An absorption enhancer for improving oral bioavailability of polypeptides and preparation method thereof

Abstract

The invention discloses an absorption enhancer and preparation method for improving the oral bioavailability of polypeptides, relates to the field of biological health care technology, including arginine grafted hyaluronic acid complex, enzyme inhibitor, and promotion auxiliary agent; the mass fraction of arginine grafted hyaluronic acid complex in the absorption enhancer for improving the oral bioavailability of polypeptides is 60-65%; the mass fraction of enzyme inhibitor in the absorption enhancer for improving the oral bioavailability of polypeptides is 5-8%; the rest are: promotion auxiliary agent; the enzyme inhibitor is pepstatin; the absorption enhancer for improving the oral bioavailability of polypeptides prepared by the method of the present invention is remarkable for the absorption effect of polypeptide oral liquid. The absorption enhancer prepared by the present invention is mainly capable of improving the absorption of components such as protein polypeptides by the gastrointestinal tract, making it easier to enter the physiological system circulation through the physiological barrier of the intestinal tract, improving absorption efficiency, and then improving oral bioavailability.

Description

Technical Field

[0001] The invention belongs to the technical field of biological health care, and in particular to an absorption promoter for improving the oral bioavailability of a polypeptide and a preparation method thereof. Background Art

[0002] Since the first successful synthesis of a biologically active peptide, the research on peptide and protein drugs has developed rapidly. With the rapid development of biotechnology, peptide and protein drugs are increasingly widely used in the treatment and health care of a variety of diseases, including blood system diseases, cancer, endocrine system diseases, cardiovascular system diseases, immune system diseases, respiratory system diseases, etc., with a wide range of applications.

[0003] Compared with traditional chemical drugs, peptide drugs have the following characteristics: ①Higher activity, a smaller dosage can achieve a certain therapeutic effect; ②Low resistance to enzymes, easily degraded by gastrointestinal digestive enzymes and hydrolases in body fluids, thus losing activity, resulting in a significant reduction in therapeutic effects; ③The relative molecular mass is large, and some drugs have strong polarity (logP<0), and the large hydrophilicity makes it difficult for them to be taken up by cells, and they cannot effectively pass through the physiological barrier of the gastrointestinal tract. In view of this, the oral bioavailability of peptide drugs is extremely low, which limits their application.

[0004] In order to ensure the highest bioavailability and activity of peptide drugs in the body, the main route of administration in the prior art is injection, which requires frequent injections. However, this greatly reduces patient compliance, while oral administration has a higher compliance. Therefore, how to improve the oral bioavailability of peptide drugs is a technical problem that needs to be solved at present.

[0005] Based on this, an absorption enhancer for improving the oral bioavailability of polypeptides and a preparation method are provided to solve the existing technical problems. Summary of the invention

[0006] The purpose of the present invention is to provide an absorption enhancer for improving the oral bioavailability of a polypeptide and a preparation method thereof, so as to solve the deficiencies in the prior art.

[0007] The technical solution adopted by the present invention is as follows:

[0008] An absorption enhancer for improving oral bioavailability of a polypeptide, comprising an arginine-grafted hyaluronic acid complex, an enzyme inhibitor, and an enhancing auxiliary;

[0009] The mass fraction of the arginine-grafted hyaluronic acid complex in the absorption enhancer for improving the oral bioavailability of the polypeptide is 60-65%;

[0010] The mass fraction of enzyme inhibitor in the absorption enhancer for improving oral bioavailability of polypeptide is 5-8%;

[0011] The rest are: promoting auxiliary agents;

[0012] The enzyme inhibitor is pepstatin.

[0013] As a further technical solution, the preparation method of the arginine-grafted hyaluronic acid complex is:

[0014] (1) First, arginine and polysialic acid are added to distilled water and stirred to mix well to obtain a composite solution;

[0015] (2) adding epichlorohydrin and sodium hydroxide to the composite liquid in step (1), stirring and reacting in a water bath for 2-4 hours, then removing epichlorohydrin by rotary evaporation, and then centrifuging and precipitating with ethanol solution, filtering, and drying to obtain a composite;

[0016] (3) adding hyaluronic acid to distilled water, stirring and mixing evenly to obtain a hyaluronic acid solution;

[0017] (4) Add the complex prepared in step (3) to the hyaluronic acid solution, keep warm in a water bath at 32° C. and stir for 2-3 days, concentrate under reduced pressure, and dialyze using a dialysis bag. Detect arginine and polysialic acid in the dialysate at the same time until arginine and polysialic acid are no longer detected in the solution. Freeze-dry the solution in the dialysis bag to obtain the arginine-grafted hyaluronic acid complex.

[0018] As a further technical solution, in step (1), 20-25 g of arginine and 4-5 g of polysialic acid are added to 30-35 mL of distilled water, and stirred at room temperature at a speed of 500 r / min for 20 min.

[0019] As a further technical solution, in step (2), 8-12 mL of epichlorohydrin and 0.6-1.0 g of sodium hydroxide are added to the composite solution;

[0020] The water bath insulation temperature is 32°C.

[0021] As a further technical solution, the ethanol solution in step (2) is a mixture of water and anhydrous ethanol in a volume ratio of 1:4;

[0022] The centrifugal speed is 5500r / min.

[0023] As a further technical solution, in step (3), 2-3 g of hyaluronic acid is added to 97-98 g of distilled water, and the mixture is stirred and mixed to obtain a hyaluronic acid solution.

[0024] As a further technical solution, in step (4), 3-3.5 g of the complex is added to 120-130 mL of hyaluronic acid solution for reaction.

[0025] As a further technical solution, the molecular retention capacity of the dialysis bag in step (4) is 480,000 Da.

[0026] As a further technical solution: the promoting auxiliary agent comprises a mixture of glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid;

[0027] The mass ratio of the glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid is 1:1:1:1-2.

[0028] A method for preparing an absorption enhancer for improving the oral bioavailability of a polypeptide comprises the following steps: adding an arginine-grafted hyaluronic acid complex, an enzyme inhibitor and an enhancing auxiliary into a mixer, stirring and mixing at a speed of 1000 r / min for 30 minutes, and after uniform mixing, sucking the mixture into a fluidized bed, using top spraying, adding anhydrous ethanol accounting for 10% of the total mass of the material, granulating the mixture to obtain granules, and then drying the mixture to remove the anhydrous ethanol to obtain the granules. Beneficial Effects

[0029] The absorption enhancer for improving the oral bioavailability of the polypeptide prepared by the method of the present invention has a significant effect of promoting the absorption of the polypeptide oral liquid.

[0030] The absorption enhancer prepared by the present invention can mainly improve the gastrointestinal absorption of components such as protein polypeptides, make it easier for them to pass through the physiological barrier of the intestine and enter the physiological system circulation, improve the absorption efficiency, and then improve the oral bioavailability.

[0031] The main mechanism of action of the absorption enhancer of the present invention is to temporarily and reversibly destroy the integrity of the intestinal barrier by opening the tight junctions between epithelial cells, thereby improving permeability and accelerating the absorption efficiency of the polypeptide. The bioavailability of the polypeptide oral solution after the introduction of the absorption enhancer of the present invention is greatly improved after oral administration compared with the polypeptide oral solution without the addition of the absorption enhancer of the present invention.

[0032] The present invention introduces an arginine-grafted hyaluronic acid complex during preparation, which can greatly improve the promoting effect of the absorption enhancer, especially can show the effect of enhancing the expression of vasodilatory neurotransmitters, thereby making it easier for the active polypeptide to pass through the physiological barrier, and under the action of enzyme inhibitors, can significantly reduce the decomposition of the active polypeptide in the polypeptide oral solution in the gastrointestinal tract, thereby reducing the decomposition loss of the active polypeptide, can keep a higher amount absorbed into the blood, and lay a foundation for subsequent improvement of the absorption rate.

[0033] Compared with arginine, the arginine-grafted hyaluronic acid complex prepared by the present invention has better mucosal adhesion, can be in close contact with the mucosal layer, can prolong the retention time of the active polypeptide, increase the concentration difference of the active polypeptide at the absorption site, and can promote the absorption and transport of the active polypeptide. In addition, the entire absorption enhancer also has the function of reversibly opening the tight junctions of epithelial cells to promote the transport of active polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a bar graph of absorbance of the embodiment and the comparative example;

[0035] Figure 2 This is a graph showing the changes in blood hydroxyproline content over time after oral administration. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] An absorption enhancer for improving oral bioavailability of a polypeptide, comprising an arginine-grafted hyaluronic acid complex, an enzyme inhibitor, and an enhancing auxiliary;

[0038] The mass fraction of the arginine-grafted hyaluronic acid complex in the absorption enhancer for improving the oral bioavailability of the polypeptide is 60-65%;

[0039] The mass fraction of enzyme inhibitor in the absorption enhancer for improving oral bioavailability of polypeptide is 5-8%;

[0040] The rest are: promoting auxiliary agents;

[0041] The enzyme inhibitor is pepstatin.

[0042] The preparation method of the arginine-grafted hyaluronic acid complex is as follows:

[0043] (1) First, arginine and polysialic acid are added to distilled water and stirred to mix well to obtain a composite solution;

[0044] (2) adding epichlorohydrin and sodium hydroxide to the composite liquid in step (1), stirring and reacting in a water bath for 2-4 hours, then removing epichlorohydrin by rotary evaporation, and then centrifuging and precipitating with ethanol solution, filtering, and drying to obtain a composite;

[0045] (3) adding hyaluronic acid to distilled water, stirring and mixing evenly to obtain a hyaluronic acid solution;

[0046] (4) Add the complex prepared in step (3) to the hyaluronic acid solution, keep warm in a water bath at 32° C. and stir for 2-3 days, concentrate under reduced pressure, and dialyze using a dialysis bag. Detect arginine and polysialic acid in the dialysate at the same time until arginine and polysialic acid are no longer detected in the solution. Freeze-dry the solution in the dialysis bag to obtain the arginine-grafted hyaluronic acid complex.

[0047] In step (1), 20-25 g of arginine and 4-5 g of polysialic acid are added to 30-35 mL of distilled water, and stirred at room temperature at a speed of 500 r / min for 20 min.

[0048] In step (2), 8-12 mL of epichlorohydrin and 0.6-1.0 g of sodium hydroxide are added to the composite solution;

[0049] The water bath insulation temperature is 32°C.

[0050] The ethanol solution in step (2) is a mixture of water and anhydrous ethanol in a volume ratio of 1:4;

[0051] The centrifugal speed is 5500r / min.

[0052] In step (3), 2-3 g of hyaluronic acid is added to 97-98 g of distilled water, and the mixture is stirred and mixed to obtain a hyaluronic acid solution.

[0053] In step (4), 3-3.5 g of the complex is added to 120-130 mL of hyaluronic acid solution to react.

[0054] The molecular cutoff of the dialysis bag in step (4) is 480,000 Da.

[0055] The promoting auxiliary agent is a mixture of glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid;

[0056] The mass ratio of the glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid is 1:1:1:1-2.

[0057] A method for preparing an absorption enhancer for improving the oral bioavailability of a polypeptide comprises the following steps: adding an arginine-grafted hyaluronic acid complex, an enzyme inhibitor and an enhancing auxiliary into a mixer, stirring and mixing at a speed of 1000 r / min for 30 minutes, and after uniform mixing, sucking the mixture into a fluidized bed, using top spraying, adding anhydrous ethanol accounting for 10% of the total mass of the material, granulating the mixture to obtain granules, and then drying the mixture to remove the anhydrous ethanol to obtain the granules.

[0058] The following are specific embodiments: Example 1

[0059] A method for preparing an absorption enhancer for improving the oral bioavailability of a polypeptide, comprising adding an arginine-grafted hyaluronic acid complex, pepstatin, and an enhancing auxiliary agent into a mixer, wherein the mass fraction of the arginine-grafted hyaluronic acid complex and the mass fraction of pepstatin in the absorption enhancer for improving the oral bioavailability of the polypeptide is 60%, the mass fraction of pepstatin is 5%, and the rest is the enhancing auxiliary agent;

[0060] The mixture is stirred at a speed of 1000 r / min for 30 minutes. After being evenly mixed, it is sucked into a fluidized bed and granulated by adding anhydrous ethanol accounting for 10% of the total mass of the material using top spraying. The granules are then dried to remove the anhydrous ethanol.

[0061] The preparation method of the arginine-grafted hyaluronic acid complex is as follows:

[0062] (1) First, add 20 g of arginine and 4 g of polysialic acid into 30 mL of distilled water, stir at 500 r / min for 20 min at room temperature, and mix evenly to obtain a composite solution;

[0063] (2) adding 8 mL of epichlorohydrin and 0.6 g of sodium hydroxide to the composite solution of step (1), stirring and reacting for 2 hours in a water bath at a temperature of 32° C., then removing epichlorohydrin by rotary evaporation, and then centrifuging with an ethanol solution, filtering, and drying to obtain a composite; the ethanol solution is a mixture of water and anhydrous ethanol in a volume ratio of 1:4;

[0064] The centrifugal speed is 5500r / min;

[0065] (3) adding 2 g of hyaluronic acid to 97 g of distilled water, stirring and mixing to obtain a hyaluronic acid solution;

[0066] (4) Add 3 g of the complex of step (3) to 120 mL of hyaluronic acid solution, keep warm and stir in a water bath at 32° C. for 2 days, concentrate under reduced pressure, and dialyze using a dialysis bag with a molecular cutoff of 480,000 Da. Simultaneously, detect arginine and polysialic acid in the dialysate until arginine and polysialic acid are no longer detected in the solution. The solution in the dialysis bag is freeze-dried to obtain an arginine-grafted hyaluronic acid complex.

[0067] The promoting auxiliary agent is a mixture of glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid in a mass ratio of 1:1:1:1. Example 2

[0068] A method for preparing an absorption enhancer for improving the oral bioavailability of a polypeptide, comprising adding an arginine-grafted hyaluronic acid complex, pepstatin, and an enhancing auxiliary agent into a mixer, wherein the mass fraction of the arginine-grafted hyaluronic acid complex and the mass fraction of pepstatin in the absorption enhancer for improving the oral bioavailability of the polypeptide is 62%, and the mass fraction of pepstatin is 6%, and the rest is the enhancing auxiliary agent;

[0069] The mixture is stirred at a speed of 1000 r / min for 30 minutes. After being evenly mixed, it is sucked into a fluidized bed and granulated by adding anhydrous ethanol accounting for 10% of the total mass of the material using top spraying. The granules are then dried to remove the anhydrous ethanol.

[0070] The preparation method of the arginine-grafted hyaluronic acid complex is as follows:

[0071] (1) First, add 22 g of arginine and 4.5 g of polysialic acid into 32 mL of distilled water, stir at 500 r / min for 20 min at room temperature, and mix evenly to obtain a composite solution;

[0072] (2) adding 10 mL of epichlorohydrin and 0.7 g of sodium hydroxide to the composite solution of step (1), stirring and reacting for 2.5 hours in a water bath at a temperature of 32° C., then removing epichlorohydrin by rotary evaporation, and then centrifuging with an ethanol solution, filtering, and drying to obtain a composite; the ethanol solution is a mixture of water and anhydrous ethanol in a volume ratio of 1:4;

[0073] The centrifugal speed is 5500r / min;

[0074] (3) adding 3 g of hyaluronic acid to 97 g of distilled water, stirring and mixing to obtain a hyaluronic acid solution;

[0075] (4) 3.2 g of the complex of step (3) was added to 122 mL of hyaluronic acid solution, and the mixture was stirred in a water bath at 32° C. for 2 days. After reduced pressure concentration, the mixture was dialyzed using a dialysis bag with a molecular cutoff of 480,000 Da. At the same time, arginine and polysialic acid in the dialysate were detected until arginine and polysialic acid were no longer detected in the solution. The solution in the dialysis bag was freeze-dried to obtain an arginine-grafted hyaluronic acid complex.

[0076] The promoting auxiliary agent is a mixture of glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid in a mass ratio of 1:1:1:1.2. Example 3

[0077] A method for preparing an absorption enhancer for improving the oral bioavailability of a polypeptide, comprising adding an arginine-grafted hyaluronic acid complex, pepstatin, and an enhancing auxiliary agent into a mixer, wherein the mass fraction of the arginine-grafted hyaluronic acid complex and the mass fraction of pepstatin in the absorption enhancer for improving the oral bioavailability of the polypeptide is 62%, and the mass fraction of pepstatin is 6%, and the rest is the enhancing auxiliary agent;

[0078] The mixture is stirred at a speed of 1000 r / min for 30 minutes. After being evenly mixed, it is sucked into a fluidized bed and granulated by adding anhydrous ethanol accounting for 10% of the total mass of the material using top spraying. The granules are then dried to remove the anhydrous ethanol.

[0079] The preparation method of the arginine-grafted hyaluronic acid complex is as follows:

[0080] (1) First, add 23 g of arginine and 4.2 g of polysialic acid into 33 mL of distilled water, stir at 500 r / min for 20 min at room temperature, and mix evenly to obtain a composite solution;

[0081] (2) adding 10 mL of epichlorohydrin and 0.8 g of sodium hydroxide to the composite solution of step (1), stirring and reacting for 3 hours in a water bath at a temperature of 32° C., then removing epichlorohydrin by rotary evaporation, and then centrifuging with an ethanol solution, filtering, and drying to obtain a composite; the ethanol solution is a mixture of water and anhydrous ethanol in a volume ratio of 1:4;

[0082] The centrifugal speed is 5500r / min;

[0083] (3) adding 2.6 g of hyaluronic acid to 97.5 g of distilled water, stirring and mixing to obtain a hyaluronic acid solution;

[0084] (4) 3.3 g of the complex of step (3) was added to 125 mL of hyaluronic acid solution, and the solution was kept warm and stirred in a water bath at 32° C. for 2.5 days. After reduced pressure concentration, the solution was dialyzed using a dialysis bag with a molecular cutoff of 480,000 Da. At the same time, arginine and polysialic acid in the dialysate were detected until arginine and polysialic acid were no longer detected in the solution. The solution in the dialysis bag was freeze-dried to obtain the arginine-grafted hyaluronic acid complex.

[0085] The promoting auxiliary agent is a mixture of glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid in a mass ratio of 1:1:1:1.6. Example 4

[0086] A method for preparing an absorption enhancer for improving the oral bioavailability of a polypeptide, comprising adding an arginine-grafted hyaluronic acid complex, pepstatin, and an enhancing auxiliary agent into a mixer, wherein the mass fraction of the arginine-grafted hyaluronic acid complex and the mass fraction of pepstatin in the absorption enhancer for improving the oral bioavailability of the polypeptide is 64%, and the mass fraction of pepstatin is 7%, and the rest is the enhancing auxiliary agent;

[0087] The mixture is stirred at a speed of 1000 r / min for 30 minutes. After being evenly mixed, it is sucked into a fluidized bed and granulated by adding anhydrous ethanol accounting for 10% of the total mass of the material using top spraying. The granules are then dried to remove the anhydrous ethanol.

[0088] The preparation method of the arginine-grafted hyaluronic acid complex is as follows:

[0089] (1) First, add 24 g of arginine and 4.8 g of polysialic acid into 34 mL of distilled water, stir at 500 r / min for 20 min at room temperature, and mix evenly to obtain a composite solution;

[0090] (2) adding 10 mL of epichlorohydrin and 0.9 g of sodium hydroxide to the composite solution of step (1), stirring and reacting for 3 hours in a water bath at a temperature of 32° C., then removing epichlorohydrin by rotary evaporation, and then centrifuging with an ethanol solution, filtering, and drying to obtain a composite; the ethanol solution is a mixture of water and anhydrous ethanol in a volume ratio of 1:4;

[0091] The centrifugal speed is 5500r / min;

[0092] (3) adding 2 g of hyaluronic acid to 97.8 g of distilled water, stirring and mixing to obtain a hyaluronic acid solution;

[0093] (4) 3.4 g of the complex of step (3) was added to 126 mL of hyaluronic acid solution, and the solution was kept warm and stirred in a water bath at 32° C. for 2.5 days. After reduced pressure concentration, the solution was dialyzed using a dialysis bag with a molecular cutoff of 480,000 Da. At the same time, arginine and polysialic acid in the dialysate were detected until arginine and polysialic acid were no longer detected in the solution. The solution in the dialysis bag was freeze-dried to obtain the arginine-grafted hyaluronic acid complex.

[0094] The promoting auxiliary agent is a mixture of glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid in a mass ratio of 1:1:1:1.8. Example 5

[0095] A method for preparing an absorption enhancer for improving the oral bioavailability of a polypeptide, comprising adding an arginine-grafted hyaluronic acid complex, pepstatin, and an enhancing auxiliary agent into a mixer, wherein the mass fraction of the arginine-grafted hyaluronic acid complex and the mass fraction of pepstatin in the absorption enhancer for improving the oral bioavailability of the polypeptide is 65%, and the mass fraction of pepstatin is 8%, and the rest is the enhancing auxiliary agent;

[0096] The mixture is stirred at a speed of 1000 r / min for 30 minutes. After being evenly mixed, it is sucked into a fluidized bed and granulated by adding anhydrous ethanol accounting for 10% of the total mass of the material using top spraying. The granules are then dried to remove the anhydrous ethanol.

[0097] The preparation method of the arginine-grafted hyaluronic acid complex is as follows:

[0098] (1) First, add 25 g of arginine and 5 g of polysialic acid into 35 mL of distilled water, stir at 500 r / min for 20 min at room temperature, and mix evenly to obtain a composite solution;

[0099] (2) adding 12 mL of epichlorohydrin and 1.0 g of sodium hydroxide to the composite solution of step (1), stirring and reacting for 4 hours in a water bath at a temperature of 32° C., then removing epichlorohydrin by rotary evaporation, and then centrifuging with an ethanol solution, filtering, and drying to obtain a composite; the ethanol solution is a mixture of water and anhydrous ethanol in a volume ratio of 1:4;

[0100] The centrifugal speed is 5500r / min;

[0101] (3) adding 3 g of hyaluronic acid to 98 g of distilled water, stirring and mixing to obtain a hyaluronic acid solution;

[0102] (4) Add 3.5 g of the complex of step (3) to 130 mL of hyaluronic acid solution, keep warm in a water bath at 32° C. and stir for 3 days. After reduced pressure concentration, dialyze using a dialysis bag with a molecular cutoff of 480,000 Da. At the same time, detect arginine and polysialic acid in the dialysate until arginine and polysialic acid are no longer detected in the solution. The solution in the dialysis bag is freeze-dried to obtain an arginine-grafted hyaluronic acid complex.

[0103] The promoting auxiliary agent is a mixture of glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid in a mass ratio of 1:1:1:2.

[0104] Comparative Example 1:

[0105] A method for preparing an absorption enhancer for improving the oral bioavailability of a polypeptide, comprising adding an arginine-grafted hyaluronic acid complex and an enhancing auxiliary agent into a mixer, wherein the mass fraction of the arginine-grafted hyaluronic acid complex in the absorption enhancer for improving the oral bioavailability of the polypeptide is 60%, and the rest is: the enhancing auxiliary agent;

[0106] The mixture is stirred at a speed of 1000 r / min for 30 minutes. After being evenly mixed, it is sucked into a fluidized bed and granulated by adding anhydrous ethanol accounting for 10% of the total mass of the material using top spraying. The granules are then dried to remove the anhydrous ethanol.

[0107] The preparation method of the arginine-grafted hyaluronic acid complex is as follows:

[0108] (1) First, add 20 g of arginine and 4 g of polysialic acid into 30 mL of distilled water, stir at 500 r / min for 20 min at room temperature, and mix evenly to obtain a composite solution;

[0109] (2) adding 8 mL of epichlorohydrin and 0.6 g of sodium hydroxide to the composite solution of step (1), stirring and reacting for 2 hours in a water bath at a temperature of 32° C., then removing epichlorohydrin by rotary evaporation, and then centrifuging with an ethanol solution, filtering, and drying to obtain a composite; the ethanol solution is a mixture of water and anhydrous ethanol in a volume ratio of 1:4;

[0110] The centrifugal speed is 5500r / min;

[0111] (3) adding 2 g of hyaluronic acid to 97 g of distilled water, stirring and mixing to obtain a hyaluronic acid solution;

[0112] (4) Add 3 g of the complex of step (3) to 120 mL of hyaluronic acid solution, keep warm and stir in a water bath at 32° C. for 2 days, concentrate under reduced pressure, and dialyze using a dialysis bag with a molecular cutoff of 480,000 Da. Simultaneously, detect arginine and polysialic acid in the dialysate until arginine and polysialic acid are no longer detected in the solution. The solution in the dialysis bag is freeze-dried to obtain an arginine-grafted hyaluronic acid complex.

[0113] The promoting auxiliary agent is a mixture of glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid in a mass ratio of 1:1:1:1.

[0114] Comparative Example 2:

[0115] A method for preparing an absorption enhancer for improving the oral bioavailability of a polypeptide, comprising adding arginine, pepstatin and an enhancing auxiliary agent into a mixer, wherein the absorption enhancer for improving the oral bioavailability of a polypeptide comprises 60% arginine, 5% pepstatin by mass fraction, and the rest being the enhancing auxiliary agent;

[0116] The mixture is stirred at a speed of 1000 r / min for 30 minutes. After being evenly mixed, it is sucked into a fluidized bed and granulated by adding anhydrous ethanol accounting for 10% of the total mass of the material using top spraying. The granules are then dried to remove the anhydrous ethanol.

[0117] test:

[0118] Mixing marine fish skin oligopeptides with water to obtain a polypeptide oral solution;

[0119] Each 100mL of peptide oral solution contains 10g of marine fish skin oligopeptide;

[0120] 1 g of the samples of the embodiment and the comparative example were added to 100 mL of the polypeptide oral solution to prepare 8 experimental groups;

[0121] This is a blank group of polypeptide oral liquids without adding the samples of the embodiments and comparative examples;

[0122] After testing in each experimental group and the blank group, the average mass fraction of hydroxyproline content was 9.25%, and the difference between the groups did not exceed 0.02%;

[0123] Adult SD rats (half male and half female, with an average weight of 300 g) were fasted but not watered for 12 h before the experiment. The Example, Comparative Example and Blank group were gavaged respectively. The control group was gavaged with physiological saline. The Example, Comparative Example and Blank group were gavaged at an amount of 50 mg of marine fish skin oligopeptide per gram of body weight.

[0124] After 24 hours of intragastric administration, the feces and urine of the rats were collected. The content of hydroxyproline was determined by the kit method, and the absorption rate was calculated.

[0125] Absorption rate = hydroxyproline absorption / body weight = (total intake - metabolic amount) / body weight;

[0126] Metabolism = Hydroxyproline content in feces + Hydroxyproline content in urine:

[0127] Table 1

[0128]

[0129] It can be seen from Table 1 that the absorption rate of the polypeptide oral solution can be greatly improved by introducing the promoter prepared by the present invention.

[0130] Determination of Hydroxyproline Content in Blood

[0131] The same method was used to gavage SD adult rats with the samples of Example 5 and Comparative Example 1-2, and blood was collected from the eye sockets at the last hour. The blood was centrifuged at high speed for 20 min at a speed of 3500 r / min, and serum was collected to determine the content of hydroxyproline using a kit method:

[0132] Table 2

[0133]

[0134] It can be seen from Table 2 that the promoter of the present invention can promote the absorption and utilization of the polypeptide oral solution. After oral administration, the hydroxyproline content in the blood is significantly increased.

[0135] The same method was used to gavage SD adult rats with the sample of Example 5, and blood was collected from the eye sockets at 0.5, 1, 2, 4, and 6 hours, respectively. The blood was centrifuged at high speed for 20 minutes at a speed of 3500 r / min, and serum was collected to determine the content of hydroxyproline using a kit method:

[0136] Table 3

[0137]

[0138] It can be seen from Table 3 that the absorption rate of the polypeptide oral liquid is relatively fast, which shows that the promoter prepared by the present invention can significantly improve the bioavailability of the polypeptide oral liquid.

[0139] The above description is only a preferred embodiment of the present invention, but the present invention is not limited to the scope of implementation shown. Any changes made in accordance with the concept of the present invention, or modifications to equivalent embodiments with equivalent changes, which still do not exceed the spirit covered by the specification, should be within the protection scope of the present invention.

Claims

1. An absorption enhancer for improving the oral bioavailability of a polypeptide, characterized in that: It includes arginine-grafted hyaluronic acid complex, enzyme inhibitor, and promoting auxiliary agent; The mass fraction of the arginine-grafted hyaluronic acid complex in the absorption enhancer for improving the oral bioavailability of the polypeptide is 60-65%; The mass fraction of the enzyme inhibitor in the absorption enhancer for improving the oral bioavailability of the polypeptide is 5-8%; The rest are: promoting auxiliary agents; The enzyme inhibitor is pepstatin; The preparation method of the arginine-grafted hyaluronic acid complex is as follows: (1) First, arginine and polysialic acid are added to distilled water, and stirred to mix evenly to obtain a composite solution; (2) adding epichlorohydrin and sodium hydroxide to the composite liquid in step (1), stirring and reacting in a water bath for 2-4 hours, then removing epichlorohydrin by rotary evaporation, and then centrifuging with an ethanol solution, filtering, and drying to obtain a composite; (3) adding hyaluronic acid to distilled water, stirring and mixing to obtain a hyaluronic acid solution; (4) adding the complex prepared in step (2) to the hyaluronic acid solution, keeping warm in a water bath at 32° C. and stirring for 2-3 days, concentrating under reduced pressure and dialyzing with a dialysis bag, while detecting arginine and polysialic acid in the dialysate until arginine and polysialic acid are no longer detected in the solution, and freeze-drying the solution in the dialysis bag to obtain the arginine-grafted hyaluronic acid complex; The promoting auxiliary agent comprises a mixture of glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid; The mass ratio of the glycodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid and glycodehydrocholic acid is 1:1:1:1-2.

2. The absorption enhancer for improving the oral bioavailability of a polypeptide according to claim 1, characterized in that: In step (1), 20-25 g of arginine and 4-5 g of polysialic acid are added to 30-35 mL of distilled water, and stirred at room temperature at a speed of 500 r / min for 20 min.

3. The absorption enhancer for improving the oral bioavailability of a polypeptide according to claim 1, characterized in that: In step (2), 8-12 mL of epichlorohydrin and 0.6-1.0 g of sodium hydroxide are added to the composite solution; The water bath insulation temperature is 32°C.

4. The absorption enhancer for improving the oral bioavailability of a polypeptide according to claim 1, characterized in that: The ethanol solution in step (2) is a mixture of water and anhydrous ethanol in a volume ratio of 1:4; The centrifugal speed is 5500r / min.

5. The absorption enhancer for improving oral bioavailability of a polypeptide according to claim 1, characterized in that: In step (3), 2-3 g of hyaluronic acid is added to 97-98 g of distilled water, and the mixture is stirred to obtain a hyaluronic acid solution.

6. The absorption enhancer for improving the oral bioavailability of a polypeptide according to claim 1, characterized in that: In step (4), 3-3.5 g of the complex is added to 120-130 mL of hyaluronic acid solution to react.

7. The absorption enhancer for improving oral bioavailability of a polypeptide according to claim 1, characterized in that: The molecular cutoff of the dialysis bag in step (4) is 480,000 Da.

8. The method for preparing an absorption enhancer for improving oral bioavailability of a polypeptide according to claim 1, characterized in that: Add arginine-grafted hyaluronic acid complex, enzyme inhibitor and promoting auxiliary agent into a mixer, stir and mix at a speed of 1000r / min for 30min, and after uniform mixing, suck into a fluidized bed, use top spray, add anhydrous ethanol that accounts for 10% of the total mass of the material, granulate to obtain granules, and then dry and remove the anhydrous ethanol to obtain the granules.

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