CN117736268A - Anglerfish fish tripe blood sugar reducing oligopeptide, preparation method and application thereof - Google Patents
Anglerfish fish tripe blood sugar reducing oligopeptide, preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a blood sugar-reducing oligopeptide of a fish maw, a preparation method and application thereof.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a anglerfish fish maw blood sugar reducing oligopeptide, a preparation method and application thereof.
Background
Diabetes is a systemic disease of disturbed glucose metabolism due to absolute or relative insufficient insulin secretion in the body. Diabetes causes metabolic disturbance of carbohydrate, fat and protein, finally causes multisystem damage, and long-term hyperglycemia can cause chronic progressive damage of a plurality of tissues and organs such as diabetic eye disease, nephropathy, nerve damage, heart disease, vascular disease and the like, and organ failure can even occur seriously. In acute cases, severe metabolic disorders such as diabetic ketoacidosis and diabetic hypertonicity can be caused. Diabetes can cause a decrease in the quality of life, a decrease in life span, and an increase in the rate of death. Therefore, the development of high-efficiency hypoglycemic drugs is important for the treatment of diabetes and complications thereof.
Disclosure of Invention
On one hand, the invention provides a fish maw oligopeptide with a blood sugar reducing effect, which can realize the blood sugar reducing effect by inhibiting the activity of dipeptidyl peptidase IV (DPP-IV) and can be applied to preparing medicines and functional products for treating or assisting in treating diabetes.
An anglerfish fish tripe hypoglycemic oligopeptide is an octapeptide compound, the amino acid sequence of which is Pro-Tyr-Gly-Gln-Ser-Gly-Phe-Pro (PYGQSFP) and the molecular weight of which is 851.9Da.
On the other hand, the invention provides a preparation method of the anglerfish fish tripe oligopeptide, which comprises the following steps:
1) Pretreatment of anglerfish tripe: thawing anglerfish tripe, removing impurities, mashing by a masher, adding isopropanol into the mashed anglerfish tripe, performing ultrasonic degreasing, centrifuging, drying solid precipitate, grinding and sieving to obtain anglerfish defatted fish tripe powder;
2) Enzymolysis of the defatted fish maw powder of the anabrous grahami: na is added into anglerfish defatted fish maw powder 2 HPO 4 -NaH 2 PO 4 Buffer solution, stirring evenly, adjusting the pH value of the solution, adding trypsin for enzymolysis, and inactivating enzyme after enzymolysis; adding fish maw powder weight flavor protease, performing enzymolysis, inactivating enzyme, centrifuging, and collecting supernatantAnglerfish fish maw proteolytic liquid;
3) Preparation of anglerfish fish tripe oligopeptide: grading the anglerfish fish tripe proteolytic liquid through an ultrafiltration membrane with the molecular weight cutoff of 1.0kDa, 3.5kDa and 5.0kDa, collecting each grading component, measuring the inhibition activity of each component on dipeptidyl peptidase IV, and selecting the component with the best activity, namely the anglerfish tripe ultrafiltration zymolyte; the ultrafiltration zymolyte is purified by gel column chromatography and reversed phase high performance liquid chromatography in sequence to obtain the PYGQSFGFP.
Preferably, the weight-volume ratio of the mashed anglerfish maw to the isopropanol in the step 1) is 1 g:10-15 mL.
Preferably, na in the step 2) 2 HPO 4 -NaH 2 PO 4 The buffer solution concentration of (2) is 0.5mol/L, the pH is 7.2, and the anglerfish defatted fish maw powder and Na 2 HPO 4 -NaH 2 PO 4 The weight-volume ratio of the buffer solution is 1 g:10-12 mL.
Preferably, in the step 2), the pH value is 6.5-8.0, the enzymolysis temperature of the trypsin is 35-40 ℃, the addition amount of the trypsin is 1.0-2.0% of the weight of the anglerfish defatted fish maw powder, and the enzymolysis time of the trypsin is 2-3 h.
Preferably, the enzymolysis temperature of the flavor protease in the step 2) is 45-55 ℃, the addition amount of the flavor protease is 1.0-2.0% of the weight of the anglerfish defatted fish maw powder, and the enzymolysis time of the flavor protease is 2-3 h.
Preferably, the specific procedures of gel column chromatography and RP-HPLC purification in the step 3) are as follows:
gel column chromatography: dissolving anglerfish fish tripe ultrafiltration zymolyte in Na 2 HPO 4 -NaH 2 PO 4 Preparing buffer solution (with the concentration of 0.5mol/L and the pH of 7.2) into solution with the concentration of 60-80 mg/mL, separating by Sephadex G-15 column chromatography, eluting by double distilled water at the flow rate of 0.5-0.8 mL/min, collecting each chromatographic peak according to a chromatographic chart under 254nm, and determining DPP-IV inhibition activity of each chromatographic peak, wherein the sample with the highest activity is gel chromatography zymolyte;
RP-HPLC purification: the gel chromatography zymolyte is prepared into a solution with the concentration of 25-30 mug/mL by double distilled water, the solution is purified by RP-HPLC, 1 oligopeptide Pro-Tyr-Gly-Gln-Ser-Gly-Phe-Pro (PYGQSFGFP) with high DPP-IV inhibitory activity is obtained according to the activity of the prepared oligopeptide, and the molecular weight of the oligopeptide is 851.9Da by ESI-MS.
Further, the RP-HPLC conditions are: the sample injection amount is 12-15 mu L; column Kromasil C18 (250 mm×4.6mm,5 μm); mobile phase: 35% acetonitrile; the elution speed is 1.0-1.2 mL/min; the ultraviolet detection wavelength is 254nm.
In a further aspect, the invention provides the use of the above-mentioned anglerfish tripe hypoglycemic oligopeptide, which can significantly inhibit dipeptidyl peptidase IV (DPP-IV) activity, and IC for DPP-IV 50 0.342mg/mL; the anglerfish fish tripe oligopeptide disclosed by the invention can obviously reduce postprandial blood sugar level of a diabetic mouse, improve oral maltose tolerance and glucose tolerance of the mouse, and reduce the contents of Total Cholesterol (TC) and Triglyceride (TG); the anglerfish tripe oligopeptide has the advantages of safety, no toxic or side effect, remarkable blood sugar reducing effect and the like, and can be applied to preparing medicaments and functional products for treating or assisting in treating diabetes.
Drawings
FIG. 1 shows the inhibitory activity (IC) of anglerfish fish maw proteolytic enzyme hydrolysate (YDH) and ultrafiltration fractions (YDH 1-YDH 4) of the invention on dipeptidyl peptidase IV (DPP-IV) 50 ,mg/mL)。
FIG. 2 is a Sephadex G-15 column chromatography chromatogram of the anglerfish fish maw proteolytic enzyme hydrolysate ultrafiltration fraction YDH1 of the present invention.
FIG. 3 shows the inhibitory activity (IC) of the components (YDH 1 a-YDH 1 c) obtained by separating the anglerfish fish maw proteolysis solution ultrafiltration component YDH1 by Sephadex G-15 column chromatography on dipeptidyl peptidase IV (DPP-IV) 50 ,mg/mL)。
FIG. 4 is a RP-HPLC chromatogram of the Sephadex G-21 column chromatography separation component YDH1b of the present invention.
FIG. 5 shows the purification of the component YDH1b of Sephadex G-21 column chromatography by RP-HPLC to obtain the components (YDP 1-YDP 10) for dipeptidylpeptidase IV (DPP-IV) inhibitory Activity (IC) 50 ,mg/mL)。
FIG. 6 is a mass spectrum of the anglerfish fish maw oligopeptide Pro-Tyr-Gly-Gln-Ser-Gly-Phe-Pro (PYGQSFP) of the present invention.
FIG. 7 shows the structure of the anglerfish fish maw oligopeptide Pro-Tyr-Gly-Gln-Ser-Gly-Phe-Pro (PYGQSFP) according to the invention.
FIG. 8 is the effect of the anglerfish fish maw oligopeptide Pro-Tyr-Gly-Gln-Ser-Gly-Phe-Pro (PYGQSFP) of the invention on the area under the postprandial blood glucose profile of diabetic mice.
FIG. 9 is the effect of the anglerfish fish maw oligopeptide Pro-Tyr-Gly-Gln-Ser-Gly-Phe-Pro (PYGQSFP) of the invention on the area under the oral glucose tolerance curve of diabetic mice.
FIG. 10 is the effect of the anglerfish fish maw oligopeptide Pro-Tyr-Gly-Gln-Ser-Gly-Phe-Pro (PYGQSFP) of the invention on the area under the oral maltose tolerance curve of diabetic mice.
FIG. 11 is the effect of the anglerfish tripeptide PYGQSFGFP of the invention on the Triglyceride (TG) and Total Cholesterol (TC) levels of diabetic mice.
Detailed Description
The invention is described in further detail below with reference to the embodiments of the drawings.
The preparation process of the anglerfish fish tripe hypoglycemic oligopeptide comprises the following steps: anglerfish fish tripe, pretreatment for ultrasonic degreasing, composite enzymolysis, ultrafiltration membrane grading, chromatographic preparation, DPP-IV oligopeptide inhibition and blood sugar reducing function evaluation.
Example 1
1) Pretreatment of anglerfish tripe: thawing anglerfish fish maw, removing impurities, homogenizing by a tissue masher, adding isopropanol solution according to a feed-liquid ratio of 1g to 15mL, degreasing by ultrasonic waves of 40KHZ and 300W for 30min, repeating for three times, centrifuging at room temperature of 6000g for 25min, drying solid precipitate, grinding, and sieving by a 200-mesh sieve to obtain defatted fish maw powder;
2) Enzymolysis of the defatted fish maw powder of the anabrous grahami: adding the defatted fish maw powder into Na according to the feed-to-liquid ratio of 1g:12mL 2 HPO 4 -NaH 2 PO 4 Adding buffer solution (0.5 mol/L, pH 7.2), stirring, regulating temperature to 37deg.C, regulating pH to 7.1, and adding fishTrypsin accounting for 1.5% of the weight of the tripe powder is subjected to enzymolysis for 2.2 hours, and enzyme activity is inactivated at 90 ℃ for 20 min; regulating the temperature of the solution to 50deg.C, adding flavourzyme accounting for 1.8% of the weight of fish maw powder, performing enzymolysis for 2.8h, inactivating enzyme at 90deg.C for 20min, centrifuging at 6000rmp for 20min, and collecting supernatant, namely anglerfish maw proteolytic liquid (YDH).
3) Preparation of anglerfish fish tripe oligopeptide: grading the anglerfish fish maw proteolytic liquid (YDH) by ultrafiltration membrane with molecular weight cut-off of 1.0kDa, 3.5kDa and 5.0kDa, and collecting graded component YDH1 (MW<1kDa)、YDH 2(1kDa<MW<3.5kDa)、YDH 3(3.5kDa<MW<5.0 kDa) and YDH4 (MW>5.0 kDa), the inhibitory activity of 4 fractions on dipeptidyl peptidase IV (DPP-IV) was determined (with half inhibitory concentration IC 50 Represented by the figure 1), the component with the best activity, namely, the ultra-filtration zymolyte (YDH 1) of the anglerfish tripe is selected and purified by gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) in sequence, so as to obtain the anglerfish tripe dipeptidyl peptidase IV (DPP-IV) inhibitory oligopeptide. The molecular weight was determined by mass spectrometry and the amino acid sequence was determined by amino acid sequence analysis.
(1) Gel column chromatography: dissolving anglerfish fish tripe ultrafiltration zymolyte (YDH 1) in Na 2 HPO 4 -NaH 2 PO 4 Preparing buffer solution (with concentration of 0.5mol/L and pH of 7.2) into solution with concentration of 75mg/mL, separating by Sephadex G-15 column chromatography, eluting with double distilled water at flow rate of 0.75mL/min, collecting chromatographic peaks (YDH 1 a-YDH 1 c) according to a chromatogram under 254nm (see figure 2), and measuring DPP-IV inhibition activity of 3 chromatographic peaks (see figure 3); wherein YDH1b shows the strongest DPP-IV inhibitory activity, namely gel chromatography zymolyte (YDH 1 b).
(2) RP-HPLC purification: preparing 30 mug/mL solution of the gel chromatography zymolyte (YDH 1 b) by double distilled water, purifying by RP-HPLC (sample injection amount is 12-15 mug; chromatographic column Kromasil C18 (250 mm multiplied by 4.6mm,5 mu m), mobile phase is 35% acetonitrile, eluting speed is 1.0-1.2 mL/min, ultraviolet detection wavelength is 254 nm) to obtain a chromatogram under 254nm (see figure 4), and preparing 10 oligopeptide components (YDP 1-YDP 10); the DPP-IV inhibitory activity of each chromatographic peak was determined (see FIG. 5), wherein YDP7 was specific for DPP-IV IC 50 0.342mg/mL, significantly lower than other oligopeptides, YDP7 was selected for structural partitioningAnalysis and functional evaluation.
(3) And (3) structural detection: the highest activity YDP7 was collected and the amino acid sequence was determined to be Pro-Tyr-Gly-Gln-Ser-Gly-Phe-Pro (PYGQSFP) using an ESI-MS protein/polypeptide sequencer with a molecular weight of 851.9Da (see FIG. 6) (see FIG. 7).
(4) Functional evaluation: referring to the experimental method of document [ Chen Mingzhu ] anti-diabetic activity of flavone component in Hangzhou white chrysanthemum and mechanism research [ D ] Tianjin university of science and technology, 2019], in vivo experiment of mice is adopted to evaluate the in vivo diabetes treatment function of anglerfish fish maw oligopeptide PYGQSFP,
preparation of diabetic mouse model: after 1 week of acclimation, the mice were randomized into two groups: the high-fat feed group and the blank group are respectively fed with high-fat feed and common feed, and drinking water is free. After 4 weeks of feeding, mice were weighed with no water on food for 6 hours. Blood glucose levels were measured in mice of the high fat diet group and the blank group by taking blood from the tail tip, and then streptozotocin was injected intraperitoneally into the mice of the high fat diet group at a dose of 100mg/kg, followed by continuing to administer the notification diet. After 1 week, the mice were weighed again, blood glucose levels were measured, and fasting blood glucose levels greater than 11.0mM were considered successful in molding.
Grouping and administration of laboratory animals
Blank group: mice fed with normal feed.
Model group: mice with fasting blood glucose values greater than 11.0mM after 5 weeks were fed with high fat diet.
PYGQSGFP group: mice after 5 weeks of modeling were successful, following 4 weeks of mice dosed daily at a dose of 50 mg/kg.
The experimental results show that: the PYGQSFGFP of the invention can obviously reduce postprandial blood sugar level of a model mouse (see figure 8), improve oral glucose tolerance (see figure 9) and maltose tolerance (see figure 10) of the mouse, and reduce the content of Triglyceride (TG) and Total Cholesterol (TC) (see figure 11).
Finally, it should be noted that the above list is only one embodiment of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (9)
1. The anglerfish fish tripe hypoglycemic oligopeptide is characterized in that the oligopeptide is an octapeptide compound, the amino acid sequence of the octapeptide compound is Pro-Tyr-Gly-Gln-Ser-Gly-Phe-Pro (PYGQSFP), and the molecular weight of the octapeptide compound is 851.9Da.
2. The method for preparing the anglerfish fish maw blood sugar-reducing oligopeptide according to claim 1, which is characterized by comprising the following steps:
1) Pretreatment of anglerfish tripe: thawing anglerfish tripe, removing impurities, mashing by a masher, adding isopropanol into the mashed anglerfish tripe, performing ultrasonic degreasing, centrifuging, drying solid precipitate, grinding and sieving to obtain anglerfish defatted fish tripe powder;
2) Enzymolysis of the defatted fish maw powder of the anabrous grahami: na is added into anglerfish defatted fish maw powder 2 HPO 4 -NaH 2 PO 4 Buffer solution, stirring evenly, adjusting the pH value of the solution, adding trypsin for enzymolysis, and inactivating enzyme after enzymolysis; adding fish maw powder weight flavor protease, performing enzymolysis, inactivating enzyme after enzymolysis, centrifuging, and collecting supernatant to obtain anglerfish fish maw protein enzymolysis liquid;
3) Preparation of anglerfish fish tripe oligopeptide: grading the anglerfish fish tripe proteolytic liquid through an ultrafiltration membrane with the molecular weight cutoff of 1.0kDa, 3.5kDa and 5.0kDa, collecting each grading component, measuring the inhibition activity of each component on dipeptidyl peptidase IV, and selecting the component with the best activity, namely the anglerfish tripe ultrafiltration zymolyte; the ultrafiltration zymolyte is purified by gel column chromatography and reversed phase high performance liquid chromatography in sequence to obtain the PYGQSFGFP.
3. The method for preparing the anglerfish fish maw blood sugar-reducing oligopeptide according to claim 2, wherein the weight-volume ratio of the mashed anglerfish maw to the isopropanol in the step 1) is 1 g:10-15 mL.
4. Anglerfish fish maw drop as claimed in claim 2A process for producing a blood glucose oligopeptide characterized in that Na is used in step 2) 2 HPO 4 -NaH 2 PO 4 The buffer solution concentration of (2) is 0.5mol/L, the pH is 7.2, and the anglerfish defatted fish maw powder and Na 2 HPO 4 -NaH 2 PO 4 The weight-volume ratio of the buffer solution is 1 g:10-12 mL.
5. The method for preparing the anglerfish fish maw blood sugar-reducing oligopeptide according to claim 2, wherein the pH value in the step 2) is 6.5-8.0, the enzymolysis temperature of trypsin is 35-40 ℃, the addition amount of trypsin is 1.0-2.0% of the weight of the anglerfish defatted fish maw powder, and the enzymolysis time of trypsin is 2-3 h.
6. The method for preparing the anglerfish fish maw blood sugar-reducing oligopeptide according to claim 2, wherein the enzymolysis temperature of the flavourzyme in the step 2) is 45-55 ℃, the addition amount of the flavourzyme is 1.0-2.0% of the weight of the anglerfish defatted fish maw powder, and the enzymolysis time of the flavourzyme is 2-3 h.
7. The method for preparing the anglerfish fish maw hypoglycemic oligopeptide according to claim 2, wherein the specific procedures of gel column chromatography and RP-HPLC purification in the step 3) are as follows:
gel column chromatography: dissolving anglerfish fish tripe ultrafiltration zymolyte in Na 2 HPO 4 -NaH 2 PO 4 Preparing buffer solution (0.5 mol/L, pH 7.2) into solution with concentration of 60-80 mg/mL, separating by Sephadex G-15 column chromatography, eluting with double distilled water at flow rate of 0.5-0.8 mL/min, collecting each chromatographic peak according to a chromatogram under 254nm, and determining DPP-IV inhibitory activity of each chromatographic peak, wherein the sample with the highest activity is gel chromatography zymolyte;
RP-HPLC purification: the gel chromatography zymolyte is prepared into a solution with the concentration of 25-30 mug/mL by double distilled water, the solution is purified by RP-HPLC, 1 oligopeptide Pro-Tyr-Gly-Gln-Ser-Gly-Phe-Pro (PYGQSFGFP) with high DPP-IV inhibitory activity is obtained according to the activity of the prepared oligopeptide, and the molecular weight of the oligopeptide is 851.9Da by ESI-MS.
8. The method for preparing the anglerfish fish maw hypoglycemic oligopeptide according to claim 8, wherein the RP-HPLC conditions are as follows: the sample injection amount is 12-15 mu L; column Kromasil C18 (250 mm×4.6mm,5 μm); mobile phase: 35% acetonitrile; the elution speed is 1.0-1.2 mL/min; the ultraviolet detection wavelength is 254nm.
9. Use of an anglerfish fish maw hypoglycemic oligopeptide according to claim 1 in the manufacture of a medicament for the treatment or co-treatment of diabetes.
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