CN118206615A

CN118206615A - Giant salamander muscle short peptide with blood sugar reducing function and preparation method thereof

Info

Publication number: CN118206615A
Application number: CN202211624267.9A
Authority: CN
Inventors: 邢新会; 王怡; 陈海红
Original assignee: Shenzhen International Graduate School of Tsinghua University
Current assignee: Shenzhen International Graduate School of Tsinghua University
Priority date: 2022-12-15
Filing date: 2022-12-15
Publication date: 2024-06-18

Abstract

The application provides a short peptide with DPP-IV (dipeptidyl peptidase IV) inhibitory activity and a composition thereof, wherein the amino acid sequence of the short peptide is selected from any one of WRPPDH, WAPPSKD, IPDSPF, IPEMIF or VPIAVPT. The short peptide is extracted from giant salamander cultivated in China, can be used as a functional component of medicines, special medical food and health food, and is applied to the prevention and treatment of hyperglycemia and related diseases of human body caused by hyperglycemia.

Description

Giant salamander muscle short peptide with blood sugar reducing function and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a short peptide with DPP-IV inhibitory activity, and a preparation method and application thereof.

Background

Diabetes mellitus is widely spread worldwide and is mainly caused by insulin metabolic disorders or hyposecretion and is characterized by high blood glucose concentrations. With the continuous research on the pathogenesis of diabetes mellitus, DPP-IV gradually becomes a new target for treating diabetes mellitus. DPP-IV is a 766 amino acid serine protease that is ubiquitously found in humans, particularly in many tissues such as the lung, brain and kidneys. Based on in vitro studies, this enzyme preferentially hydrolyzes Xaa-Pro (proline) or Xaa-Ala (alanine) on the N-terminus of the short peptide, where Xaa represents any of the 20 natural amino acids. Proline residues are present in the second position at its N-terminus, as an evolutionarily conserved strategy, to protect several bioactive peptides from general proteolytic attack. And the second position of the N end of GLP-1 is Ala, and the GLP-1 is rapidly subjected to enzymolysis and inactivation by DPP-IV after being released, so that the function of promoting blood sugar reduction is lost. DPP-IV inhibitors increase GLP-1 levels in vivo by inactivating DPP-IV, resulting in a glycemic modulating effect. Therefore, research and development of DPP-IV inhibitors are important directions for preventing and treating type 2 diabetes and obesity.

In recent years, researchers separate and purify various active peptides from various natural raw materials, and the active peptides have the advantages of mild action, definite function, high safety, small toxic and side effects and the like, and are a new research hotspot. The bioactive peptide with DPP-IV inhibition effect has wide application prospect as medicines, special medical food or health food. ,

Disclosure of Invention

Giant salamander (Latin school name: andrias davidianus) is the largest amphibian existing in the world. The artificial propagation technology of the giant salamander is widely popularized and applied in the 70 s of the last century while the resource protection of the giant salamander is enhanced. Nowadays, the country has formulated relevant laws for the eating and industrial application of the cultured Chinese giant salamander after the second generation of children, so that the development of nutrition and medical health value of the giant salamander is possible. The giant salamander muscle protein is a high-quality protein, has high content of essential amino acids and good composition ratio, and completely meets the requirement mode of human body. The giant salamander muscle protein is rich in 18 amino acids, wherein 6 flavor-developing amino acids account for 42.77% of the total amino acids, 8 human essential amino acids account for 40.72% of the total amino acids, and the ratio of essential amino acids to non-essential amino acids is 68.68%. The giant salamander muscle has low fat content but high unsaturated fatty acid content, especially rich docosahexaenoic acid (DHA), and has the characteristics of similar unsaturated fatty acid composition and higher DHA content as those of deep sea fish. The development of giant salamander-derived medicinal, food and health care products can fully utilize giant salamander resources, and can provide a foundation for the development of new bioactivity and medicinal functions.

The application aims to provide an active short peptide extracted from giant salamander muscle and having DPP-IV inhibition effect.

Specifically, the present application provides a short peptide having DPP-IV inhibitory activity, which has the amino acid sequence: any one selected from WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5).

The application provides nucleic acid sequences encoding the short peptides.

The present application provides a composition comprising one or more short peptides of amino acid sequence WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5).

Preferably, the composition provided by the application further comprises a pharmaceutically, edible or health-care acceptable carrier, auxiliary material or second active ingredient.

Preferably, the second active ingredient in the compositions provided herein is selected from the group consisting of DPP-IV inhibitors, SGLT-2 inhibitors, GLP-1 receptor agonists, insulin secretagogues, α -glucosidase inhibitors, angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, calcium channel blockers, β -receptor blockers and the like.

The application provides a giant salamander muscle enzymolysis extract which consists of polypeptides with weight average molecular weight less than 3000 Da.

The giant salamander muscle enzymolysis extract provided by the application comprises one or more than two of the following short peptides: WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5).

The giant salamander muscle enzymolysis extract provided by the application is prepared by a method comprising the following steps:

removing lipid substances in giant salamander muscle powder by using an organic solvent;

centrifugal layering is carried out on the giant salamander muscle powder without lipid by using a chloroform-n-butanol mixed solution, and saccharide substances in the giant salamander muscle powder are removed;

carrying out enzymolysis on giant salamander muscle powder without saccharide substances by using protease, centrifuging, supernatant and separating to obtain giant salamander muscle peptide;

Preferably, the organic solvent is selected from the group consisting of dehydrated ether, isobutanol or n-hexane;

Preferably, the protease is selected from the group consisting of the enzymes NY100, AXH or ProtA, further preferably the enzyme NY100;

Preferably, the mass ratio of the protease to the giant salamander muscle powder is 1:15-1:50, and more preferably 1:50;

Preferably, the enzymolysis time is 6 to 72 hours, more preferably 71.12 hours;

Preferably, the pH value of the enzymolysis is 6-8, and more preferably 7.0;

preferably, the separation is performed using a 3kDa ultrafiltration centrifuge tube.

The application provides a method for preparing giant salamander muscle enzymolysis extract, which is characterized by comprising the following steps:

Preferably, the enzymolysis time is 6 to 72 hours, more preferably 71.12 hours;

Preferably, the pH value of the enzymolysis is 6-8, and more preferably 7.0;

Further, in the method for preparing the giant salamander muscle enzymolysis extract, the giant salamander muscle enzymolysis extract is provided by the application.

The application provides the use of a short peptide with an amino acid sequence selected from any one of WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5) or a composition comprising one or more of said short peptides in the preparation of a DPP-IV inhibitor medicament.

The application provides the use of a short peptide with an amino acid sequence selected from any one of WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5) or a composition comprising one or more of the short peptides in the preparation of a medicament, a special medical food or a health food for preventing, alleviating or treating hyperglycemia and diseases related to the human body caused by hyperglycemia.

Preferably, the composition further comprises a pharmaceutically, dietetic or health-care acceptable carrier, auxiliary material or a second active ingredient.

Preferably, the second active ingredient in the above composition is selected from the group consisting of DPP-IV inhibitors, SGLT-2 inhibitors, GLP-1 receptor agonists, insulin secretagogues, α -glucosidase inhibitors, angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, calcium channel blockers, β -receptor blockers and the like.

The present application provides a method of inhibiting DPP-IV comprising administering to a subject an effective amount of a short peptide having the amino acid sequence: any one selected from WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5).

The present application provides a method for preventing, alleviating or treating hyperglycemia and diseases associated with the human body, the method comprising administering to a subject an effective amount of a short peptide having the amino acid sequence: any one selected from WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5).

The present application provides a medicament for preventing, alleviating or treating hyperglycemia and related diseases, which comprises a short peptide having an amino acid sequence of any one selected from WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5) or a composition comprising one or two or more of the short peptides.

The application provides application of the giant salamander muscle enzymolysis extract or the giant salamander muscle enzymolysis extract prepared by the method in preparation of medicines for reducing blood sugar, reducing body fat rate or regulating intestinal flora.

The application provides a medicament for reducing blood sugar, reducing body fat rate or regulating intestinal flora, which comprises the giant salamander muscle enzymolysis extract or the giant salamander muscle enzymolysis extract prepared by the method.

Furthermore, the short peptide and giant salamander muscle enzymolysis extract provided by the application is extracted from cultured giant salamander.

Effects of the invention

The short peptide provided by the application has remarkable DPP-IV inhibition activity and potential hypoglycemic effect, and can be applied to the development of DPP-IV inhibitor drugs and other drugs related to hyperglycemia. The short peptide only contains 6-7 amino acid sequences, has a short structure, is more beneficial to human body absorption, can achieve the effect of reducing blood sugar through oral administration, is safer and more effective, and provides more various sources and research references for DPP-IV inhibitory peptide. Meanwhile, the giant salamander muscle enzymolysis extract provided by the application can effectively relieve the influence of high-fat diet on overweight and fasting blood glucose increase, has the function of assisting in blood glucose control, and can obviously reduce insulin resistance level; furthermore, the giant salamander muscle enzymolysis extract can obviously reduce body fat rate, has the function of regulating intestinal flora, and can improve the in-vivo healthy environment by increasing the relative content of probiotics.

In addition, the short peptide and the enzymolysis extract come from giant salamanders, the production process of the application also increases the development strength of the giant salamander breeding resources, improves the added value of giant salamander products, and is beneficial to the healthy development of the giant salamander industry in China.

Drawings

FIG. 1 shows the inhibition of DPP-IV by supernatant in 20. Mu.L of enzymatic hydrolysate under different enzymatic hydrolysis conditions.

FIG. 2 shows the enzymatic hydrolysis conditions for three enzymes (a. Enzyme NY100; b. Enzyme AXH; c. Enzyme ProtA).

FIG. 3 shows a response surface optimization graph (A.Time interacts with E/S; B.Time interacts with pH; C.pH interacts with E/S; D.optimization calculation).

FIG. 4 shows the inhibition curves of DPP-IV by M0-3, M3-10 at various concentrations.

FIG. 5 shows the liquid chromatograms of M0-3 at different sample volumes.

FIG. 6 shows the inhibition of DPP-IV by M0-3 and its different retention time separation fractions at different concentrations.

FIG. 7 is a diagram showing the double reciprocal of Lineweaver-Burk for inhibition of DPP-IV by five short peptides.

FIG. 8 is a graph showing changes in body weight of mice.

FIG. 9 is a graph showing changes in blood glucose in mice.

Fig. 10 (left) shows the change in glucose concentration during the glucose tolerance test in mice, and fig. 10 (right) shows the area schematic under the line.

FIG. 11 shows a HOMA-IR pattern of mice.

FIG. 12 is a graph showing body fat percentage of mice.

FIG. 13 is a graph showing abundance of fecal flora in mice.

Detailed Description

The present application is described in further detail below in conjunction with the detailed description of the application, examples are given to provide a better understanding of the present application and to fully convey the scope of the application to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The specification and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As referred to throughout the specification and claims, the terms "include" or "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth a preferred embodiment for practicing the application, but is not intended to limit the scope of the application, as the description proceeds with reference to the general principles of the description. The scope of the application is defined by the appended claims.

The application provides a short peptide extracted from giant salamander muscle cultivated by the method, and in particular relates to the short peptide which can prevent GLP-1 from being rapidly degraded by DPP-IV by inhibiting enzymolysis activity of DPP-IV, so that acting time of GLP is prolonged, and forward action of the short peptide in the process of reducing blood sugar and the like is enhanced. It has inhibitory activity against DPP-IV.

In a specific embodiment of the application, the short peptide is selected from one of WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5).

In the foregoing short peptide sequences, I, P, V, Q, E, S, T, W and like capital letters each represent an amino acid or an amino acid residue thereof, and the correspondence between each capital letter and an amino acid is shown in Table 1.

Table 1 amino acids and their corresponding capital letters

Chinese name	Letter abbreviations	Chinese name	Letter abbreviations
				Glycine (Gly)	G	Serine (serine)	S
Alanine (Ala)	A	Threonine (Thr)	T
				Valine (valine)	V	Cysteine (S)	C
Leucine (leucine)	L	Asparagine derivatives	N
				Isoleucine (Ile)	I	Glutamine	Q
Proline (proline)	P	Aspartic acid	D
				Phenylalanine (Phe)	F	Glutamic acid	E
Tryptophan	W	Lysine	K
				Methionine (methionine)	M	Arginine (Arg)	R
Tyrosine	Y	Histidine	H

In an embodiment of the application, a nucleic acid sequence encoding the short peptide is also included.

In some embodiments, the short peptide may be extracted from an organism or obtained via enzymatic hydrolysis. In some embodiments, the short peptide may be produced by chemical synthesis. In some embodiments, the short peptide may be produced by biosynthesis.

The application also provides a composition comprising one or more than two short peptides of amino acid sequence WRPPDH, WAPPSKD, IPDSPF, IPEMIF or VPIAVPT. In some embodiments, the composition comprises one, two or more short peptides as described previously that inhibit DPP-IV activity.

In some embodiments, the composition may be a pharmaceutical composition, or a special medical use food, or a health food. Further, the composition also comprises a pharmaceutically, edible or health acceptable carrier, auxiliary materials or a second active ingredient.

Preferably, the second active ingredient in the compositions provided herein is selected from a DPP-IV inhibitor, an SGLT-2 inhibitor or a GLP-1 receptor agonist.

In some embodiments, the compositions of the present application further comprise a pharmaceutically acceptable excipient.

In a specific embodiment, the composition may be prepared in the following form: the short peptide is mixed with a pharmaceutically acceptable carrier, for example, to obtain an oral preparation such as a tablet (including sugar-coated tablet, film-coated tablet, sublingual tablet, orally disintegrating tablet), a capsule (including soft capsule, microcapsule), a granule, a powder, a lozenge, a syrup, an emulsion, a suspension, a film (e.g., orally disintegrating film), etc., a parenteral preparation such as an injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, instillation), an external preparation (e.g., skin preparation, ointment), a suppository (e.g., rectal suppository, vaginal suppository), a pill, a nasal drop, a respiratory preparation (inhalant), an eye drop, etc. In addition, these formulations may be used as controlled release formulations (e.g., sustained release microcapsules), such as immediate release formulations, sustained release formulations, and the like. Such formulations may be obtained by preparation methods conventionally used in the art.

In particular, examples of such pharmaceutically acceptable carriers include excipients (e.g., starches,

Lactose, sucrose, calcium carbonate, calcium phosphate, etc.), binders (e.g., starch, acacia, carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, alginic acid, gelatin, polyvinylpyrrolidone, etc.), lubricants (e.g., magnesium stearate, calcium stearate, talc, etc.), disintegrants (e.g., carboxymethylcellulose calcium, talc, etc.), diluents (e.g., water for injection, saline, etc.), additives (e.g., stabilizers, preservatives, colorants, flavoring agents, dissolution aids, emulsifiers, buffers, isotonic agents, etc.), and the like.

Embodiments of the present application also provide the use of a short peptide having an amino acid sequence selected from any one of WRPPDH, WAPPSKD, IPDSPF, IPEMIF or VPIAVPT or a composition comprising one or more of said short peptides in the manufacture of a medicament for use as a DPP-IV inhibitor.

In some embodiments, there is also provided the use of a short peptide or a composition comprising one or more of said short peptides, wherein said amino acid sequence is selected from any of WRPPDH, WAPPSKD, IPDSPF, IPEMIF or VPIAVPT, in the manufacture of a medicament for the prevention, alleviation or treatment of hyperglycemia and hyperglycemia-related human diseases.

In some embodiments, there is also provided the use of a short peptide or a composition comprising one or more of said short peptides, wherein said amino acid sequence is selected from any of WRPPDH, WAPPSKD, IPDSPF, IPEMIF or VPIAVPT, for the preparation of a special medical use food and a health food for ameliorating hyperglycemia and hyperglycemia-related human diseases.

In some embodiments, the compositions are useful in the prevention, treatment, and amelioration of type 2 diabetes. In other embodiments, the composition may reduce the risk of cardiovascular disease caused by type 2 diabetes, hyperglycemia, hypertension, hyperlipidemia, and the like.

The present application provides a method for preventing, alleviating or treating hyperglycemia and related diseases, comprising administering to a subject an effective amount of a short peptide having the amino acid sequence: any one selected from WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5).

The short peptides or compositions thereof referred to in the present application may also be used with other currently known drugs for the treatment of hyperglycemia, type 2 diabetes. When used together, there is no limitation on the administration time of the respective drugs, two or more different drugs may be administered simultaneously, and the respective drugs may be administered at different times. The dosage of the known drug may be determined in accordance with the number of administrations used clinically, and appropriately selected according to the administration patient, the administration route, and the like.

The medicament or composition containing the short peptide of the present application may be administered to a mammal (e.g., human, mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey). The mode of administration may be oral or parenteral (e.g., intravenous, intramuscular, subcutaneous, intra-organ, intranasal, intradermal, instillation, intracerebral, rectal, vaginal, intraperitoneal, etc.).

The amount of the short peptide of the present application to be administered to a subject varies depending on the administration route, symptoms, age of patient, etc., and can be practically determined by a clinician.

The application also provides a giant salamander muscle enzymolysis extract which consists of polypeptides with weight average molecular weight less than 3000 Da.

In some embodiments, the giant salamander muscle enzyme extract comprises one or more short peptides having the amino acid sequence WRPPDH, WAPPSKD, IPDSPF, IPEMIF or VPIAVPT.

In some embodiments provided by the application, the giant salamander muscle enzymolysis extract is prepared by a method comprising the following steps:

And (3) carrying out enzymolysis on giant salamander muscle powder without carbohydrate substances by using protease, centrifuging, supernatant and separating to obtain the giant salamander muscle peptide.

In some embodiments, the giant salamander muscle powder is derived from cultured giant salamander.

In some embodiments, the organic solvent may be selected from the group consisting of dehydrated ether, isobutanol, and n-hexane.

In some embodiments, the protease may be selected from the group consisting of enzymes NY100, AXH, or ProtA, preferably enzyme NY100.

In some embodiments, the mass ratio of protease to giant salamander muscle powder is 1:15 to 1:50, for example, 1:15,1:20,1:25,1:30,1:35,1:40,1:45,1:50.

In some embodiments, the time of the enzymatic hydrolysis is 6 to 72 hours, for example, 6, 8, 10, 12, 18, 24, 30, 40, 50, 60, 70, 71, 72 hours, etc.

In some embodiments, the pH of the enzymatic hydrolysis is between 6 and 8, e.g., the pH may be 6, 6.5, 7, 7.5, 8, etc.;

In some embodiments, the separation is performed using a 3kDa ultrafiltration centrifuge tube. .

The method for removing the grease in the giant salamander meat powder is not limited. In some specific embodiments, soxhlet extraction may be used to remove lipids from the muscle powder of cultured giant salamanders. The specific method comprises the following steps: the giant salamander muscle powder is weighed and moved into a filter paper tube, the filter paper tube is placed into an extraction tube of a Soxhlet extractor, a clean and dry receiving bottle is connected, anhydrous diethyl ether is added from the upper end of a condenser tube of the extractor to the position of two thirds of the inner volume of the bottle, the bottle is placed on a fat analyzer for heating, the temperature is kept at about 40-60 ℃, and the reflux is carried out for 6-8 times per hour, and the extraction is carried out for 3 hours. And taking down the receiving bottle, recovering the solid sample in the filter paper tube, drying and collecting the solid sample to obtain the de-lipidated giant salamander muscle powder. In other embodiments, organic solvents are used to leach lipid from the muscle powder of the cultured giant salamander. The specific method comprises the following steps: weighing giant salamander muscle powder, placing the giant salamander muscle powder into a conical flask, adding isobutanol or n-hexane according to the mass ratio of the meat powder to the solvent of 1:10, shaking and shaking uniformly, and standing at room temperature for 24 hours. Centrifuging at 10000rpm for 5min, pouring out the upper organic solvent, drying the lower precipitate, and collecting to obtain lipid-removed giant salamander muscle powder.

In some embodiments, the giant salamander muscle enzyme extract is capable of alleviating the effects of a high-fat diet on overweight, alleviating the effects of a high-fat diet on elevated abdominal blood glucose, and has an auxiliary blood glucose control effect. In other embodiments, the giant salamander muscle enzyme extract significantly reduces insulin resistance levels, indicating that it helps to alleviate insulin levels.

In some embodiments, the giant salamander muscle enzyme extract is capable of significantly reducing body fat rate.

In some embodiments, the giant salamander muscle enzymolysis extract has a regulating function on intestinal flora, and can improve the in-vivo healthy environment by increasing the relative content of probiotics.

Further, the application provides a method for preparing the giant salamander muscle enzymolysis extract, which comprises the following steps:

preferably, the mass ratio of the protease to the giant salamander muscle powder is 1:15-1:50;

Preferably, the enzymolysis time is 6-72 hours;

Preferably, the pH value of the enzymolysis is 6-8;

The application also provides application of the giant salamander muscle enzymolysis extract or the giant salamander muscle enzymolysis extract prepared by the method in preparation of medicines for reducing blood sugar, reducing body fat rate or regulating intestinal flora.

The application also provides a medicine for reducing blood sugar, reducing body fat rate or regulating intestinal flora, which comprises the giant salamander muscle enzymolysis extract or the giant salamander muscle enzymolysis extract prepared by the method.

Examples

Example 1 selection and optimization of giant salamander muscle enzymatic hydrolysis conditions

(1) The enzymolysis conditions of giant salamander muscles are selected and optimized through orthogonal experiments

Orthogonal experiments were designed using SPSS 22.0.0.0 software. And (3) performing enzymolysis experiments according to the conditions designed in the table 1, performing general linear model univariate analysis on experimental results, and drawing a marginal value estimation graph of the fixed factors on the dependent variables. The experimental results are shown in FIG. 1. It can be seen that under different conditions, the inhibition rate of DPP-IV by the enzymatic hydrolysate obtained by using NY100 is basically the highest, followed by AXH and then ProtA.

TABLE 1 level of orthogonal experimental factors

The results of the orthogonal experiment of the enzyme NY100 were analyzed as shown in table 2 (the numbers under each variable correspond to the values under the number in table 1, and tables 3 and 4 are the same). Since the magnitude of the R value reflects the sensitivity of the dependent variable to the independent variable, the primary and secondary relationship of the enzymolysis effect is as follows for the enzyme NY 100: a (enzymatic time) > C (solution pH) > B (enzyme to substrate ratio).

TABLE 2L ₉(3⁴ of enzyme NY 100) orthogonal experiment results

Similarly, the results of the orthogonal experiments with enzyme AXH were analyzed as shown in Table 3. For the enzyme AXH, the primary and secondary relationship of the enzymolysis effect is as follows: a (enzymatic time) > B (enzyme to substrate ratio) > C (solution pH).

TABLE 3L ₉(3⁴) orthogonal experiment results for enzyme AXH

Similarly, the results of the orthogonal experiments with the enzyme ProtA were analyzed as shown in table 4. For the enzyme ProtA, the primary and secondary relation of the enzymolysis effect is as follows: b (enzyme to substrate ratio) > C (solution pH) > a (enzymatic time).

TABLE 4L ₉(3⁴) orthogonal experiment results for the enzyme ProtA

* K is the sum of the results at each level of each factor, R is the range of the K value

Single factor analysis in a general linear model was performed using SPSS as shown in fig. 2. For enzyme NY100, the optimal combination of conditions for single-factor analysis is a ₃B₃C₃; for enzyme AXH, the optimal combination of conditions for single-factor analysis is a ₃B₂C₂; for the enzyme ProtA, the optimal combination of conditions for one-way analysis is a ₂B₁C₁.

(2) Response surface experiments optimize conditions of enzyme NY100 on muscle enzymolysis of giant salamander

Response surface experiments were designed and statistically analyzed using Design-Expert 12.0.3 software, and the factor level table is shown in table 5. And performing enzymolysis experiments according to the conditions of software design, and performing second-order multiple regression analysis on experimental results.

TABLE 5 response surface method factor level Table

A response surface optimization chart of the giant salamander muscle proteolytic condition is shown in figure 3. Wherein the graph A shows a three-dimensional curved surface graph of the Inhibition ratio of enzyme to substrate ratio E/S and Time Time interaction, and the Inhibition reaches a theoretical maximum value due to the fact that the curved surface is a concave function in the experimental range and an AC local optimal solution exists. B. In the graph C, the curved surface is a non-concave function in the experimental range, and boundary values (enzymolysis time 71.12 hours, enzyme-substrate mass ratio 1:50 and solvent pH 7.0) of AB and BC are taken to obtain giant salamander muscle proteolytic liquid with highest DPP-IV inhibition activity, and inhibition under the predicted and optimized condition is 0.816.

Example 2 preparation of giant salamander muscle enzymatic extract

(1) Organic solvent leaching to remove lipid in giant salamander muscle powder

About 5g of giant salamander muscle powder is weighed, placed in a conical flask, 50g of isobutanol (mass ratio of meat powder to solvent 1:10) is added, shaken and shaken uniformly, and then kept stand for 24 hours at room temperature. Centrifuge at 10000rpm for 5min. Layering the solution, pouring out the upper organic solvent, drying the lower sediment, and collecting the sediment to obtain the lipid-removed giant salamander muscle powder.

(2) Removing saccharides in giant salamander muscle powder

Preparing giant salamander muscle powder obtained in the step (1) into giant salamander muscle powder solution with the concentration of 5mg/mL in an conical flask, wherein the solvent is water. Chloroform-n-butanol mixed solution (volume ratio 4:1) was prepared as a Sevag reagent.

And mixing the giant salamander meat powder solution with the Sevag reagent according to a volume ratio of 4:1. Adding rotor, and placing the conical flask on an electromagnetic stirrer to stir for 30min. And centrifuged at 5000rpm for 15min. The solution is layered, the upper layer is organic solution, the middle layer is protein, and the next layer is water layer containing polysaccharide. The organic and aqueous layers were aspirated using a pipette or syringe to give the remaining protein layer. And repeating the centrifugation operation for 3-4 times on the protein layer, and drying and collecting the protein layer substance obtained by final treatment to obtain giant salamander muscle powder without saccharides.

(3) Pretreatment before enzymolysis

Dissolving giant salamander muscle powder obtained in the step (2) in PBS buffer solution at the concentration of 100mg/mL, and placing the giant salamander muscle powder in an ultrasonic cleaner for ultrasonic treatment for 10min so as to improve the solubility of giant salamander muscle protein.

(4) Enzymolysis extract of giant salamander muscle obtained by enzymolysis

The protease (NY 100 enzyme) was dissolved in PBS to prepare an enzyme solution having a concentration of 20 mg/mL. Uniformly mixing the giant salamander meat powder solution pretreated in the step (3) with the enzyme solution according to the mass ratio of 50:1 to prepare an enzymolysis system, and adjusting the pH of the system to 7.0. The enzymolysis system is placed in a preheated 50 ℃ constant temperature shaking table, the rotating speed is set to 200rpm, and the reaction time is set to 72h. And (3) placing the enzymolysis system after the reaction in a water bath kettle at 95 ℃ and inactivating for 15min. After the inactivation is finished, the ice bath is carried out to room temperature, and then the ice bath is transferred to a refrigerator with the temperature of minus 80 ℃ to be rapidly cooled for 5min. And centrifuging the enzymolysis liquid at 8000rpm, and performing vacuum freeze-drying on the supernatant to obtain the giant salamander muscle enzymolysis extract.

Example 3 determination of DPP-IV inhibitory Activity of giant salamander muscle enzyme extract

The giant salamander muscle enzymolysis liquid in example 2 was centrifuged in stages according to molecular weight using ultrafiltration centrifuge tubes of 3kDa and 10kDa to obtain components having molecular weights of 0-3kDa (M0-3), 3-10kDa (M3-10) and molecular weights of more than 10kDa (M10+).

M0-3, M3-10, M10+ component solution (20. Mu.L), 2mM substrate Gly-Pro-pNA solution (20. Mu.L) and pH8.0 Tris-HCl buffer (20. Mu.L) at concentrations of 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL were premixed in 96-well microplates, and finally DPP-IV (40. Mu.L, final concentration 0.025U/mL) was added to initiate the reaction at a final volume of 100. Mu.L. Incubate at 37℃for 60min. The absorbance of the system at 405nm was measured using a microplate reader. The negative control system included Tris-HCl (20. Mu.L) at pH8.0, gly-Pro-pNA solution (20. Mu.L) at a concentration of 2mM substrate, PBS buffer (20. Mu.L) and DPP-IV (40. Mu.L, final concentration 0.025U/mL). The blank system was Tris-HCl buffer (60. Mu.L) at pH8.0, gly-Pro-pNA solution (20. Mu.L) as a 2mM substrate, and PBS buffer (20. Mu.L).

The DPP-IV inhibition of the samples was calculated according to the following formula:

Wherein Abs _Negative represents the absorbance of the negative control mixture with PBS added; abs _Sample represents the absorbance of the added short peptide samples in the experimental group; abs _Blank represents the absorbance of the blank control system. The DPP-IV inhibition results of the individual components are shown in FIG. 4.

Concentration as independent variable and DPP-IV inhibition as dependent variable were plotted and fitted to a curve. And calculating the corresponding concentration when the DPP-IV inhibition ratio is 0.5 according to the fitting formula, wherein the concentration is the estimated half inhibition concentration IC ₅₀. The IC ₅₀ of each component is shown in Table 6. Wherein, the inhibition activity of the low molecular weight group M0-3 on the enzyme DPP-IV is obviously higher than that of the high molecular weight groups M3-10 and M10+.

TABLE 6 IC of M0-3, M3-10 and M10+ components ₅₀

Example 4 screening and assay of short peptides with DPP-IV inhibitory Activity

(1) Short peptide screening with DPP-IV inhibitory activity in giant salamander muscle enzymolysis liquid

Since the results of example 3 show that the inhibitory activity of the low molecular weight group M0-3 on the enzyme DPP-IV is significantly higher than that of the high molecular weight groups M3-10 and M10+, the composition of the group M0-3 was further analyzed. Giant salamander muscle peptide samples of M0-3 groups were prepared as 30mg/mL solutions and analyzed by reverse phase liquid chromatography to give a graph as shown in FIG. 5, wherein the horizontal axis represents retention time and the vertical axis represents response value of the UV detector. The sample volumes were measured at 5. Mu.L and 100. Mu.L, respectively. When the sample injection volume is 100 mu L, part of peak value exceeds the detection limit, which is unfavorable for component separation. The chromatographic condition can be adopted without exceeding the detection limit when the sample injection volume is 5 mu L. According to FIG. 5, the material flowing out in the first 5min of the gradient treatment is inorganic salts, and a large amount of organic matters flow out in 12.5-25 min. It is divided into four segments, namely, retention time 12.5-14 minutes (T12.5-14), retention time 14-18 minutes (T14-18), retention time 18-21 minutes (T18-21), and retention time 21-25 minutes (T21-25). The fractions of each retention period were collected into centrifuge tubes, and lyophilized, and DPP-IV inhibitory activity was measured in the same manner as in example 2, and the results are shown in FIG. 6 and Table 7.

TABLE 7 half inhibitory concentration IC ₅₀ values for DPP-IV for M0-3 and different retention time separation fractions

Experimental results show that the separated components with retention time within the range of 18-25 minutes have higher DPP-IV inhibition activity. Subsequently, the isolated fractions in the 18-25 min interval were subjected to protein N-terminal sequencing using a liquid chromatography/mass spectrometry system (LTQ-Orbitrap Velos Mass Spectrometer, Q-Exactive). Short peptides in the giant salamander muscle proteolytic liquid are detected through analysis, and the total number of the short peptides is 2126. According to the report of the literature, the short peptide with the structure that the amino acid at the second position of the N end is Pro or Ala, the first position is Trp or the first two positions are Val-Pro or Ile-Pro is more likely to have high-efficiency DPP-IV inhibition activity, so that five short peptide sequences WRPPDH, WAPPSKD, IPDSPF, IPEMIF, VPIAVPT are obtained as candidates by screening according to the condition, the synthesis is carried out by entrusting the Kirsrui biotechnology (the purity and the correctness of each short peptide are proved by adopting an HPLC and MS detection, and the purity of a sample provided by the HPLC detection is required to be more than 98 percent), and then the subsequent in vitro DPP IV inhibition activity verification is carried out.

(2) Screening short peptides for inhibitory activity and inhibition type assay.

DPP-IV inhibitory activity of 5 short peptide samples was determined by the method of example 2.

Inhibition type assay: each of the short peptide test solutions of 100. Mu.M was prepared, and DPP IV inhibitory activity was measured, and the concentration of the chromogenic substrate Gly-Pro-PNA was changed to 0.1, 0.2, 0.3, 0.4 and 0.5mM, respectively. Meanwhile, tris-HCl with pH of 8.0 was used as a negative control sample. After incubation at 37℃for 60min, the absorbance at 405nm was recorded with a microplate reader. The reciprocal of Gly-Pro-PNA concentration was plotted as independent variable and the reciprocal of absorbance as dependent variable, and a curve was fitted. Competitive inhibition if the intercept of the fitted curve of the experimental group and the negative control is the same and the slope is different; if the slope of the fitting curve of the experimental group and the negative control is the same, the experimental group is anti-competitive inhibition; if the intersection of the fitted curve of the experimental group and the negative control and the horizontal axis is the same and the slope is different, the inhibition is non-competitive.

The inhibitory activity and inhibition type analysis results of the 5 short peptide samples are shown in fig. 7 and table 8.

TABLE 8 half inhibitory concentration IC ₅₀ of five short peptides on DPP-IV and type of inhibition on DPP-IV

The experimental results show that the IC ₅₀ values of the 5 short peptide samples are below 0.5mM, which indicates that the short peptide sequence WRPPDH, WAPPSKD, IPDSPF, IPEMIF, VPIAVPT provided by the application can inhibit DPP-IV more effectively, more safely and effectively than the peptide with hypoglycemic effect in the prior report.

Example 5 blood sugar-reducing, body fat-reducing and intestinal flora-regulating functional test of giant salamander muscle enzymolysis extract

Experimental protocol: 60C 57BL/6J male mice (purchased from Zhejiang Veitz Lihua laboratory animal technologies Co., ltd.) were selected at 6 weeks of age (weight 18-22 g). Adaptive cultures (fed with standard feed) were performed for the first week. The second week is pre-fed: 60 mice were randomly divided into 6 groups of 10 mice each. The six groups were Control (Control), model (HFD, high fat diet), low Dose (LD), medium Dose (MD), high Dose (HD), positive Control (SITAGLPTIN, sitagliptin drug). Each group was fed standard feed during the pre-feed period. The LD group was supplemented with 200 mg/(kg mouse body weight) of the muscle unfractionated enzymatic extract of giant salamander dissolved in sterile water daily by the feeding method. The MD group was supplemented with 400 mg/(kg of mouse body weight) of the giant salamander muscle enzyme-hydrolyzed extract in sterile water daily by the feeding method. The HD group was supplemented with 800 mg/(kg mouse body weight) of giant salamander muscle enzyme extract dissolved in sterile water daily by the gavage method. Group SITAGLIPTIN was supplemented daily with 12.5 mg/(kg of mouse body weight) of sitagliptin phosphate (stratinol) in sterile water by the method of gavage. Establishment of a high fat diet-induced hyperglycemia model was performed from week 3 to week 11: each group was fed a sufficient amount of high fat diet (20 kcal of protein, 20kcal of carbohydrate, 60kcal of fat in the diet) except for the control group. Animals were sacrificed on the 11 th weekend. The following experiments were modeled using the above experimental protocol.

(1) Weight loss effect of giant salamander muscle enzymolysis extract

Mice body weight was tested once a week during the experiment and the average of each group was plotted as shown in figure 8. Welch and BrownForsythe one-way anova and Dunnett T3 post hoc test (average, n=10) were used. Significant differences (P < 0.05) represent the method: * P <0.05; * P <0.01; * P <0.001; * P < 0.0001. After 11 weeks, the HD group had significantly lower body weight than the HFD group, indicating that feeding with high doses of giant salamander muscle peptide significantly reduced the effect of the high fat diet on overweight in mice. MD and SITAGLIPTIN groups are somewhat lower than HFD groups, which indicates that the effect of high-fat diet on overweight of mice is relieved by feeding giant salamander muscle peptide at a medium dose, and the effect of feeding sitagliptin phosphate is achieved.

(2) Giant salamander muscle enzymolysis extract for reducing fasting blood sugar

Mice were tested weekly for fasting blood glucose during the experiment, and the graph shown in fig. 9 was plotted after each group was averaged. Welch and BrownForsythe one-way anova and Dunnett T3 post hoc test (average, n=10) were used. Significant differences (P < 0.05) represent the method: * P <0.05; * P <0.01; * P <0.001; * P < 0.0001. After 11 weeks, the fasting blood glucose of the HD group is significantly lower than that of the HFD group to a certain extent, indicating that the effect of high-fat diet on the increase of the fasting blood glucose of the mice is relieved by the administration of the high-dose giant salamander muscle peptide, and the effect of the administration of the high-fat diet is superior to that of the administration of sitagliptin phosphate.

(3) Auxiliary blood sugar regulating and controlling function of giant salamander muscle enzymolysis extract

Oral glucose tolerance test was performed at week 9 of animal experiment. Mice were transferred to clean cages for a fasting period of 16 hours prior to testing. The mice body weight was initially recorded and fasting blood glucose was measured. After 30min of adaptation, mice were initially prepared for gastric lavage glucose (OGTT). Glucose solution (0.01 ml/g of 20% glucose solution of mouse body weight) was administered to mice following standard gavage procedures with a 1ml syringe connection gavage needle. Blood glucose was measured for each mouse at 15min,30min,60min,90min,120min, plotted as the curve in FIG. 10 (left), and the area under the calculated curve is shown as the bar graph in FIG. 10 (right). Compared with HFD groups, the sample groups (LD, MD and HD) fed with the giant salamander muscle enzymolysis extract reduce the peak blood sugar after glucose is orally taken, and reduce the area under the curve of the change of blood sugar along with time, which indicates that the giant salamander muscle enzymolysis extract has the function of assisting blood sugar control.

(4) Action of giant salamander muscle enzymolysis extract for relieving insulin resistance

Mice were measured for fasting blood glucose and serum was taken for insulin content determination prior to sacrifice. HOMA-IR was calculated using fasting insulin and fasting blood glucose data and plotted as in fig. 11 using Welch and BrownForsythe one-way anova and Dunnett T3 post hoc test (mean ± s.e.m, n=8). Significant differences (P < 0.05) represent the method: * P <0.05; * P <0.01; * P <0.001; * P <0.0001. Insulin resistance is a target organ for insulin action, including muscle, liver, and even adipose tissue, with reduced action on insulin. The insulin resistance index, also called the insulin resistance index of the steady state model, the english abbreviation HOMA-IR, is a mathematical model created by foreign scholars, the simple formula is fasting insulin x fasting blood glucose ≡22.5, if the value exceeds 2.69, the presence of insulin resistance is considered.

HD group significantly reduced insulin resistance levels compared to HFD group and was significantly stronger than SITAGLIPTIN group. The high-dose giant salamander muscle enzymolysis extract is helpful for relieving insulin level, and the effect is better than that of sitagliptin phosphate at the concentration in the embodiment.

(5) Verification of body fat rate reducing function of giant salamander muscle enzymolysis extract

The sacrificed mice were dissected, fat was collected from the mice and weighed, the ratio of fat to Body weight (g fat/100 g Body weight) was calculated, recorded as Body fat rate (Body fat rate), and a bar graph was drawn as shown in fig. 12. Welch and BrownForsythe one-way anova and Dunnett T3 post hoc test (mean ± s.e.m., n=8). Significant differences (P < 0.05) represent the method: * P <0.05; * P <0.01; * P <0.001; * P <0.0001.

HD significantly reduced body fat rate compared to HFD and significantly stronger than SITAGLIPTIN, MD significantly reduced body fat rate to some extent compared to HFD. The results show that the medium-dose and high-dose giant salamander muscle enzymolysis extracts are favorable for reducing the body fat rate, and the effect of the high-dose giant salamander muscle enzymolysis extracts on reducing the body fat rate is better than that of sitagliptin phosphate at the concentration in the embodiment.

(6) Intestinal flora regulation method by giant salamander muscle enzymolysis extract

Fresh feces from mice were collected at week 10 of the experiment. The DNA fragments of the mouse fecal microbiota were subjected to double-ended (Paired-end) sequencing using an Illumina platform. The original data is denoised by a DADA2 method of QIIME2 (2019.4) software, a characteristic sequence is obtained, and a Greengenes database is selected for species annotation, so that the visualization of the composition distribution of each sample on six classification levels of phylum, class, order, family, genus and species is realized. Visualization at the genus classification level as shown in fig. 13, it can be seen that: the relative content of the intestinal probiotics Lactobacillus of the HD group is higher than that of HFD, and the Lactobacillus has the effects of preventing and relieving diabetes, enhancing the health of liver mitochondria and improving the way of metabolism of glucose and lipid by a host. The relative content of the intestinal probiotics of the HD group, namely the tremella (Oscillospira) is higher than that of the Control group, oscillospira can produce butyrate, which indicates that the organism can play an important role in various aspects of functions and health, and the abundance of the organism is positively related to the diversity of microorganisms, high-density lipoprotein and sleeping time. According to the change conditions of the bacteria, the giant salamander muscle enzymolysis extract can be presumed to have the function of regulating intestinal flora, and the in-vivo healthy environment is improved by increasing the relative content of probiotics.

The above description is only of the preferred embodiments of the present application, and is not intended to limit the present application in any way. Any person skilled in the art may make variations or modifications to the equivalent embodiments using the teachings disclosed above. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.

Claims

1. A short peptide having DPP-IV inhibitory activity, characterized by the amino acid sequence: any one selected from WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5).

2. A nucleic acid sequence encoding the short peptide of claim 1.

3. A composition comprising one or more short peptides of amino acid sequence WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5).

4. The short peptide of claim 1 or the composition of claim 3, wherein the short peptide is extracted from giant salamander.

5. The giant salamander muscle enzymolysis extract is characterized by comprising polypeptides with weight average molecular weight less than 3000 Da.

6. The giant salamander muscle enzyme-hydrolyzed extract according to claim 5, comprising one or more short peptides selected from the group consisting of: WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5).

7. A method for preparing giant salamander muscle enzymolysis extract, comprising the steps of:

Preferably, the enzymolysis time is 6-72 hours;

Preferably, the pH value of the enzymolysis is 6-8;

8. Use of the short peptide of claim 1 or the composition of claim 3 for the preparation of a DPP-IV inhibitor medicament.

9. Use of the short peptide of claim 1 or the composition of claim 3 for the preparation of a medicament, special medical use food and health food for preventing, alleviating or treating hyperglycemia and diseases related to the human body caused by hyperglycemia.

10. A method of inhibiting DPP-IV comprising administering to a subject an effective amount of a short peptide having the amino acid sequence: any one selected from WRPPDH (Seq ID No. 1), WAPPSKD (Seq ID No. 2), IPDSPF (Seq ID No. 3), IPEMIF (Seq ID No. 4) or VPIAVPT (Seq ID No. 5).