阅读说明：本技术 一种鳕鱼皮寡肽及其分离纯化和在制备ɑ-葡萄糖苷酶抑制剂及抗Ⅱ型糖尿病药物中的应用 (Codfish skin oligopeptide, separation and purification thereof, and application of codfish skin oligopeptide in preparation of alpha-glucosidase inhibitor and anti-type II diabetes drug ) 是由 贾建萍 包国良 李青青 于 2019-01-30 设计创作，主要内容包括：本发明公开了一种鳕鱼皮寡肽及其分离纯化方法和应用,该鳕鱼皮寡肽的氨基酸序列为Tyr-Val-Arg。分离纯化方法包括：以阿拉斯加狭鳕鱼皮为原料,蛋白酶酶解法制备鳕鱼皮胶原肽混合肽；再依次进行超滤处理、葡聚糖凝胶色谱柱粗分以及高效液相色谱分离；葡聚糖凝胶色谱柱由葡聚糖凝胶树脂Sephadex G-25和葡聚糖凝胶树脂Sephadex G-50串联而成。经活性试验发现,该鳕鱼皮寡肽具有ɑ-葡萄糖苷酶抑制活性,能辅助降血糖,可用于制备抗Ⅱ型糖尿病药物。(The invention discloses a cod skin oligopeptide, a separation and purification method and application thereof. The separation and purification method comprises the following steps: taking Alaska pollack skin as a raw material, and preparing the cod skin collagen peptide mixed peptide by a protease enzymolysis method; sequentially carrying out ultrafiltration treatment, crude separation by a sephadex chromatographic column and high performance liquid chromatography separation; the Sephadex chromatographic column is formed by serially connecting Sephadex G-25 and Sephadex G-50. Activity tests show that the codfish skin oligopeptide has alpha-glucosidase inhibition activity, can assist in reducing blood sugar, and can be used for preparing anti-type II diabetes drugs.)

1. The cod skin oligopeptide is characterized in that the amino acid sequence is Tyr-Val-Arg.

2. A method for isolating and purifying cod skin oligopeptide according to claim 1, comprising:

(1) taking Alaska pollack skin as a raw material, and preparing the cod skin collagen peptide mixed peptide by a protease enzymolysis method;

the protease enzymolysis method specifically comprises the following steps:

mixing Alaska pollack skin, pancreatin and water, and carrying out enzymolysis for 6-10 h at the temperature of 52-58 ℃ and under the pH value of 5.5-6.5;

the adding amount of the pancreatin is 0.15-0.25 wt% based on the mass of Alaska pollack skin;

the mass ratio of Alaska pollack skin to water is 1: 4-8;

(2) carrying out ultrafiltration treatment on the cod skin collagen peptide mixed peptide prepared in the step (1) by adopting an ultrafiltration membrane with the molecular weight cutoff of 3000Da, and then concentrating and drying to obtain cod skin collagen peptide;

(3) roughly separating the cod skin collagen peptide prepared in the step (2) by using water as a mobile phase and adopting a sephadex chromatographic column;

the filler of the Sephadex chromatographic column is formed by connecting Sephadex G-25 and Sephadex G-50 in series;

(4) and (4) further separating the crude fraction product obtained in the step (3) by utilizing a high performance liquid chromatography technology to obtain the cod skin oligopeptide.

3. The method for separating and purifying cod skin oligopeptide according to claim 2, wherein in the step (1), the protease hydrolysis method comprises:

mixing Alaska pollack skin, pancreatin and water, and performing enzymolysis at 55 deg.C and pH of 6.0 for 8 hr;

the adding amount of the pancreatin is 0.20 wt% based on the mass of Alaska pollack skin;

the mass ratio of Alaska pollack skin to water is 1: 6.

4. the method for separating and purifying cod skin oligopeptide according to claim 2, wherein in the step (2), the ultrafiltration membrane is selected from a hollow fiber polysulfone ultrafiltration membrane.

5. The method for separating and purifying cod skin oligopeptide according to claim 2, wherein in the step (3), Sephadex G-25 is selected from 100 mesh, Sephadex G-50 is selected from 60 mesh;

the flow rate of the mobile phase is 0.8-1.4 mL/min.

6. The method for separating and purifying cod skin oligopeptide according to claim 2, wherein in the step (4), the separation conditions of the high performance liquid chromatography are as follows:

a chromatographic column: angioent Eclipse XDB-C₁₈A column;

mobile phase: solution A: 0.05% trifluoroacetic acid-water solution; and B, liquid B: 0.05% trifluoroacetic acid in acetonitrile;

linear gradient elution is adopted, 0-20min, and 5% -20% of B is adopted; 20-25min, 20% -100% B;

the flow rate was 1.0mL/min, the column temperature was 30 ℃ and the detection wavelength was 220 nm.

7. Use of the cod skin oligopeptide of claim 1 in the preparation of an alpha-glucosidase inhibitor.

8. Use of a cod skin oligopeptide according to claim 1 in the preparation of a medicament for the treatment of type ii diabetes.

Technical Field

The invention relates to the field of separation and purification and application of fish skin oligopeptides, in particular to Alaska pollock skin oligopeptide, separation and purification of the Alaska pollock skin oligopeptide and application of the Alaska pollock skin oligopeptide in preparation of alpha-glucosidase inhibitors and anti-II diabetes drugs.

Background

Alaska Pollock. The fish is named as Theragra chalcogramma, lives in the northern part of Atlantic ocean, is a cold water deep sea fish, has tender meat quality and light meat flavor, and due to the special long-term growth environment (low temperature and high pressure), the amino acid composition and the amino acid sequence of fish protein (fish meat, fish skin and the like) of the fish are possibly different from other shallow sea fish, freshwater fish and the like.

The cod skin collagen peptide is a mixture of protein, polypeptide, oligopeptide and amino acid (the content is mainly oligopeptide) prepared by an enzyme method by taking cod skin as a raw material. Wherein the oligopeptide is a small peptide with 2-12 amino acid residues.

For example, chinese patent publication No. CN 108530530 a discloses a method for preparing cod skin collagen peptide, which comprises: 1) removing impurities from fish skin, cleaning, crushing into slurry, and washing with acid, alkali and water to neutrality; 2) adding the fish skin treated in the step 1) into hot water for heat preservation; 3) adding protease into the protein extracting solution obtained in the step 2), and performing enzymolysis treatment twice; 4) removing solid residues from the slurry after enzymolysis by using a centrifugal machine, removing a small amount of macromolecular impurities from the obtained clear liquid by using an ultrafiltration membrane, removing inorganic salts and micromolecular impurities by using a nanofiltration membrane, concentrating to obtain a fish skin collagen peptide solution, and performing spray drying to obtain fish skin collagen peptide powder.

Further, as disclosed in chinese patent publication No. CN 104152518A, a method for preparing cod skin collagen peptide as a food supplement for liver diseases comprises: (1) pretreating cod skin; (2) and (3) carrying out enzymolysis reaction: adding trypsin for enzymolysis reaction; (3) performing ultrafiltration to obtain GM2 component; (4) separating by DEAE-Sepharose FF ion exchange chromatography to obtain GM2-2 component, and freeze drying to obtain collagenase.

However, the collagen peptide or the collagen protease obtained by the technical scheme belongs to macromolecular peptide or protein, and the separation method is not suitable for separation and purification of the cod skin oligopeptide.

The cod skin oligopeptides with small molecular weight are relatively close in molecular weight, and the differences of other physical properties such as electrification property, hydrophobicity and the like are not obvious, so that components with high biological activity cannot be separated by utilizing a traditional membrane separation mode or a chromatographic separation method adopting a single filler.

The biological activities of the presently reported fish skin oligopeptides include antioxidation, blood sugar reduction, immunity improvement and the like, for example, WANG and the like (WANG T Y, HSIEH C H, HUNG CC, et al. Fish skin collagen hydrolases as active peptides IV inhibitors and glucose-like peptides-1 peptide having a complex beta peptide in diabetics, a complex beta peptide-and cold-water peptides) hydrolyze the fish skin of flatfish and tilapia, and a dipeptidyl peptidase is obtained after separation and purification, and is found to promote the secretion of glucagon-like peptide and insulin and further play a role in regulating blood sugar.

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia, which is caused by defects in insulin secretion or impaired biological action, or both. Hypoglycemic agents are roughly divided into three types according to the hypoglycemic mechanism: (1) stimulating insulin secretion or insulin preparations, such as sulfonylureas; (2) increase the utilization of glucose by peripheral tissues, such as biguanide hypoglycemic agents; (3) alpha-glucosidase inhibitors such as acarbose which is commonly used clinically.

The presently disclosed fish skin oligopeptides with blood sugar reducing effect all play a role in regulating blood sugar according to a mechanism (1), and other blood sugar reducing mechanisms are not disclosed.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides three cod-skin oligopeptides with novel amino acid sequences and a separation and purification process thereof, and activity tests show that the three cod-skin oligopeptides have alpha-glucosidase inhibition activity, can assist in reducing blood sugar and can be used for preparing anti-type II diabetes drugs.

The specific technical scheme is as follows:

the amino acid sequence of the cod skin oligopeptide is Glu-Gly-Gly-Tyr-Thr-Arg, Tyr-Val-Arg or Phe-Tyr-Glu respectively.

The invention also discloses a separation and purification method of the cod skin oligopeptide, which comprises the following steps:

(1) taking Alaska pollack skin as a raw material, and preparing the cod skin collagen peptide mixed peptide by a protease enzymolysis method;

the protease enzymolysis method specifically comprises the following steps:

mixing Alaska pollack skin, pancreatin and water, and carrying out enzymolysis for 6-10 h at the temperature of 52-58 ℃ and under the pH value of 5.5-6.5;

the adding amount of the pancreatin is 0.15-0.25 wt% based on the mass of Alaska pollack skin;

the mass ratio of Alaska pollack skin to water is 1: 4-8;

(2) carrying out ultrafiltration treatment on the cod skin collagen peptide mixed peptide prepared in the step (1) by adopting an ultrafiltration membrane with the molecular weight cutoff of 3000Da, and then concentrating and drying to obtain cod skin collagen peptide;

(3) roughly separating the cod skin collagen peptide prepared in the step (2) by using water as a mobile phase and adopting a sephadex chromatographic column;

the filler of the Sephadex chromatographic column is formed by connecting Sephadex G-25 and Sephadex G-50 in series;

(4) and (4) further separating the crude products obtained in the step (3) by utilizing a high performance liquid chromatography technology to obtain three kinds of cod skin oligopeptides.

Preferably, in step (1), the protease enzymolysis method:

mixing Alaska pollack skin, pancreatin and water, performing enzymolysis at 55 deg.C and pH 6.0 for 8 hr, and heating to 90 deg.C for enzyme deactivation;

the adding amount of the pancreatin is 0.20 wt% based on the mass of Alaska pollack skin;

the mass ratio of Alaska pollack skin to water is 1: 6.

by adopting the optimized enzymolysis process, the yield of the cod skin collagen peptide mixed peptide obtained after enzymolysis is higher and can reach 75.0%, and activity tests show that the cod skin collagen peptide mixed peptide has α -glucosidase inhibition activity IC₅₀It was 50.4 mg/mL.

Tests prove that in the cod skin collagen peptide mixed peptide obtained by the enzymolysis process:

88.08% of substances with molecular mass of 180-1000 Da and 7.37% of substances with molecular mass of less than 180 Da.

The contents of macromolecular protein, peptide and free amino acid are respectively 0.53g/100mL, 5.20g/100mL and 0.38g/100mL, and the mass ratio of the macromolecular protein, the peptide and the free amino acid is 9: 85: 6.

therefore, the obtained cod skin collagen peptide mixed peptide is mainly oligopeptide with 2-8 amino acid residues.

Preferably, in step (2), the ultrafiltration membrane is selected from a hollow fiber polysulfone ultrafiltration membrane.

In the step (3), the separation is carried out by adopting a mode of serially connecting the Sephadex resins, the specification of a chromatographic column is 2.6 multiplied by 50cm, wherein the Sephadex resin Sephadex G-25(20cm) is filled in the upper layer, the Sephadex resin Sephadex G-50(15cm) is filled in the lower layer, and the middle layer is isolated by quantitative filter paper.

Experiments show that in the separation and purification process, the selection of a Sephadex chromatographic column is particularly critical, and the cod skin collagen peptide with the molecular weight of less than 3000Da after ultrafiltration can be effectively separated only by selecting a mode of serially connecting Sephadex G-25 and Sephadex G-50.

Effective separation of cod skin collagen peptides cannot be achieved when a single Sephadex column, such as Sephadex G-25 or Sephadex G-50 as used in the present invention, or other types of Sephadex columns, such as Sephadex G-10 and Sephadex G-15, are used.

Preferably, in the step (3), Sephadex G-25 is selected from 100 meshes, and Sephadex G-50 is selected from 60 meshes.

Further experiments show that the thickness degree of the gel resin also has influence on experimental results, and when 60-mesh Sephadex G-25 and 60-mesh Sephadex G-50 are used in series, effective separation cannot be realized.

Preferably, in the step (3), the flow rate of the mobile phase is 0.8-1.4 mL/min, and more preferably 1.2 mL/min.

After the crude separation by sephadex chromatography, two separated components are obtained by separation according to the elution sequence and are marked as a separated component A and a separated component B.

Preferably, in the step (4), the separation conditions of the high performance liquid chromatography are as follows:

a chromatographic column: angioent Eclipse XDB-C₁₈A column;

mobile phase: solution A: 0.05% trifluoroacetic acid-water solution; and B, liquid B: 0.05% trifluoroacetic acid in acetonitrile;

linear gradient elution is adopted, 0-20min, and 5% -20% of B is adopted; 20-25min, 20% -100% B;

the flow rate was 1.0mL/min, the column temperature was 30 ℃ and the detection wavelength was 220 nm.

The amino acid sequences of the cod skin oligopeptides prepared by the separation and purification process are Glu-Gly-Gly-Tyr-Thr-Arg, Tyr-Val-Arg and Phe-Tyr-Glu respectively through MALDI-TOF-MS/MS analysis and identification.

Activity tests show that α -glucosidase inhibitory activity IC of the three₅₀Respectively 5.2mg/mL and 7.59mg/mL，13.4mg/mL。

Therefore, all three cod skin oligopeptides can be applied to preparation of the alpha-glucosidase inhibitor. Preferably, the cod skin oligopeptide with the amino acid sequence of Glu-Gly-Gly-Tyr-Thr-Arg has better alpha-glucosidase inhibition effect.

The action mechanism of the alpha-glucosidase inhibitor is as follows: competitively inhibits various alpha-glucosidase in small intestine to reduce the speed of starch decomposition into glucose, thereby slowing the absorption of glucose in intestinal tract and reducing postprandial hyperglycemia. Type ii diabetes is characterized by postprandial hyperglycemia with glucose toxicity that can exacerbate insulin resistance and insulin secretion deficiencies, and when only about 50% of the islet beta cell function remains, an increase in fasting glucose occurs and impaired glucose tolerance occurs.

Based on the research, the three cod skin oligopeptides disclosed by the invention can be further used for preparing anti-type II diabetes drugs, and preferably, the cod skin oligopeptides with the amino acid sequences of Glu-Gly-Gly-Tyr-Thr-Arg have better blood sugar inhibition effect.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses three cod skin oligopeptides with novel amino acid sequence structures;

aiming at the characteristics that the cod skin collagen peptide prepared by an enzymatic hydrolysis method is small in molecular weight, concentrated in molecular weight of a mixture and small in dispersity, the cod skin collagen peptide is roughly divided in a manner that Sephadex G-25 and Sephadex G-50 are connected in series, and then is further separated by a high performance liquid chromatography technology to finally obtain three novel cod skin oligopeptides;

activity tests show that the three codfish skin oligopeptides have alpha-glucosidase inhibition activity, can assist in reducing blood sugar, and can be used for preparing anti-type II diabetes drugs.

Drawings

FIG. 1 is an elution curve of a separated fraction obtained by separation through a Sephadex column in example 1, with an elution volume (mL) on the abscissa and an absorbance at a wavelength of 220nm on the ordinate;

FIG. 2 is an elution curve of a separated fraction obtained by separation through a Sephadex column in comparative example 1, with the abscissa being the elution volume (mL) and the ordinate being the absorbance at a wavelength of 220 nm;

FIG. 3 is an elution curve of a separated fraction obtained by separation through a Sephadex column in comparative example 2, with the abscissa being the elution volume (mL) and the ordinate being the absorbance at a wavelength of 220 nm;

FIG. 4 is an elution curve of a separated fraction obtained by separation through a Sephadex column in comparative example 3, with an elution volume (mL) on the abscissa and an absorbance at a wavelength of 220nm on the ordinate;

FIG. 5 is an elution curve of a separated fraction obtained by separation through a Sephadex column in comparative example 4, with an elution volume (mL) on the abscissa and an absorbance at a wavelength of 220nm on the ordinate;

FIG. 6 is an elution curve of a separated fraction obtained by separation through a Sephadex column in comparative example 5, with an elution volume (mL) on the abscissa and an absorbance at a wavelength of 220nm on the ordinate;

FIG. 7 is an elution curve of a fraction separated by Sephadex column chromatography in example 2, with elution volume (mL) on the abscissa and absorbance at a wavelength of 220nm on the ordinate.

The specific implementation mode is as follows: