Bovine bone collagen active small peptide, composition and application

Abstract

The invention provides a bovine bone collagen active small peptide, a composition and application, and belongs to the technical field of bioactive peptides. The small peptide obtained in the invention still has high biological activity after gastrointestinal digestion, and does not have toxic effect on cells. Animal experiments prove that the obtained active small peptide can reduce fasting blood glucose of animals and control postprandial blood glucose, can be further used for preparing products for controlling glucose or treating diabetes, and has huge application value.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, and particularly relates to a bovine bone collagen active small peptide, its composition, and its application. Background Technology

[0002] The statements in this section are merely background information related to the present invention and do not necessarily constitute prior art.

[0003] In recent years, the number of obese people and diabetic patients has increased, and the general public commonly tries to control blood sugar and lose weight by controlling sugar intake or taking medication. Glucosidase inhibitors are a class of widely used hypoglycemic drugs in clinical practice, primarily working by inhibiting the activity of glucose in the small intestine. This method increases the activity of glycosidases, thereby slowing down the breakdown and absorption of carbohydrates and thus lowering postprandial blood glucose. It is particularly suitable for diabetic patients with a high-carbohydrate diet or elevated postprandial blood glucose, especially those with type 2 diabetes.

[0004] Currently, [the following] have been isolated and purified from multiple species. Substances that inhibit glucosidase activity, such as the three bioactive peptides with hypoglycemic effects isolated and purified from oysters disclosed in CN120829477A; and the inhibitory peptides obtained from defatted *Sapindus mukorossi* pulp disclosed in CN119978054A, are examples of substances that inhibit glucosidase activity. Small peptides with glucosidase activity; prepared from camel blood in CN 120795065 A Glucosidase inhibitors. And can glucosidase inhibitors be isolated and purified from the bones of cattle, which have significant production value? The substance that inhibits glucosidase activity is unknown. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a method having Small peptides of bovine bone collagen with glucosidase inhibitory activity.

[0006] To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions: In a first aspect, the present invention provides a bovine bone collagen active peptide with the amino acid sequence GPQGEAGPSI or GSTAAPCI.

[0007] In a specific embodiment of the present invention, the small peptide is obtained by solid-phase synthesis.

[0008] In a second aspect, the present invention provides the above-mentioned bovine bone collagen active peptides in the preparation of... Applications of glucosidase inhibitors.

[0009] A third aspect of the present invention provides the application of the above-mentioned bovine bone collagen active small peptides in the preparation of drugs or health products that help lower blood sugar in animals.

[0010] In a fourth aspect, the present invention provides the use of the above-mentioned bovine bone collagen active peptides in the preparation of drugs for the prevention or treatment of diabetes or obesity.

[0011] In a specific embodiment of the present invention, the diabetes is type 2 diabetes.

[0012] A fifth aspect of the present invention provides a composition comprising the above-described active small peptide.

[0013] In specific embodiments of the present invention, the composition includes other blood glucose-lowering components and / or pharmaceutically acceptable carriers.

[0014] In specific embodiments of the present invention, the other blood sugar-lowering components include metformin hydrochloride, liraglutide, acarbose, or sitagliptin.

[0015] The above one or more technical solutions have the following beneficial effects: In this invention, bovine bones are hydrolyzed using multiple enzymes, and then separated and purified using ultrafiltration, liquid chromatography, and other techniques to obtain a product with... Small peptides with glucosidase inhibitory activity were obtained. These peptides retained high biological activity after gastrointestinal digestion and showed no toxic effects on cells. Animal experiments demonstrated that the obtained active peptides could lower fasting blood glucose and control postprandial blood glucose, and can be further used to prepare products for blood sugar control or treatment of diabetes, demonstrating significant application value.

[0016] Advantages of additional aspects of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0018] Figure 1 The images show the mass spectra of the active small peptides of bovine bone collagen in this invention, where A is the LR-1 mass spectrum and B is the LR-2 mass spectrum.

[0019] Figure 2 The active small peptide pair in Example 2 of this invention Inhibitory activity of glucosidase, where A represents the inhibitory effect of LR-1 at different concentrations. Inhibitory activity of glucosidase, B being the effect of LR-2 at different concentrations. Inhibitory activity of glucosidase.

[0020] Figure 3 The active small peptides LR-1 and LR-2 in Example 3 of this invention, after being digested in the gastrointestinal tract, have the following effects: Effect of glucosidase-inhibiting peptide activity ( The values ​​indicate significant differences between the gastrointestinal digestion products of each peptide and their corresponding control groups. * indicates p < 0.05, ** indicates p < 0.01, and ns indicates no significant difference.

[0021] Figure 4 Examples 3 of the present invention show different concentrations of LR-1 and LR-2. Effects on cell viability ( The values ​​indicate significant differences between each experimental group and its control group. * indicates p < 0.05, and ** indicates p < 0.01. (This indicates no significant difference).

[0022] Figure 5 The graph shows the effect of different doses of LR-1 on fasting blood glucose levels in type 2 diabetic rats in Example 4 of this invention. The values ​​indicate significant differences between each experimental group and its control group. * indicates p < 0.05, and ** indicates p < 0.01. (This indicates no significant difference).

[0023] Figure 6 Example 4 of this invention illustrates the effect of different doses of LR-1 on type 2 diabetic rats at different time points after glucose ingestion ('*' indicates a significant difference between each experimental group and its control group, * indicates p < 0.05, ** indicates p < 0.01). (This indicates no significant difference).

[0024] Figure 7 The graph shows the effect of different doses of LR-1 on oral glucose tolerance in type 2 diabetic rats in Example 4 of this invention. The values ​​indicate significant differences between each experimental group and its control group. * indicates p < 0.05, and ** indicates p < 0.01. (This indicates no significant difference). Detailed Implementation

[0025] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0026] The composition may also contain wetting agents, emulsifiers and lubricants such as sodium dodecyl sulfate and magnesium stearate, as well as colorants, releasing agents, coating agents, sweeteners, flavoring agents and aroma agents, preservatives and antioxidants.

[0027] It should be noted that any aspects of this invention not described in detail are well known to those skilled in the art. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0028] Unless otherwise specified, all materials and reagents used in the following embodiments were obtained commercially.

[0029] In this invention, the complex protease was purchased from Shanghai Yuanye Biotechnology, catalog number S10155; the trypsin was purchased from Shanghai Yuanye Biotechnology, catalog number S10032; and the alkaline protease was purchased from Shanghai Yuanye Biotechnology, catalog number S10154.

[0030] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0031] Example 1 This embodiment provides an active bovine bone collagen peptide and its preparation method, which includes the following steps: (1) Pretreatment: Crush the beef bones to 70-100 mesh using a pulverizer, and mix 200g of beef bones according to the feed-to-water ratio. Under the conditions of 121℃ and 0.1 Pretreatment for 1 h yielded bovine bone collagen solution; Enzymatic hydrolysis: After cooling, add 0.3% of trypsin and alkaline protease (equal mass) by weight of bovine bone, and hydrolyze at 37℃ for 2 hours. After hydrolysis, inactivate the enzyme at 95℃ for 15 minutes. Add 0.2% of compound protease by weight of bovine bone, and hydrolyze at 50℃ and pH 7.5 for 2 hours. After hydrolysis, inactivate the enzyme at 95℃ for 15 minutes.

[0032] (2) Ultrafiltration: After cooling the inactivated enzyme hydrolysate, it is then subjected to ultrafiltration. Centrifugation under the specified conditions yielded the enzymatic supernatant. The molecular weight cutoff of the ultrafiltration membrane was [value missing]. The crude polypeptide solution was then concentrated and lyophilized. (3) Gel chromatography: using Separation was performed using a chromatographic column, with the following specific conditions: each sample was separated using... The concentration was dissolved in a 30% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid (TFA), and the sample loading volume was... Use a 30% acetonitrile aqueous solution to Elution at a flow rate of 215, absorbance was 215. Collect the second elution peak (F2).

[0033] Detection of the above separated components The inhibitory effect on glucosidase was shown in Table 1, and the method was as follows: Will Glucosidase, p-nitrobenzene Galactoside and the test sample (enzymatic digest) were dissolved separately in 0.10 M sodium phosphate buffer (pH = 6.8), and added to a 96-well plate. 0.10M sodium phosphate buffer (pH 6.8), then add solution ( ),as well as Sample solution Mix well, then preheat in a 37°C constant temperature water bath for 5 minutes, then immediately add... glucosidase solution To start the reaction, mix well and incubate in a 37°C water bath for 30 minutes; immediately after incubation, add... 1M The solution terminates the reaction. Absorbance was measured at a specific wavelength. The glucosidase inhibitory activity was calculated using formula (1). A blank control group (containing only the substrate) was also included in the experiment. The experiment consisted of three replicates: a control group (peptide solution replaced with buffer), a background group (peptide solution only, the rest water), and a background group (peptide solution only, the rest water).

[0034] Inhibition rate of glucosidase activity Equation (1) In the formula: —The absorbance of sodium phosphate buffer solution is used instead of the sample solution; —The absorbance of the sample solution during the reaction; —The absorbance of the background group, which contains only the absorbance of the peptide solution; —Absorbance of the blank group, absorbance of the PNPG solution during the reaction.

[0035] Table 1. Pairs of different separated components Inhibition of glucosidase (4) Reversed-phase high-performance liquid chromatography (RP-HPLC) separation and purification of F2 component: The chromatographic column is... BEH C18 column, column temperature 28℃, flow rate: Each injection ,exist The detection wavelength was set at [wavelength]. Mobile phase A was an aqueous solution containing 0.1% TFA; mobile phase B was an acetonitrile solution containing 0.1% TFA. The gradient elution conditions were: 0–5 min, 2% B; 5–20 min, 2%–15% B; 20–40 min, 15% B; 40–60 min, 2% B. Peptide solutions with retention times of 5–6, 8–9, and 12–13 min were collected.

[0036] (5) Perform high performance liquid chromatography-tandem mass spectrometry separation and identification on the product obtained in step (4): Liquid chromatography conditions include: chromatographic column is Column temperature 28℃; Sample dissolved in 0.1% formic acid, sample loading volume was... The gradient elution conditions were: 0–3 min, 5% B; 3–30 min, 5%–10% B; 30–40 min, 10%–30% B; 40–60 min, 90% B. Mass spectrometry conditions included: ion source: electrospray ionization (ESI); scanning mode: full mass spectrometry; mass range: 100-1500 m / z; ionization mode: positive ion; ion source voltage: 5000 V; ion source temperature: 250 °C.

[0037] (6) Utilize the mass spectrometry data obtained in step (5) The software performs the filtering.

[0038] Two small peptides with potential functional bioactive collagen were finally obtained, and their secondary mass spectra corresponded to... Figure 1 A in Figure 1 The amino acid sequence of B in the sequence is GPQGEAGPSI and GSTAAPCI, which are named LR-1 and LR-2, respectively.

[0039] Example 2 This embodiment provides the active small peptide pair obtained in Example 1. The application of glucosidase activity inhibition includes the following steps: 1. The above peptides were synthesized in a solid phase, specifically by Shanghai Sangon Biotech Co., Ltd.

[0040] 2. Study the effects of different concentrations of LR-1 and LR-2 on... The inhibitory activity of glucosidase was determined using the same method as in Example 1, and their IC50 values ​​were calculated.

[0041] The results showed that the IC50 values ​​for LR-1 and LR-2 were respectively: , In addition, different concentrations of LR-1 and LR-2 have different effects on... Inhibitory activity of glucosidase, such as Figure 2As shown in Figures A and B, respectively, it is demonstrated that with the increase of LR-1 and LR-2 concentrations, the effect on... The stronger the inhibitory effect on glucosidase, the more potent these two peptides are considered novel. Glucosidase inhibitory peptide; in addition, Glucosidase is a glycoside hydrolase that can catalyze... Glucosyl groups are derived from different substrates (such as maltodextrin, ... The non-reducing ends of glycosidic bonds (such as glycosides) hydrolyze to release... Therefore, the results above also indicate that LR-1 and LR-2 can effectively delay the absorption of carbohydrates and effectively control blood sugar, and can be used to prepare auxiliary hypoglycemic products.

[0042] Example 3 This embodiment provides verification of the stability and effect on cell activity of the active small peptide obtained in Example 1.

[0043] (a) Stability verification after digestion and absorption To further verify that the obtained small peptides can exist stably in vivo and exert their biological functions, gastrointestinal digestion was simulated in vitro. Artificial gastric digestive fluid and artificial small intestinal digestive fluid were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0044] The small peptide was dissolved in gastric juice to a final concentration of [missing value]. The sample was incubated on a shaker at 37°C for 2 hours. After digestion, the pH was adjusted to 7.0, and then an equal volume of preheated artificial small intestine digestion solution (37°C) was added. The sample was then incubated on a shaker at 37°C for 4 hours. The digested sample was then heated in boiling water for 10 minutes to terminate the reaction. The digested product was collected, lyophilized, and the effects of the product on the digestion process were determined. The inhibition rate of glucosidase was measured. A control group was also set up, which used buffer solution instead of gastric juice and small intestinal digestive juice. The relative activity of glucosidase was calculated using formula (2). The experiment was conducted in triplicate.

[0045] Relative activity Equation (2) Where A1 and A2 represent the gastrointestinal digestive products (experimental group) and the undigested gastrointestinal digestive products (control group), respectively. Glucosidase inhibition rate.

[0046] The results are as follows Figure 3 As shown, the active small peptides LR-1 and LR-2 have good gastrointestinal stability and, after digestion, are... The glucosidase inhibitory activity remains above 90%.

[0047] (ii) Cell viability test To verify whether the obtained small peptides had any effect on cell growth, a cell viability test was performed. Cells were cultured in DMEM medium... HepG2 cells were cultured at cell / well density. Different concentrations of LR-1 or LR-2 were used. Culture cells for 24 hours, with five replicates for each concentration. After 24 hours of incubation, ... (Cell Counting Kit-8 was purchased from Beyotime) Add to each well, then return to the incubator and incubate at 37°C for 2 hours. Then measure the absorbance at 450 nm using a microplate reader. Positive controls were set up: metformin hydrochloride, a hypoglycemic drug that improves insulin sensitivity and is used to treat type II diabetes; the control group was given serum-free DMEM medium containing cells but not the drug, and the blank control group was given only serum-free DMEM medium without cells. Cell viability was calculated using formula (3): Cell viability Equation (3) Where A0 represents the absorbance value of the blank control group, A1 represents the absorbance value of the control group, and A2 represents the absorbance value of the experimental group.

[0048] The results are as follows Figure 4 As shown, after 24 hours of co-culture, different concentrations of LR-1 or LR-2 had no negative impact on the growth of HepG2 cells.

[0049] Example 4 This embodiment provides the application of the active small peptide obtained in Example 1 in reducing blood glucose in animals. Specifically, the hypoglycemic effect of LR-1 is verified using a type 2 diabetic rat model.

[0050] Forty-five rats were randomly divided into five groups of nine each after acclimatization. These groups included a normal control group, a model group, and a metformin hydrochloride group. (day), LR-1 low-dose group ( (days), LR-1 high-dose group ( (Days). Except for the normal group, the other groups were fed a high-fat diet (20% lard, 18% sucrose, 3% egg yolk powder, 10% casein, 48.5% basal diet) for 8 consecutive weeks. In the 5th week, a low dose of streptozotocin was injected intraperitoneally. Modeling was performed on a 3-day period. The normal group was fed normal feed for 8 consecutive weeks, and in the 5th week, an equal volume of physiological saline was injected intraperitoneally.

[0051] For 8 consecutive weeks after successful model establishment, rats in the metformin hydrochloride group and LR-1 group were orally administered the corresponding dose of metformin hydrochloride or LR-1 daily. Rats in the model group and normal group were orally administered physiological saline for 8 consecutive weeks. On days 0, 14, 28, 42, and 56 after model establishment, after a 12-hour fast with unlimited water intake, blood was collected from the tail vein, and fasting blood glucose concentration was measured using a glucometer and blood glucose test strips according to the manufacturer's (Omron) instructions. On day 56 after model establishment, after a 12-hour fast with unlimited water intake, rats were administered glucose solution (2 g / kg) by gavage, and blood glucose levels were measured at 0, 30, 60, and 120 minutes after oral glucose administration using a glucometer and blood glucose test strips to evaluate the rats' glucose tolerance.

[0052] The result was that fasting blood glucose was as follows Figure 5 As shown, compared with the normal group, blood glucose levels were elevated in all other groups before administration. Compared with the model group, both metformin hydrochloride and the low-dose LR-1 group reduced blood glucose, while the high-dose LR-1 group significantly reduced blood glucose, indicating that LR-1 can effectively reduce fasting blood glucose levels in type 2 diabetic rats, and that a dose-effect relationship exists. Postprandial blood glucose levels are as follows: Figure 6 As shown, the blood glucose level in the normal group did not fluctuate significantly after glucose administration, while the blood glucose levels in other groups increased significantly. Compared with the model group, LR-1 can significantly improve postprandial blood glucose levels and prevent rapid blood glucose rise that could cause bodily damage. An oral glucose tolerance curve was plotted based on blood glucose levels, and the area under the glucose tolerance curve was calculated. The result is as follows Figure 7 As shown, compared with the model group, the area under the curve (AUC) of the high- and low-dose LR-1 groups was significantly reduced, indicating enhanced glucose tolerance. This demonstrates that LR-1 effectively lowers fasting blood glucose and controls postprandial blood glucose compared to the model group, and its effect is no worse than metformin hydrochloride, and even superior to metformin hydrochloride at high doses.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bovine bone collagen active small peptide, characterized in that, The amino acid sequence of the small peptide is GPQGEAGPSI or GSTAAPCI.

2. The collagen active peptide as described in claim 1, characterized in that, Obtained through solid-phase synthesis.

3. The bovine bone collagen active peptides according to any one of claims 1-2 in the preparation Applications of glucosidase inhibitors.

4. The use of the bovine bone collagen active peptide according to any one of claims 1-2 in the preparation of a drug or health product that helps lower blood sugar in animals.

5. The use of the bovine bone collagen active peptide according to any one of claims 1-2 in the preparation of a drug for the prevention or treatment of diabetes or obesity.

6. The application as described in claim 5, characterized in that, The diabetes mellitus mentioned is type 2 diabetes mellitus.

7. A composition, characterized in that, The composition comprises the active small peptide of claim 1.

8. The composition as described in claim 7, characterized in that, The composition includes other blood glucose-lowering ingredients and / or pharmaceutically acceptable carriers.

9. The composition as described in claim 8, characterized in that, Other blood sugar-lowering ingredients include metformin hydrochloride, liraglutide, acarbose, or sitagliptin.

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