阅读说明：本技术 一种从蛤蟆油中提取硫酸软骨素/硫酸皮肤素/透明质酸的方法 (Method for extracting chondroitin sulfate/dermatan sulfate/hyaluronic acid from oviductus ranae ) 是由 魏峥 黄海月 林江慧 梁群焘 于 2019-10-18 设计创作，主要内容包括：本发明提供一种从蛤蟆油中提取硫酸软骨素/硫酸皮肤素/透明质酸的方法。所述方法先将蛤蟆油充分溶胀后加入的胰蛋白酶酶解蛤蟆油,再通过DEAE阴离子交换色谱柱提取蛤蟆油中的糖胺聚糖OR-GAG,然后经透析、冻干、浓缩后,用硫酸软骨素ABC酶特异性酶解OR-GAG,足量的酶可将长链酶解至硫酸软骨素/硫酸皮肤素/透明质酸(CS/DS/HA)二糖单元。CS/DS/HA二糖单元经2-氨基吖啶酮荧光标记后,利用LC/MS-ITTOF进行结构分析,得到蛤蟆油中各二糖组分的结构与摩尔百分比。(The invention provides a method for extracting chondroitin sulfate, dermatan sulfate and hyaluronic acid from oviductus ranae. The method comprises the steps of firstly adding trypsin into fully swelled oviductus ranae to carry out enzymolysis on the oviductus ranae, then extracting glycosaminoglycan OR-GAG in the oviductus ranae through a DEAE anion exchange chromatographic column, then carrying out dialysis, freeze-drying and concentration, carrying out specific enzymolysis on the OR-GAG by using chondroitin sulfate ABC enzyme, wherein a sufficient amount of enzyme can carry out enzymolysis on a long chain to a chondroitin sulfate/dermatan sulfate/hyaluronic acid (CS/DS/HA) disaccharide unit. After the CS/DS/HA disaccharide unit is fluorescently labeled by 2-aminoacridone, the structure analysis is carried out by utilizing LC/MS-ITTOF to obtain the structure and the mole percentage of each disaccharide component in the oviductus ranae.)

1. A method for extracting chondroitin sulfate/dermatan sulfate/hyaluronic acid from oviductus ranae is characterized by comprising the following steps:

(1) enzymolysis of the toad oil by trypsin: soaking oviductus Ranae, performing specific enzymolysis with trypsin, performing enzymolysis, centrifuging, and collecting supernatant;

(2) extracting glycosaminoglycan from the oviductus ranae by anion exchange chromatography: replacing glycosaminoglycan in the oviductus ranae supernatant obtained in the step (1) by eluent with different high salt concentrations by using the anion exchange effect of DEAE cellulose resin, removing NaCl in the eluent by dialysis, and freeze-drying and concentrating to obtain oviductus ranae glycosaminoglycan OR-GAG;

(3) obtaining disaccharide components by hydrolyzing the toad oil glycosaminoglycan with chondroitin sulfate ABC enzyme: taking the oviductus ranae glycosaminoglycan OR-GAG in the step (2), adding chondroitin sulfate ABC enzyme, and performing enzymolysis on the oviductus ranae glycosaminoglycan OR-GAG long chain specificity to a CS/DS/HA disaccharide unit;

(4) purification of the disaccharide component by size exclusion chromatography: separating and purifying the CS/DS/HA disaccharide by using a liquid phase molecular exclusion chromatographic column peptide column;

(5) LC/MS-ITTOF analysis of CS/DS/HA disaccharide structure in oviductus ranae: and (3) after the CS/DS/HA disaccharide obtained after liquid phase purification is subjected to fluorescence labeling by 2-aminoacridone and sodium cyanoborohydride, and the disaccharide structure and the component mole percentage thereof are obtained through LC/MS-ITTOF analysis.

2. The method of claim 1, wherein the method comprises the steps of:

(1) enzymolysis of the toad oil by trypsin: grinding oviductus Ranae into powder, soaking in ultrapure water, adding trypsin for enzymolysis of oviductus Ranae protein, and centrifuging to obtain supernatant;

(2) extracting glycosaminoglycan from the oviductus ranae by anion exchange chromatography: washing a DEAE column with water to remove ethanol in the resin, adding a balance liquid to enable the chromatographic column to reach a stable state, adding the supernatant obtained in the step (1), adding the balance liquid to remove protein impurities, eluting oviductus ranae glycosaminoglycan OR-GAG by using eluents with different high salt concentrations, carrying out ultraviolet detection on the collected sample, and finally removing a large amount of salt in glycosaminoglycan by dialysis and freeze-drying;

(3) obtaining disaccharide components by hydrolyzing the toad oil glycosaminoglycan with chondroitin sulfate ABC enzyme: taking the oviductus ranae glycosaminoglycan OR-GAG in the step (2), adding chondroitin sulfate ABC enzyme, carrying out enzymolysis on the oviductus ranae glycosaminoglycan OR-GAG long chain specificity to a CS/DS/HA disaccharide unit, inactivating with boiling water, and centrifuging at a high speed to obtain a supernatant;

(4) purification of the disaccharide component by size exclusion chromatography: taking the supernatant in the step (3), and separating and purifying the sample through a molecular exclusion chromatography column peptide column to obtain a pure CS/DS/HA disaccharide component of the oviductus ranae;

(5) LC/MS-ITTOF analysis of CS/DS/HA disaccharide structure in oviductus ranae: performing fluorescence labeling on CS/DS/HA disaccharide in the oviductus ranae by using 2-aminoacridinone and sodium cyanoborohydride, analyzing the structure of the labeled CS/DS/HA disaccharide by using LC/MS-ITTOF, and obtaining the component content and the mole percentage of CS/DS/HA in the oviductus ranae according to an established standard curve.

3. The method of claim 2, wherein the chondroitin sulfate/dermatan sulfate/hyaluronic acid is extracted from oviductus ranae, and the method comprises the following steps: the specific steps of the step (1) are as follows: grinding oviductus Ranae into powder in a mortar, soaking 100-200 mg of the powder oviductus Ranae in 20 mL of ultrapure water at normal temperature for 24-48 h to fully swell the powder to prepare an oviductus Ranae sample, and adding 100 mu L of 10 mu g/uL of trypsin solution for enzymolysis under the reaction condition of 36-40 ℃ and 24-48 h; after the enzymolysis is finished, centrifuging at 10000rpm for 10-20min, and taking supernatant.

4. The method of claim 2, wherein the chondroitin sulfate/dermatan sulfate/hyaluronic acid is extracted from oviductus ranae, and the method comprises the following steps: the concrete steps of the step (2) are that 5-20 mL of DEAE resin washed by water is taken firstly and filled into a column, then 0.2M NaCl with ten to twenty times column volume and 10-50 mM phosphoric acid equilibrium buffer solution are used for balancing the DEAE column, after the column is stabilized, the sample is loaded, and the equilibrium buffer solution is added for removing protein impurities, then, the glycosaminoglycan in the oviductus ranae is extracted by a sectional elution method, and the sectional elution high-salt concentration eluent is respectively 0.5M NaCl and 10-50 mM phosphoric acid buffer solution; 1.0M NaCl, 10-50 mM phosphate buffer; 1.5M NaCl, 10-50 mM phosphate buffer; 2.0M NaCl, 10-50 mM phosphate buffer; collecting each fraction by using a 3 mL/tube, and carrying out ultraviolet detection, wherein the wavelength is 212 nm; dialyzing the fraction collected by DEAE with 1000-5000 Da dialysis membrane to remove NaCl in the eluate, changing deionized water every two hours, dialyzing for 2-4 days, and freeze-drying for 2-4 days to remove water to obtain pure oviductus Ranae glycosaminoglycan OR-GAG.

5. The method of claim 2, wherein the chondroitin sulfate/dermatan sulfate/hyaluronic acid is extracted from oviductus ranae, and the method comprises the following steps: the specific steps of the step (3) are as follows: and (3) taking 1-100 ng of the oviductus ranae glycosaminoglycan OR-GAG in the step (2), adding 10 mu L of 30-50 mIU/mu L of chondroitin sulfate ABC for enzymolysis, carrying out enzymolysis on the oviductus ranae glycosaminoglycan OR-GAG long-chain specific enzyme to a CS/DS/HA disaccharide unit, wherein the enzymolysis temperature is 36-40 ℃, the enzymolysis time is 24-48 h, after the enzymolysis is finished, inactivating with boiling water, and centrifuging at 10000rpm for 10-20min to obtain supernatant.

6. The method of claim 2, wherein the chondroitin sulfate/dermatan sulfate/hyaluronic acid is extracted from oviductus ranae, and the method comprises the following steps: the specific steps of the step (4) are as follows: taking the supernatant in the step 3 toThe sample loading volume of less than or equal to 200 mu L is divided by a size exclusion chromatographic column peptide column; separating and purifying CS/DS/HA disaccharide with peptide column, and mobile phase is 0.1-0.3 MNH₄HCO₃The flow rate is 0.4 mL/min, and CS/DS/HA disaccharide peaks at 35-44 min and is collected; NH in mobile phase₄HCO₃It is removed by evaporation at 55-65 ℃.

7. The method of claim 2, wherein the chondroitin sulfate/dermatan sulfate/hyaluronic acid is extracted from oviductus ranae, and the method comprises the following steps: the specific steps of the step (5) are as follows: fluorescence labeling purified CS/DS/HA disaccharide with 5-10 μ L2-aminoacridinone<Passing 20 μ L through reversed phase chromatography ODS column, mobile phase A phase 20-80 mM ammonium acetate, pH =5-7, mobile phase B phase 100% methanol, flow rate 0.3 mL/min, CDL 100-₂The flow rate is 1.5 mL/min, and the detection voltage is 1.6-1.8 kV; according to the established standard curve, the content and the mole percentage of the disaccharide component are obtained.

Technical Field

The invention belongs to the field of natural product preparation and purification, and particularly relates to a method for extracting chondroitin sulfate/dermatan sulfate/hyaluronic acid from oviductus ranae.

Background

Oviductus Ranae, also called oviductus Ranae, and oviductus Ranae, is a dry product of female frog oviduct. About 0.5 g of crude protein, 0.3 g of nitrogen-free extract, 0.1 g of ash, 0.05 g of fat and 0.02 g of water are contained in 1g of oviductus ranae. Oviductus Ranae is recorded in Ben Cao Tu Jing and Ben Cao gang mu, and in the Ming and Qing dynasty, medical experts are more advocating oviductus Ranae which is regarded as a high-quality tribute offering palace and has the nutritional ingredients not inferior to tonics such as ginseng, bird's nest and the like. The traditional efficacy of the oviductus ranae is to tonify kidney, replenish vital essence, nourish yin and moisten lung, and is mainly used for deficiency of kidney and marrow, dizziness and tinnitus, weakness after illness or postpartum. Modern medicine finds that the traditional Chinese medicine has the effects of enhancing the immunity of organisms, improving the oxidation resistance, reducing blood fat, resisting cancers, assisting in the treatment and the like. Wherein, the effects of oxidation resistance, cancer resistance and the like can be related to the chondroitin sulfate/dermatan sulfate/hyaluronic acid contained in the composition.

Chondroitin Sulfate (CS), Dermatan Sulfate (DS) and Hyaluronic Acid (HA) all belong to glycosaminoglycans (GAG), wherein CS/DS is a polyanionic long chain composed of acetylgalactosamine (GalNAc) and hexuronic acid (GlcA/IdoA) as disaccharide units, and HA is the only one glycosaminoglycan without sulfation modification formed by acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) as disaccharide units. The CS/DS modification sites mainly comprise the oxysulfation at the 2-position of GlcA, the oxysulfation at the 4-position and the oxysulfation at the 6-position of GalNAc, and the biological functions of the modified products are different due to different degrees and sulfation modifications at different positions. The biological functions of CS/DS mainly include promoting cartilage regeneration, anticoagulation, anti-thrombosis, anti-aging, improving organism immunity and the like, and HA is generally used in moisturizing cosmetics such as skin aging delaying, skin moistening and the like due to a large number of hydroxyl groups on a long chain and strong hydrophilic effect.

As relevant reports on the extraction and the structural analysis of the CS/DS/HA in the oviductus ranae are not found at home and abroad, the invention creatively establishes a method for extracting the CS/DS/HA in the oviductus ranae, carries out structural analysis on the CS/DS/HA in the oviductus ranae for the first time, and provides an original extraction approach and a structural analysis means for further researching the structure and the efficacy of the CS/DS/HA in the oviductus ranae.

Disclosure of Invention

The invention aims to provide a method for extracting chondroitin sulfate, dermatan sulfate and hyaluronic acid from oviductus ranae. The method comprises the steps of taking oviductus ranae as a raw material, specifically hydrolyzing oviductus ranae protein by trypsin, separating polyanion long-chain GAG by anion exchange chromatography, removing NaCl in the GAG by dialysis, freeze-drying and concentrating to be in an anhydrous state, adding chondroitin sulfate ABC enzyme to specifically hydrolyze CS/DS/HA long chains to disaccharide units, and purifying and separating the obtained CS/DS/HA disaccharide units by molecular exclusion chromatography. Finally, the CS/DS/HA disaccharide unit is marked by a fluorescent substance AMAC (2-aminoacridone), and the CS/DS/HA disaccharide unit is subjected to structural analysis by utilizing LC/MS-ITTOF to obtain the component content and the mole percentage of the CS/DS/HA disaccharide.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for extracting chondroitin sulfate/dermatan sulfate/hyaluronic acid from oviductus Ranae comprises the following steps:

(1) enzymolysis of the toad oil by trypsin: soaking oviductus Ranae, performing specific enzymolysis with trypsin, performing enzymolysis, centrifuging, and collecting supernatant.

(2) Extracting glycosaminoglycan from the oviductus ranae by anion exchange chromatography: replacing glycosaminoglycan in the oviductus ranae supernatant obtained in the step (1) by eluent with different high salt concentrations by using the anion exchange effect of DEAE cellulose resin, removing NaCl in the eluent by dialysis, and freeze-drying and concentrating to obtain oviductus ranae glycosaminoglycan OR-GAG.

(3) Obtaining disaccharide components by hydrolyzing the toad oil glycosaminoglycan with chondroitin sulfate ABC enzyme: and (3) adding chondroitin sulfate ABC enzyme into the oviductus ranae glycosaminoglycan OR-GAG obtained in the step (2), and performing enzymolysis on the oviductus ranae glycosaminoglycan OR-GAG long chain specificity to the CS/DS/HA disaccharide unit.

(4) Purification of the disaccharide component by size exclusion chromatography: and separating and purifying the CS/DS/HA disaccharide by using a liquid phase molecular exclusion chromatographic column peptide column.

(5) LC/MS-ITTOF analysis of CS/DS/HA disaccharide structure in oviductus ranae: the CS/DS/HA disaccharide obtained by enzymolysis is treated by AMAC (2-aminoacridinone) and NaBH₃After CN fluorescence labeling, the disaccharide structure and the mole percentage of the components thereof are obtained by LC/MS-ITTOF analysis.

A method for extracting chondroitin sulfate/dermatan sulfate/hyaluronic acid from oviductus ranae is characterized by comprising the following specific steps:

(1) enzymolysis of the toad oil by trypsin: grinding oviductus Ranae into powder, soaking in ultrapure water, adding trypsin for enzymolysis of oviductus Ranae protein, and centrifuging to obtain supernatant.

(2) Extracting glycosaminoglycan from the oviductus ranae by anion exchange chromatography: washing DEAE column with water to remove ethanol in resin, adding balance liquid to make chromatographic column reach stable state, adding supernatant obtained in step (1), adding balance liquid to remove protein impurities, eluting with eluents with different high salt concentrations to obtain oviductus Ranae glycosaminoglycan (OR-GAG), performing ultraviolet detection on collected sample, removing a large amount of salt in glycosaminoglycan by dialysis, and lyophilizing.

(3) Obtaining disaccharide components by hydrolyzing the toad oil glycosaminoglycan with chondroitin sulfate ABC enzyme: and (3) taking the toad oil glycosaminoglycan OR-GAG1-100 ng in the step (2), adding 10 mu L of 30-50 mIU/mu L of chondroitin sulfate ABC, performing enzymolysis on the long chain specificity of the toad oil glycosaminoglycan OR-GAG to a CS/DS/HA disaccharide unit, inactivating with boiling water, and performing high-speed centrifugation to obtain a supernatant.

(4) Purification of the disaccharide component by size exclusion chromatography: and (4) taking the supernatant obtained in the step (3), and separating and purifying the sample through a molecular exclusion chromatography column peptide column to obtain a pure CS/DS/HA disaccharide component of the oviductus ranae.

(5) LC/MS-ITTOF analysis of CS/DS/HA disaccharide structure in oviductus ranae: performing fluorescent labeling on CS/DS/HA disaccharide in the oviductus ranae by using AMAC (2-aminoacridinone) and sodium cyanoborohydride, analyzing the structure of the labeled CS/DS/HA disaccharide by using LC/MS-ITTOF, and obtaining the component content and the mole percentage of CS/DS/HA in the oviductus ranae according to a well established standard curve.

The specific steps of the step (1) are as follows: grinding oviductus Ranae into powder in a mortar, soaking 100-200 mg of the powder oviductus Ranae in 20 mL of ultrapure water at normal temperature for 24-48 h to fully swell the powder to prepare an oviductus Ranae sample, and adding 100 mu L of 10 mu g/uL of trypsin solution for enzymolysis under the reaction condition of 36-40 ℃ and 24-48 h; after the enzymolysis is finished, centrifuging at 10000rpm for 10-20min, and taking supernatant.

The concrete steps of the step (2) are that 5-20 mL of DEAE resin washed by water is firstly taken to be filled into a column, the DEAE column is balanced by 0.2M NaCl which is ten to twenty times of the column volume and 10-50 mM phosphoric acid equilibrium buffer solution, the sample is loaded after the column is stabilized, the equilibrium buffer solution is added to remove protein impurities, then the glycosaminoglycan in the oviductus ranae is extracted by a sectional elution method, and the sectional elution high-salt concentration eluent is respectively 0.5M NaCl and 10-50 mM phosphoric acid buffer solution; 1.0M NaCl, 10-50 mM phosphate buffer; 1.5M NaCl, 10-50 mM phosphate buffer; 2.0M NaCl, 10-50 mM phosphate buffer; collecting each fraction by using a 3 mL/tube, and carrying out ultraviolet detection, wherein the wavelength is 212 nm; dialyzing the fraction collected by DEAE with 1000-5000 Da dialysis membrane to remove NaCl from the eluate, changing deionized water every two hours, dialyzing for 2-4 days, and lyophilizing for 2-4 days to remove water to obtain pure oviductus Ranae glycosaminoglycan (OR-GAG).

The specific steps of the step (3) are as follows: taking the oviductus ranae glycosaminoglycan OR-GAG1-100 ng obtained in the step (2), adding 10 mu L of 30-50 mIU/mu L of chondroitin sulfate ABC for enzymolysis, carrying out enzymolysis on the oviductus ranae glycosaminoglycan OR-GAG to a CS/DS/HA disaccharide unit under the enzymolysis condition of 36-40 ℃, carrying out enzymolysis for 24-48 h, inactivating with boiling water after the enzymolysis is finished, centrifuging at 10000r for 10-20min, and taking supernatant.

The specific steps of the step (4) are as follows: taking the supernatant in the step 3, and passing the supernatant through a size exclusion chromatographic column peptide column by using the sample volume of less than or equal to 200 mu L; separating and purifying CS/DS/HA disaccharide by peptide column with mobile phase of 0.1-0.3M NH₄HCO₃The CS/DS/HA disaccharide peaks at 35-44 min and is collected at a flow rate of 0.4 mL/min. NH in mobile phase₄HCO₃It is removed by evaporation at 55-65 ℃.

The specific steps of the step (5) are as follows: fluorescence labeling purified oviductus Ranae CS/DS/HA disaccharide with 5-10 μ L AMAC (2-aminoacridinone)<Passing 20 μ L through reversed phase chromatography ODS column, mobile phase A phase 20-80 mM ammonium acetate, pH =5-7, mobile phase B phase 100% methanol, flow rate 0.3 mL/min, CDL 100-₂The flow rate is 1.5 mL/min, and the detection voltage is 1.6-1.8 kV. According to the established standard curve, the content and the mole percentage of the disaccharide component are obtained.

The invention has the advantages that: the method takes oviductus ranae as a raw material, performs enzymolysis on oviductus ranae protein by trypsin, separates polyanion long-chain GAG by anion exchange chromatography, then adds chondroitin sulfate ABC enzyme to perform specific enzymolysis on CS/DS/HA long chains to a disaccharide unit, purifies and separates the obtained CS/DS/HA disaccharide unit by using molecular exclusion chromatography, finally marks the CS/DS/HA disaccharide unit by using a fluorescent substance AMAC (2-aminoacridinone), and performs structural analysis on the CS/DS/HA disaccharide unit by using LC/MS-ITTOF to obtain the component content and the mole percentage of CS/DS/HA disaccharide. The method of the invention provides a chemical component basis for researching the medicinal value of the oviductus ranae and is beneficial to developing new health-care and medicinal functions of the oviductus ranae.

Description of the drawings:

FIG. 1 UV-detectable map of fractions obtained by stepwise elution on a DEAE column: the eluent concentration corresponding to C1 was 0.5M NaCl, the eluent concentration corresponding to C2 was 1.0M NaCl, the eluent concentration corresponding to C3 was 1.5M NaCl, and the eluent concentration corresponding to C4 was 2.0M NaCl.

FIG. 2 is a diagram of the separation and purification of the CS/DS/HA disaccharide component by Peptide column-HPLC method. The short horizontal line represents the range of collection time of the disaccharide component.

FIG. 3 CS/DS standard disaccharide structure.

Figure 4 HA standard disaccharide structure.

FIG. 5 ODS-MS method A CS/DS/HA disaccharide standard curve was established.

FIG. 6 EIC chart of CS/DS/HA disaccharide in oviductus Ranae: a is an EIC map of 40ng each of 8 standard disaccharides, B is an EIC map of the disaccharide contained in C1, C is an EIC map of the disaccharide contained in C2, D is an EIC map of the disaccharide contained in C3, and E is an EIC map of the disaccharide contained in C4. 1 is 2S4S6S, 2 is 2S4S, 3 is 2S6S, 4 is 4S6S, 5 is 2S, 6 is 4S, 7 is 6S, 8 is 0S.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.