Dipeptidyl peptidase IV inhibition oligopeptide for tuna roe
阅读说明：本技术 一种金枪鱼鱼卵二肽基肽酶ⅳ抑制寡肽 (Dipeptidyl peptidase IV inhibition oligopeptide for tuna roe ) 是由 陈如萍 王玉梅 王斌 于 2021-11-09 设计创作，主要内容包括：本发明公开了一种金枪鱼鱼卵二肽基肽酶Ⅳ抑制寡肽及其用途,该二肽基肽酶Ⅳ(DPP-Ⅳ)抑制寡肽的氨基酸序列为Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu,分子量为939.07Da。本发明制备的金枪鱼鱼卵的二肽基肽酶Ⅳ(DPP-Ⅳ)抑制寡肽可显著抑制DPP-Ⅳ活力,降低模型小鼠餐后血糖水平,改善小鼠口服葡萄糖糖耐量和麦芽糖糖耐量,降低模型小鼠的甘油三酯(TG)和胆固醇(TC)的含量,且安全无毒副作用；可应用于制备治疗或者辅助治疗II型糖尿病的特殊医学用途食品、保健产品和药物。(The invention discloses a dipeptidyl peptidase IV (DPP-IV) inhibition oligopeptide with the amino acid sequence of Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu and the molecular weight of 939.07Da and application thereof. The dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide of the tuna roe prepared by the invention can obviously inhibit DPP-IV activity, reduce postprandial blood glucose level of a model mouse, improve oral glucose tolerance and maltose tolerance of the mouse, reduce the contents of Triglyceride (TG) and cholesterol (TC) of the model mouse, and is safe and has no toxic or side effect; can be used for preparing food, health product and medicine for treating or adjunctively treating type II diabetes.)
1. The dipeptidyl peptidase IV inhibiting oligopeptide is characterized in that the oligopeptide is a nonapeptide compound, the amino acid sequence of the oligopeptide is Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu, and the molecular weight of ESI-MS determination is 939.07 Da.
2. The method for preparing the dipeptidyl peptidase IV inhibitory oligopeptide of tuna eggs according to claim 1, which comprises the following steps:
1) pretreatment of fish eggs: unfreezing tuna roes, removing impurities, mashing the tuna roes by using a tissue mashing machine, adding an acetone solution, carrying out ultrasonic treatment at 200W for 15-20min for degreasing, repeating the steps for three times, centrifuging the tuna roes at 4 ℃ and 9000rmp for 15-20min, and drying solid precipitates to obtain degreased roes;
2) enzymolysis of fish eggs: adding the defatted roe into a buffer solution with the pH value of 1.5-2.5, uniformly stirring, adjusting the temperature of the solution to 37 ℃, adding protease A accounting for 2.0-2.5% of the weight of the defatted roe, and performing enzymolysis for 3-4 hours; then adjusting the pH value of the solution to 6.5-7.5, adding protease B accounting for 2.0-2.5% of the weight of the defatted fish eggs, and carrying out enzymolysis for 4-5 h; after enzymolysis, cooling the solution to room temperature, centrifuging at 12000rmp for 10-15 min, and collecting supernatant to obtain tuna roe protein enzymolysis solution;
3) preparation of fish egg dipeptidyl peptidase IV inhibitory oligopeptide: and (2) classifying the tuna roe proteolytic solution by an ultrafiltration membrane with the molecular weight cutoff of 3.0 and 1.0kDa, collecting classified components, determining the inhibition effect of each component on dipeptidyl peptidase IV, and purifying the component with the best activity by gel column chromatography and reversed phase high performance liquid chromatography (RP-HPLC) in sequence to obtain the dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide.
3. The method for preparing dipeptidyl peptidase IV inhibitory oligopeptide of tuna eggs according to claim 2, wherein the tuna in the step 1) is bonito Katsuwonus pelamis.
4. The method for preparing the dipeptidyl peptidase IV inhibitory oligopeptide of tuna roe according to claim 2, wherein the weight-to-volume ratio of the mashed roe and acetone in the step 1) is 1g: 8-10 mL.
5. The method for preparing the dipeptidyl peptidase IV inhibitory oligopeptide of tuna eggs according to claim 2, wherein the weight-to-volume ratio of the defatted fat fish to the buffer in the step 2) is 1 g/10-12 mL.
6. The method for preparing the dipeptidyl peptidase IV inhibitory oligopeptide of tuna eggs according to claim 2, wherein the buffer in the step 2) is a phosphate buffer.
7. The method for preparing the dipeptidyl peptidase IV inhibitory oligopeptide of tuna eggs according to claim 2, wherein the protease A in the step 2) is pepsin, and the enzyme activity is not less than 1.5X 104U/g。
8. The method for preparing the dipeptidyl peptidase IV inhibitory oligopeptide of tuna eggs according to claim 2, wherein the protease B in the step 2) is trypsin with the enzyme activity of not less than 2.0 x 104U/g。
9. The method for preparing the dipeptidyl peptidase IV inhibitory oligopeptide of tuna eggs according to claim 2, wherein the gel column chromatography step in the step 3) is:
dissolving the ultrafiltration zymolyte with the best activity in double distilled water to prepare a solution with the concentration of 35-40 mg/mL, carrying out Sephadex G-15 column chromatography separation, eluting with double distilled water at the flow rate of 0.6mL/min, collecting chromatographic peaks according to a chromatogram under 215nm, and determining the dipeptidyl peptidase IV inhibition effect of each chromatographic peak; selecting a sample with the highest chromatographic peak activity to prepare a solution with the concentration of 15-20 mg/mL, and performingAnd (3) separating Peptide10/300GL, eluting with double distilled water at the flow rate of 0.6mL/min, collecting chromatographic peaks according to a chromatogram under 215nm, and determining the dipeptidyl peptidase IV inhibition effect of each chromatographic peak to obtain the gel chromatography zymolyte.
The RP-HPLC purification step in the step 3) is as follows: preparing the gel chromatography zymolyte into a solution with the concentration of 35-40 mu g/mL by using double distilled water, purifying by using RP-HPLC, and obtaining 1 oligopeptide Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu with high dipeptidyl peptidase IV inhibition effect according to the activity of the prepared oligopeptide, wherein the molecular weight is 939.07Da by ESI-MS (electronic signature analysis).
Further preferably, the RP-HPLC conditions are: the sample volume is 10 mu L; chromatography column Kromasil C-18(250 mm. times.4.6 mm, 5 μm); mobile phase: 50% acetonitrile; the elution speed is 0.6 mL/min; the ultraviolet detection wavelength is 215 nm.
10. Use of the dipeptidyl peptidase IV inhibitory oligopeptide of tuna roe according to claim 1 for the preparation of special medical foods, health products and medicaments for the treatment or adjuvant treatment of type II diabetes.
Technical Field
The invention relates to the technical field of polypeptides, and particularly relates to a dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide which is derived from tuna roes and has the purpose of treating type II diabetes.
Background
Diabetes is a metabolic disease characterized by hyperglycemia. Hyperglycemia is caused by a defect in insulin secretion or an impaired biological action, or both. Type II diabetes (diabetes mellitus type 2, T2DM), known as non-insulin dependent diabetes mellitus or adult-onset diabetes, is a chronic metabolic disease that frequently occurs after the age of 35-40 years. The latest published data of the national health council show that the diabetes in China reaches 1.14 hundred million people, and more than 90 percent of the diabetes II accounts for patients.
Glucagon-like peptide 1(GLP-1) is a ghrelin consisting of 30 amino acids secreted by intestinal tract L cells, and has biological effects of promoting insulin secretion, inhibiting glucagon secretion, stimulating islet beta cell proliferation, and the like. GLP-1 has effects of regulating blood sugar and energy metabolism, protecting cardiac muscle cells, improving cardiac function, dilating blood vessel, and directly or indirectly protecting cardiovascular system. However, GLP-1 is very easily degraded by dipeptidyl peptidase IV (DPP-IV) in vivo, and the plasma half-life is very short (1-2 min). Thus, there are two main directions for GLP-1 based type II diabetes drug development: GLP-1 receptor agonists and dipeptidyl peptidase IV (DPP-IV) inhibitors that are resistant to DPP-IV degradation.
Tuna is an important fish species for ocean fishery in the world, and accounts for over 70 percent of the total yield of the fishery in open sea. Byproducts which account for about 50 to 70 percent of the total weight are generated in the processing process of the tuna and mainly comprise viscera, minced meat, fish heads, fish skins, fish bones and the like of the tuna, and the byproducts are used as feed raw materials or primary feed in a large amount, thereby causing great waste of resources of the tuna and bringing great pressure to the ecological environment.
Based on the above, the applicant selects tuna processing by-product-roe as a raw material, designs enzymolysis and chromatographic preparation processes to prepare dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide which is obvious in activity, safe and nontoxic, and can be applied to special medical food, health products and medicines for treating or assisting in treating type II diabetes.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a tuna roe dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide which can be applied to the preparation of special medical food, health-care products and medicines for treating or assisting in treating type II diabetes.
A dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide of tuna roe is a nonapeptide compound, the amino acid sequence of the oligopeptide is Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu (EIPGTRGPL), and the molecular weight of ESI-MS is 939.07 Da.
A preparation method of dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide of tuna roes is characterized by comprising the following steps:
1) pretreatment of fish eggs: unfreezing tuna roes, removing impurities, mashing the tuna roes by using a tissue mashing machine, adding an acetone solution, carrying out ultrasonic treatment at 200W for 15-20min for degreasing, repeating the steps for three times, centrifuging the tuna roes at 4 ℃ and 9000rmp for 15-20min, and drying solid precipitates to obtain degreased roes;
2) enzymolysis of fish eggs: adding the defatted roe into a buffer solution with the pH value of 1.5-2.5, uniformly stirring, adjusting the temperature of the solution to 37 ℃, adding protease A accounting for 2.0-2.5% of the weight of the defatted roe, and performing enzymolysis for 3-4 hours; then adjusting the pH value of the solution to 6.5-7.5, adding protease B accounting for 2.0-2.5% of the weight of the defatted fish eggs, and carrying out enzymolysis for 4-5 h; after enzymolysis, cooling the solution to room temperature, centrifuging at 12000rmp for 10-15 min, and collecting supernatant to obtain tuna roe protein enzymolysis solution;
3) preparation of fish egg dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide: the above protease hydrolysate has a cut-off molecular weight of 3.0 andfractionating with 1.0kDa ultrafiltration membrane, collecting fraction, and determining the inhibitory effect of each fraction on dipeptidyl peptidase IV (DPP-IV) (half inhibitory concentration IC)50And (3) selecting the component with the best activity, and purifying the component with the best activity by gel column chromatography and reversed phase high performance liquid chromatography (RP-HPLC) in sequence to obtain the dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide.
Preferably, the tuna in step 1) is bonito (Katsuwonus pelamis).
Preferably, the weight volume ratio of the mashed fish eggs to the acetone in the step 1) is 1g: 8-10 mL.
Preferably, the weight-volume ratio of the defatted fat fish to the buffer solution in the step 2) is 1g: 10-12 mL.
Preferably, the buffer in step 2) is a phosphate buffer.
Preferably, the protease A in the step 2) is pepsin, and the enzyme activity is more than or equal to 1.5 multiplied by 104U/g；
Preferably, the protease B in the step 2) is trypsin, and the enzyme activity is more than or equal to 2.0 multiplied by 104U/g；
Preferably, the gel column chromatography step in step 3) is:
dissolving the ultrafiltration zymolyte with the best activity in double distilled water to prepare a solution with the concentration of 35-40 mg/mL, carrying out Sephadex G-15 column chromatography separation, eluting with double distilled water at the flow rate of 0.6mL/min, collecting chromatographic peaks according to a chromatogram under 215nm, and determining the dipeptidyl peptidase IV (DPP-IV) inhibition effect of each chromatographic peak; selecting a sample with the highest chromatographic peak activity to prepare a solution with the concentration of 15-20 mg/mL, and performingSeparating Peptide10/300GL, eluting with double distilled water at flow rate of 0.6mL/min, collecting chromatographic peak according to chromatogram under 215nm, and determining dipeptidyl peptidase IV (DPP-IV) inhibition of each chromatographic peak to obtain gel chromatography zymolyte.
Preferably, the RP-HPLC purification step in the step 3) is as follows: preparing the gel chromatography zymolyte into a solution with the concentration of 35-40 mu g/mL by using double distilled water, purifying by using RP-HPLC, and obtaining 1 oligopeptide Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu (EIPGTRGPL) with high dipeptidyl peptidase IV (DPP-IV) inhibition effect according to the activity of the prepared oligopeptide, wherein the molecular weight is 939.07Da by ESI-MS (ESI-MS).
Further preferably, the RP-HPLC conditions are: the sample volume is 10 mu L; chromatography column Kromasil C-18(250 mm. times.4.6 mm, 5 μm); mobile phase: 50% acetonitrile; the elution speed is 0.6 mL/min; the ultraviolet detection wavelength is 215 nm.
The tuna roe oligopeptide Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu (EIPGTRGPL) provided by the invention can obviously inhibit DPP-IV activity, obviously reduce the postprandial blood sugar level of diabetic mice, improve the oral glucose tolerance and maltose tolerance of the mice and reduce the contents of triglyceride and cholesterol. The EIPGTRGPL has the advantages of safety, no toxic or side effect, remarkable hypoglycemic activity and the like, and can be applied to preparing special medical food, health-care products and medicines for treating or assisting in treating type II diabetes.
Drawings
FIG. 1 is a Sephadex G-15 column chromatography chromatogram of an ultrafiltration fraction TRH-I in an example of the present invention.
FIG. 2 shows Sephadex LH-20 column chromatography fractions TRH-I-2 in an example of the present inventionPeptide10/300GL separation chromatogram.
FIG. 3 shows an embodiment of the present inventionRP-HPLC chromatogram of Peptide10/300GL preparation fraction TRH-I-2C.
FIG. 4 Structure of Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu (EIPGTRGPL).
FIG. 5 Mass Spectrum of Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu (EIPGTRGPL).
FIG. 6 is the effect of Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu (EIPGTRGPL) in the examples on the area under the postprandial blood glucose curve of diabetic mice.
FIG. 7 is a graph of the effect of Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu (EIPGTRGPL) on the area under the oral glucose tolerance curve in diabetic mice in the examples.
FIG. 8 is a graph showing the effect of Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu (EIPGTRGPL) on the area under the oral maltose tolerance curve in diabetic mice.
Detailed Description
The following examples are intended to further illustrate the present invention, but they are not intended to limit or restrict the scope of the invention.
The tuna used in the experiments of the present invention was bonito (Katsuwonus pelamis) supplied by Ningbo today food Co., Ltd.
Preparation of phosphate buffer (ph 2.0):
liquid A: 16.6ml of phosphoric acid is taken, water is added to 1000ml, and the mixture is shaken up. B, liquid B: 71.63g of disodium hydrogen phosphate was taken and dissolved in 1000ml of water. Mixing the first solution 72.5ml with the second solution 27.5ml, and shaking.
Examples
1) Pretreatment of tuna roes: thawing skipjack roe, removing impurities, weighing 1000g, mashing with a tissue mashing machine, adding acetone solution according to the ratio of material to liquid of 1g:8mL, defatting with 200W ultrasound for 20min, repeating for three times, centrifuging at 4 deg.C and 9000rmp for 15min, and drying solid precipitate to obtain defatted roe;
2) enzymolysis of fish eggs: adding the defatted roe into pH2.0 buffer solution at a ratio of 1g to 12mL, stirring, adjusting temperature to 37 deg.C, adding pepsin (1.5 × 10) 2.5 wt% of defatted roe4U/g), and enzymolysis is carried out for 3.5 h; then, the pH of the solution was adjusted to 7.0, and trypsin (2.0X 10% by weight of defatted roe) was added thereto in an amount of 2.0% by weight4U/g), and carrying out enzymolysis for 4 h; after enzymolysis, cooling the solution to room temperature, centrifuging for 10-15 min at 12000rmp, and collecting supernatant, namely the roe protein enzymolysis solution;
3) preparation of fish egg dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide: classifying the fish roe protease hydrolysate with ultrafiltration membrane with cut-off molecular weight of 3.0 and 1.0kDa, and collecting fraction TRH-I (MW)<1.0kDa)、TRH-II(1.0<MW<3kDa) and TRH-III (MW)>3kDa) of the fractions, and determining the inhibitory effect of the fractions on dipeptidyl peptidase IV (DPP-IV) (half inhibitory concentration IC)50Expressed), selecting a component TRH-I with the best activity, sequentially carrying out gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) purification to obtain high-activity dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide TRP-6, and determining the TRP-6 structure by using an amino acid sequence analyzer and a mass spectrum, wherein the specific process comprises the following steps:
gel chromatography: dissolving the TRH-I in double distilled water to prepare a solution with the concentration of 35mg/mL, removing insoluble substances through a 0.45-micron microporous filter membrane, carrying out chromatographic separation by using a Sephadex G-15 column (2.0 multiplied by 100cm), eluting by using double distilled water at the flow rate of 0.6mL/min, preparing a gel chromatographic chromatogram according to the absorbance at 215nm, collecting various chromatographic peaks TRH-I-1-TRH-I-3 (shown in a figure 1), and determining the inhibition effect of each chromatographic peak on dipeptidyl peptidase IV (DPP-IV) (shown in a table 1); dissolving TRH-I-2 in double distilled water to obtain solution with concentration of 20mg/mL, removing insoluble substances with 0.45 μm microporous membraneSeparating Peptide10/300GL, eluting with double distilled water at flow rate of 0.6mL/min, collecting chromatographic peaks TRH-I-2A-TRH-I-2D (see figure 2) according to chromatogram under 215nm, and determining dipeptidyl peptidase IV (DPP-IV) Inhibition (IC) of each chromatographic peak50) Obtaining gel chromatography zymolyte TRH-I-2C.
TABLE 1
Components
IC50(mg/mL)
Components
IC50(mg/mL)
TRH
2.372
TRH-I-3
2.698
TRH-I
0.3876
TRH-I-2A
3.216
TRH-II
2.109
TRH-I-2B
0.978
TRH-III
6.746
TRH-I-2C
0.176
TRH-I-1
1.347
TRH-I-2D
1.397
TRH-I-2
0.269
② RP-HPLC refining: preparing the TRH-I-2C into a solution with the concentration of 35 mu g/mL by using double distilled water, removing insoluble substances through a 0.45 mu m microporous membrane, purifying by using RP-HPLC (the sample injection amount is 10 mu L; a chromatographic column Kromasil C-18(250mm multiplied by 4.6mm, 5 mu m), a mobile phase of 50% acetonitrile and ultraviolet detection wavelength is 215nm, collecting oligopeptides TRP-1-TRP-9 (shown in a figure 3) according to an absorbance curve under 215nm, and determining the inhibition effect of 9 components of oligopeptides on dipeptidyl peptidase IV (DPP-IV) (shown in a table 2) to obtain the high-activity dipeptidyl peptidase IV (DPP-IV) inhibition oligopeptide TRP-6.
TABLE 2
Components
IC50(mg/mL)
Components
IC50(mg/mL)
TRP-1
1.837
TRP-5
1.087
TRP-2
0.698
TRP-6
0.093
TRP-3
3.574
TRP-7
0.905
TRP-4
0.538
TRP-8
2.367
TRP-9
1.542
Structure detection: collecting dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide TRP-6 with highest activity, determining the amino acid sequence of the dipeptidyl peptidase IV to be Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu (EIPGTRGPL) (shown in figure 4) by using a protein/polypeptide sequence analyzer, and determining the molecular weight of the dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide TRP-6 by using ESI-MS (shown in figure 5).
Fourthly, evaluating the function: the effect of inhibiting oligopeptide EIPGTRGPL by using fish egg dipeptidyl peptidase IV (DPP-IV) to treat diabetes is evaluated by adopting a mouse in vivo experiment, and the experimental method refers to a literature [ shown in the specification, anti-diabetic activity of flavone components in Hangzhou white chrysanthemum and mechanism research thereof [ D ]. Tianjin science and technology university, 2019, P18-19 ]. The acarbose is adopted as a positive control, and the experimental result shows that: Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu (EIPGTRGPL) can remarkably reduce postprandial blood glucose level (see figure 6), improve oral glucose tolerance (see figure 7) and maltose tolerance (see figure 8) of mice, and reduce the content of Triglyceride (TG) and cholesterol (TC) (see Table 3).
TABLE 3
- -indicates no drug was given, blank: mice were normally bred, model group: mice were fed a high fat diet but without any drug.
In conclusion, Glu-Ile-Pro-Gly-Thr-Arg-Gly-Pro-Leu (EIPGTRGPL) can obviously inhibit the activity of dipeptidyl peptidase IV (DPP-IV) and reduce the blood sugar level of mice with type II diabetes, has no obvious toxic or side effect, and can be applied to special medical application food, health care products and medicines for treating or assisting in treating the type II diabetes.
Finally, it should be noted that the above-mentioned list is only one specific embodiment of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.