An oligopeptide from the swim bladder of miichthys miiuy and its preparation method and use

Abstract

The present application belongs to the field of bioengineering technology, and particularly relates to a Miichthys miiuy swim-bladder oligopeptide and a preparation method and application thereof. The present application takes Miichthys miiuy swim-bladder as raw material, and obtains dipeptidyl peptidase IV (DPP-IV) inhibiting oligopeptide Glu-Gly-Pro-Gly-Trp-Ser (EGPGWS) through ultrasonic degreasing, complex enzymolysis, ultrafiltration membrane fractionation and chromatography preparation. Mass spectrometry determines that the molecular weight of the oligopeptide is 631.6 Da. The EGPGWS of the present application can significantly inhibit dipeptidyl peptidase IV (DPP-IV) activity, reduce postprandial blood glucose level of model mice, improve oral glucose tolerance and maltose tolerance of mice, reduce triglyceride (TG) and cholesterol (TC) levels of model mice, and is safe and has no toxic side effects. The EGPGWS of the present application can be applied to preparation of special medical food, health products and drugs for treating or adjuvant treating diabetes.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to an oligopeptide from the swim bladder of a croaker, its preparation method, and its uses. Background Technology

[0002] Diabetes mellitus is a systemic disease caused by an absolute or relative deficiency of insulin secretion, leading to disordered glucose metabolism. Clinically, the typical symptoms of diabetes are the so-called "three highs and one low": polyuria, polydipsia, polyphagia, and weight loss, often accompanied by weakness and fatigue. Many patients also experience itchy skin. Long-term high blood sugar can lead to a series of complications, including chronic damage and dysfunction of various tissues, particularly the eyes, kidneys, heart, blood vessels, and nerves.

[0003] Glucagon-like peptide-1 (GLP-1) is a gut-brain peptide secreted by ileal endocrine cells. It lowers blood sugar by stimulating insulin, inhibiting glucagon, suppressing gastric emptying, and promoting pancreatic islet cell regeneration. However, the body's own GLP-1 is readily degraded by dipeptidyl peptidase IV (DPP-IV), with a plasma half-life of less than 2 minutes. Therefore, finding DPP-IV inhibitors to reduce the rate of GLP-1 degradation and maintain adequate GLP-1 levels can inhibit glucagon secretion and thus lower blood sugar. This approach has become one of the main directions in the development of drugs for treating diabetes.

[0004] Based on this, the applicant used fish scales, a byproduct of croaker processing, as raw material and employed enzymatic hydrolysis and chromatographic preparation techniques to prepare oligopeptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity. These oligopeptides can be used to prepare special medical foods, health products, and drugs for the treatment of diabetes. Summary of the Invention

[0005] This invention provides an oligopeptide from the swim bladder of croaker fish that has a hypoglycemic effect. This oligopeptide has significant dipeptidyl peptidase IV (DPP-IV) inhibitory activity and can be used to prepare special medical foods, health products and drugs for the treatment or adjuvant treatment of diabetes.

[0006] An oligopeptide from the swim bladder of a croaker fish, which is a hexapeptide compound with the amino acid sequence Glu-Gly-Pro-Gly-Trp-Ser (EGPGWS) and a molecular weight of 631.6 Da.

[0007] A method for preparing oligopeptides from croaker swim bladder, comprising the following steps:

[0008] 1) Pretreatment of croaker swim bladder: Thaw the croaker swim bladder, remove impurities, crush it with a tissue homogenizer, add isopropanol, sonicate at 40KHZ and 200 W for 15-20 min to defatt it, repeat three times, centrifuge at 9000g at room temperature for 15-20 min, dry the solid precipitate, grind it to obtain defatted swim bladder powder;

[0009] 2) Enzymatic hydrolysis of defatted swim bladder powder of croaker: Add the above defatted swim bladder powder to pH 2.0 phosphate buffer, stir well, adjust the solution temperature to 35-40℃, adjust the pH value to 1.0-2.0, add pepsin, and hydrolyze for 4-6 h. Inactivate the enzyme at 95℃ for 15 min. Adjust the solution temperature to 45-50℃, adjust the pH value to 6.5-8.0, add neutral protease, and hydrolyze for 4-6 h. Inactivate the enzyme at 95℃ for 15 min. Centrifuge at 8000 rpm for 15-20 min and collect the supernatant, which is the croaker swim bladder protease hydrolysate.

[0010] 3) Preparation of oligopeptides from croaker swim bladder: The above-mentioned croaker swim bladder protein hydrolysate was fractionated using ultrafiltration membranes with molecular weight cutoffs of 1 kDa, 5.0 kDa, and 10.0 kDa. The fractionated components were collected, and the inhibitory effect of each component on dipeptidyl peptidase IV (DPP-IV) was determined (using the half-maximal inhibitory concentration, IC50). 50 (Indicated), the component with the best activity, namely the ultrafiltration hydrolysate of croaker swim bladder, was selected and purified sequentially by macroporous resin column chromatography, gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) to obtain croaker swim bladder dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide.

[0011] In some embodiments of the present invention, the weight-to-volume ratio of croaker swim bladder to isopropanol in step 1) is 1 g: 6-8 mL.

[0012] In some embodiments of the present invention, the weight-to-volume ratio of defatted fish maw powder to pH 2.0 phosphate buffer in step 2) is 1 g: 8-10 mL.

[0013] In some embodiments of the present invention, the amount of pepsin added in step 2) is 1.5 to 2.0% of the weight of defatted fish bladder powder.

[0014] In some embodiments of the present invention, the amount of neutral white enzyme added in step 2) is 1.5 to 2.0% of the weight of defatted fish bladder powder.

[0015] In some embodiments of the present invention, the specific processes of macroporous resin column chromatography, gel column chromatography, and RP-HPLC purification in step 3) are as follows:

[0016] Macroporous resin column chromatography: The above-mentioned croaker swim bladder ultrafiltration enzymatic hydrolysate was dissolved in double-distilled water to prepare a solution with a concentration of 25-30 mg / mL. The solution was slowly added to a pretreated D101 macroporous resin column and eluted with 3-5 column volumes of double-distilled water, 25% ethanol, 75% ethanol, and 95% ethanol, respectively, at a flow rate of 1.5-2.5 mL / min. The eluted fractions were collected, and their inhibitory activity against DPP-Ⅳ was determined. The fraction with the strongest inhibitory activity was selected, lyophilized, and the croaker swim bladder macroporous resin enzymatic hydrolysate was obtained.

[0017] Gel column chromatography: The enzymatic hydrolysate of croaker swim bladder macroporous resin was dissolved in double-distilled water to prepare a solution with a concentration of 25-30 mg / mL. The solution was separated by Sephadex G-25 column chromatography, eluted with double-distilled water at a flow rate of 0.5-0.8 mL / min. Each chromatographic peak was collected based on the chromatogram at 214 nm, and the DPP-IV inhibition of each chromatographic peak was determined. The sample with the highest activity peak was the gel chromatography enzymatic hydrolysate.

[0018] RP-HPLC purification: The above gel chromatography enzymatic hydrolysate was prepared into a solution of 25-30 μg / mL with double-distilled water and purified by RP-HPLC. Based on the activity of the prepared oligopeptide, an oligopeptide with high DPP-IV inhibitory activity, Glu-Gly-Pro-Gly-Trp-Ser (EGPGWS), was obtained. The molecular weight was determined to be 631.6 Da by ESI-MS.

[0019] Preferably, the RP-HPLC conditions are as follows: injection volume 12-15 μL; chromatographic column Hypeil ODS C18 (250 mm × 4.6 mm, 5 μm); mobile phase: 60% acetonitrile; elution rate 0.8-1.0 mL / min; UV detection wavelength 214 nm.

[0020] On the other hand, the present invention provides the application of the above-mentioned croaker swim bladder oligopeptide EGPGWS in the preparation of special medical foods, health products and drugs for the treatment or adjuvant treatment of diabetes.

[0021] Compared with existing technologies, the croaker swim bladder oligopeptide Glu-Gly-Pro-Gly-Trp-Ser (EGPGWS) provided by this invention can significantly inhibit DPP-IV activity, significantly reduce postprandial blood glucose levels in diabetic mice, improve oral maltose and glucose tolerance in mice, and reduce total cholesterol (TC) and triglyceride (TG) levels. EGPGWS has the advantages of being safe, non-toxic, and having significant hypoglycemic effects, and can be used to prepare special medical foods, health products, and drugs for the treatment or adjuvant treatment of diabetes. Attached Figure Description

[0022] Figure 1The present invention relates to the inhibitory activity (IC50) of the swim bladder hydrolysate (MSH) of croaker and its ultrafiltration components (MSH1-MSH4) on dipeptidyl peptidase IV (DPP-IV). 50 (mg / mL).

[0023] Figure 2 The inhibitory activity (IC50) of the fractions (MRF1-MRF4) obtained by separating the ultrafiltration fraction MSH1 of the swim bladder hydrolysate of croaker fish using D101 macroporous resin against dipeptidyl peptidase IV (DPP-IV) is shown in the embodiment of the present invention. 50 (mg / mL).

[0024] Figure 3 This is a Sephadex G-25 column chromatography chromatogram of macroporous resin separation component MRF3 in an embodiment of the present invention.

[0025] Figure 4 The inhibitory activity (IC50) of the macroporous resin-separated fraction MRF3 obtained by Sephadex G-25 column chromatography on dipeptidyl peptidase IV (DPP-IV) in this embodiment of the invention is shown in the figure. 50 (mg / mL).

[0026] Figure 5 This is an RP-HPLC chromatogram of GF3 separated by Sephadex G-25 column chromatography in an embodiment of the present invention.

[0027] Figure 6 The inhibitory activity (IC50) of the fractions (MSP1-MSP8) obtained by RP-HPLC purification of GF3 separated by Sephadex G-25 column chromatography in this embodiment of the invention against dipeptidyl peptidase IV (DPP-IV) is shown in the figure. 50 (mg / mL).

[0028] Figure 7 This is the mass spectrum of the oligopeptide Glu-Gly-Pro-Gly-Trp-Ser (EGPGWS) from the swim bladder of the croaker, as described in this embodiment of the invention.

[0029] Figure 8 This is the structure of the croaker swim bladder oligopeptide Glu-Gly-Pro-Gly-Trp-Ser (EGPGWS) in the embodiments of the present invention.

[0030] Figure 9 This invention relates to the effect of the swim bladder oligopeptide Glu-Gly-Pro-Gly-Trp-Ser (EGPGWS) on the area under the postprandial blood glucose curve in diabetic mice.

[0031] Figure 10This invention relates to the effect of the oligopeptide Glu-Gly-Pro-Gly-Trp-Ser (EGPGWS) from croaker swim bladder on the area under the oral glucose tolerance curve in diabetic mice.

[0032] Figure 11 This invention relates to the effect of the oligopeptide Glu-Gly-Pro-Gly-Trp-Ser (EGPGWS) from croaker swim bladder on the area under the curve of oral maltose tolerance in diabetic mice. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0034] There are no particular restrictions on the solvents used in this invention; commercially available conventional solvents can be used.

[0035] 1. Preparation of a diabetic mouse model

[0036] After mice acclimatized to their environment for one week, they were randomly divided into two groups: a high-fat diet group and a control group. The high-fat diet group was fed a high-fat diet, while the control group received a normal diet. Water was freely available. Mice were weighed weekly, and their food and water intake were recorded. After four weeks of high-fat diet feeding, mice were fasted for six hours but allowed free access to water. Their weight was measured, and blood glucose levels were measured using tail blood samples. Mice in the high-fat diet group were then intraperitoneally injected with streptozocin (STZ) at a dose of 100 mg / kg, followed by continued high-fat diet feeding. One week later, mice were weighed again, and blood glucose levels were measured. A fasting blood glucose level greater than 11.0 mM was considered a successful model establishment.

[0037] 2. Animal experiments:

[0038] The blank group refers to mice fed with ordinary feed.

[0039] The model group refers to mice fed a high-fat diet according to the above modeling method, with a fasting blood glucose level of approximately 11.0 mM.

[0040] Positive control group: Mice that successfully developed the model were given acarbose (50 mg / kg) by gavage for 28 consecutive days.

[0041] EGPGWS group: Mice that successfully developed the model were administered oligopeptide EGPGWS (50 mg / kg) by gavage for 28 consecutive days.

[0042] The preparation process of an oligopeptide from croaker swim bladder with hypoglycemic effect is as follows: croaker swim bladder "ultrasonic degreasing" + compound enzymatic hydrolysis + ultrafiltration membrane fractionation + chromatographic preparation + DPP-IV inhibitory oligopeptide + hypoglycemic function evaluation.

[0043] The specific steps are as follows:

[0044] 1) Pretreatment of Miichthys miiuy swim bladder: The Miichthys miiuy swim bladder was thawed, impurities were removed, and it was mashed with a tissue homogenizer. An isopropanol solution was added at a material-liquid ratio of 1 g:8 mL, and ultrasonic degreasing was carried out at 40 KHZ and 200 W for 20 min. This was repeated three times, and then centrifuged at 9000 g for 15 min at room temperature. The solid precipitate was dried and ground to obtain defatted swim bladder powder;

[0045] 2) Enzymolysis of defatted Miichthys miiuy swim bladder powder: The above-mentioned defatted swim bladder powder was added to a buffer solution at a material-liquid ratio of 1 g:9 mL, stirred evenly, the temperature of the solution was adjusted to 37 °C, the pH value was adjusted to 1.2, 1.8% pepsin based on the weight of the swim bladder powder was added, and enzymolysis was carried out for 4 h. The enzyme activity was inactivated at 95 °C for 15 min; the temperature of the solution was adjusted to 47 °C, the pH value was adjusted to 7.3, 1.8% neutral protease based on the weight of the swim bladder powder was added, and enzymolysis was carried out for 5 h. The enzyme activity was inactivated at 95 °C for 15 min, and then centrifuged at 8000 rmp for 20 min. The supernatant was collected, which was the Miichthys miiuy swim bladder protease hydrolysate (MSH);

[0046] 3) Preparation of Miichthys miiuy swim bladder oligopeptides: The above-mentioned Miichthys miiuy swim bladder protease hydrolysate MSH was fractionated by ultrafiltration membranes with a molecular weight cut-off of 1 kDa, 5 kDa, and 10 kDa. The fractionated components MSH1 (MW < 1 kDa), MSH2 (1 kDa < MW < 5 kDa), MSH3 (5 kDa < MW < 10 kDa), and MSH4 (MW > 10 kDa) were collected. Referring to the DPP-IV inhibitory activity determination method in the literature [Liu Zhitong, Zheng Lin, Wang Chenyang, Zhao Mouming. Enzymatic preparation and structure identification of sea cucumber dipeptidyl peptidase IV inhibitory peptides [J]. Modern Food Science and Technology, 2020, 36(8): 166 - 174], the inhibitory effects of each component on dipeptidyl peptidase IV (DPP-IV) were measured (expressed by the half inhibitory concentration IC 50 ), and the component MSH1 with the best activity was selected and purified successively by macroporous resin column chromatography, gel column chromatography, and RP-HPLC to obtain the Miichthys miiuy swim bladder dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide. Its molecular weight was measured by mass spectrometry, and its amino acid sequence was analyzed by an amino acid sequence analyzer. The specific process is as follows: Figure 1 ) The component with the best activity was selected and purified successively by macroporous resin column chromatography, gel column chromatography, and RP-HPLC to obtain the Miichthys miiuy swim bladder dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide. Its molecular weight was measured by mass spectrometry, and its amino acid sequence was analyzed by an amino acid sequence analyzer. The specific process is as follows:

[0047] ① Macroporous resin column chromatography: The above-mentioned Miichthys miiuy swim bladder ultrafiltration enzymolysate MSH1 was dissolved in double-distilled water to prepare a solution with a concentration of 30 mg / mL, and slowly added to the pretreated D101 macroporous resin chromatography column (5.0 × 120 cm). Elution was carried out with 3 column volumes of double-distilled water, 25% ethanol, 75% ethanol, and 95% ethanol respectively, with a flow rate of 1.8 mL / min. Four elution components MRF1, MRF2, MRF3, and MRF4 were collected, and the inhibitory activities of the four components on DPP-IV were measured (see Figure 2The component with the strongest inhibitory activity, MRF3, was selected, freeze-dried, and the macroporous resin hydrolysate of croaker swim bladder was obtained.

[0048] ② Gel chromatography: The above MRF3 was dissolved in double-distilled water to prepare a solution with a concentration of 30 mg / mL. Separation was performed by gel chromatography on a Sephadex G-25 column (2.0 × 100 cm), eluted with double-distilled water at a flow rate of 0.6 mL / min. A gel chromatography chromatogram was prepared based on the absorbance at 214 nm, and the chromatographic peaks GF1–GF4 were collected (see…). Figure 3 The inhibitory effects of GF1-GF4 on DPP-IV were determined (see...). Figure 4 ), the DPP-IV inhibitory effect of GF3 (IC50) 50 The strongest is the gel chromatography enzymatic hydrolysate.

[0049] ③RP-HPLC purification: The above GF3 was dissolved in double-distilled water to prepare a solution with a concentration of 30 μg / mL, and purified by RP-HPLC (injection volume 12 μL; column: Hypeil ODS C18 (250 mm × 4.6 mm, 5 μm); mobile phase: 60% acetonitrile; UV detection wavelength: 214 nm; oligopeptides MSP1~MSP8 were collected according to the absorbance curve at 214 nm (see...). Figure 5 The inhibitory effects of eight oligopeptides on DPP-IV were determined (see...). Figure 6 ), and obtained the highly active DPP-Ⅳ inhibitory oligopeptide MSP6.

[0050] ④ Structural analysis: The most active MSP6 was collected, and its molecular weight was determined to be 631.6 Da using ESI-MS (see [link to ESI-MS]). Figure 7 The amino acid sequence determined by the protein / peptide sequencer is Glu-Gly-Pro-Gly-Trp-Ser (EGPGWS) (see...). Figure 8 ).

[0051] ⑤ Functional Evaluation: Following the experimental methods described in the literature [Chen Mingzhu. Study on the antidiabetic activity and mechanism of flavonoid components in Hangzhou white chrysanthemum [D]. Tianjin University of Science and Technology, 2019], the in vivo therapeutic function of EGPGWS oligopeptide from croaker swim bladder in mice was evaluated. The results showed that EGPGWS could significantly reduce postprandial blood glucose levels in model mice (see...). Figure 9 ), improve oral glucose tolerance in mice (see Figure 10 ) and maltose tolerance (see Figure 11 This reduces triglyceride (TG) and total cholesterol (TC) levels (see Table 1).

[0052] Table 1

[0053] Group Dosage (mg / kg) TG (mmol / mL) TC (mmol / mL) Blank group -- 1.67 5.86 Model group -- 3.49 10.06 Positive control group (acarbose) 100 2.36 8.29 EGPGWS 100 2.51 8.34

[0054] In summary, Glu-Gly-Pro-Gly-Trp-Ser (EGPGWS) significantly inhibits DPP-Ⅳ activity and reduces blood glucose levels in type II diabetic mice, and is safe and free of toxic side effects. It can be used to prepare special medical foods, health products and drugs for the treatment or adjuvant treatment of diabetes.

[0055] Finally, it should be noted that the above-described embodiments are merely one specific example of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A mioga oligopeptide, characterized in that The oligopeptide is a hexapeptide compound, the amino acid sequence of which is Glu-Gly-Pro-Gly-Trp-Ser, and the molecular weight of which is 631.6 Da. The oligopeptide is a hexapeptide compound, the amino acid sequence of which is Glu-Gly-Pro-Gly-Trp-Ser, and the molecular weight of which is 631.6 Da.

Images