chloroform, methanol and ethyl acetate, wherein the ratio of chloroform: methanol: volume of ethyl acetate 9: 3: 2; and (4) adopting chloroform, methanol and water as a thin-layer chromatography developing agent, wherein the chloroform: methanol: volume of water 4:1: 0.1.
7. the extraction method according to claim 1, wherein the concentration in step (3) and step (4) is performed by vacuum rotary concentration, and the concentration temperature is 40 ℃.
8. The extraction method according to claim 1, wherein the ratio of chloroform: methanol: the volume ratio of water is 10:1:0.1, 9:1:0.1, 4.5:1:0.1, 4:1:0.1, 3:1:0.1, 2:1:0.1 and 1:1:0.1 in sequence.
9. The extraction method according to claim 1, wherein the elution solvents for the two reverse silica gel column chromatographies in the step (4) are methanol and water in a volume ratio of 5:1 and methanol and water in a volume ratio of 1:1, respectively.
10. The extraction method as claimed in claim 1, wherein the particle size of the filler in the forward silica gel column in step (4) is 200-300 mesh, and the particle size of the filler in the reverse silica gel column is 50 μm.
CN201911039830.4A 2019-10-29 2019-10-29 Method for extracting sea cucumber saponin Cladoloside A Active CN110698532B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911039830.4A CN110698532B (en) 2019-10-29 2019-10-29 Method for extracting sea cucumber saponin Cladoloside A

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911039830.4A CN110698532B (en) 2019-10-29 2019-10-29 Method for extracting sea cucumber saponin Cladoloside A

Publications (2)

Publication Number Publication Date
CN110698532A true CN110698532A (en) 2020-01-17
CN110698532B CN110698532B (en) 2020-11-03

Family

ID=69203808

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911039830.4A Active CN110698532B (en) 2019-10-29 2019-10-29 Method for extracting sea cucumber saponin Cladoloside A

Country Status (1)

Country Link
CN (1) CN110698532B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114437150A (en) * 2022-01-20 2022-05-06 辽宁省海洋水产科学研究院 Method for separating and extracting stichopus japonicus cerebroside from stichopus japonicus

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102060905A (en) * 2010-11-12 2011-05-18 山东省科学院生物研究所 Technology method for preparing sea cucumber saponin Holotoxin A1 comparison product by utilizing fresh sea cucumber processing waste liquid
CN106636286A (en) * 2016-12-28 2017-05-10 中国海洋大学 Deglycosylated holothurian secondary saponin and preparation method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102060905A (en) * 2010-11-12 2011-05-18 山东省科学院生物研究所 Technology method for preparing sea cucumber saponin Holotoxin A1 comparison product by utilizing fresh sea cucumber processing waste liquid
CN106636286A (en) * 2016-12-28 2017-05-10 中国海洋大学 Deglycosylated holothurian secondary saponin and preparation method thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AVILOV,S.A.等: "New triterpene glycosides from the holothurian Cladolabes sp", 《KHIMIYA PRIRODNYKH SOEDINENII》 *
I. I. MALTSEV等: "TRITERPENE GLYCOSIDES FROM STICHOPUS JAPONICUS SELENKASEA CUCUMBER", 《COMP.BIOCHEM.PHYSIOL.》 *
翁怡毅 等: "沙海参中的三个海参皂苷", 《中国天然药物》 *
苏永昌 等: "海参皂苷的分离提取与生物活性", 《福建水产》 *
薛长湖 等: "青岛仿刺参中3 种海参皂苷的分离纯化及结构鉴定", 《中国海洋大学学报》 *
袁炜辉 等: "海参皂苷的分离提取及分析方法研究进展", 《食品研究与开发》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114437150A (en) * 2022-01-20 2022-05-06 辽宁省海洋水产科学研究院 Method for separating and extracting stichopus japonicus cerebroside from stichopus japonicus

Also Published As

Publication number Publication date
CN110698532B (en) 2020-11-03

Similar Documents

Publication Publication Date Title
CN110563781B (en) Preparation method of albizzia bark neolignan monomer compound
CN106916193A (en) A kind of method that afzclin is extracted in the leaf from cercis
CN106317148B (en) A method of extracting cordycepin from Cordyceps militaris
CN110698532B (en) Method for extracting sea cucumber saponin Cladoloside A
CN107698510B (en) Alkaloid compound extracted from green bamboo label and extraction method
CN103304490A (en) Method for separating and purifying five purine and pyrimidine bases from trichosanthes bark
CN113264974A (en) Preparation of type B cardiac glycoside and anti-angiogenesis application thereof
CN109879921B (en) Compound separated from rhizoma anemarrhenae and having antitumor activity and preparation method thereof
CN108191933B (en) Method for preparing new astilbin by taking rhizoma smilacis glabrae as raw material
CN111187317A (en) Preparation method of glycosidation glabridin
CN104892642A (en) Method for purifying vindesine
CN1318438C (en) Method for preparing tanning material bonducin
CN109796509B (en) Iridoid compound and preparation method and application thereof
CN103483410B (en) Xanthoceraside preparation method
CN104592180B (en) Method for extracting 5-methyl aureusidin from eleocharis tuberosa peels
CN105037124B (en) A kind of preparation method of selaginellin N
CN110075145B (en) Method for extracting total cardiac glycosides from oleander leaves
CN109541063B (en) Method for extracting kaempferol glucoside compounds from Nanshan tea
CN113943332B (en) Method for preparing high-purity paeoniflorin by dynamic axial compression chromatography
CN111363000B (en) Method for separating and preparing two trace components from sodium aescinate
CN112608306B (en) Preparation method and application of flavonoid saponin new ketone A in spina gleditsiae
CN109956984B (en) Method for extracting and separating nicotiflorin from China rose
CN110003293B (en) Method for extracting parasitoside from Chinese rose
CN111233944B (en) High performance liquid phase method for simultaneously preparing and separating four lignans components
CN111675717B (en) Tetrandra monomer compound and its extraction method and use

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant