Oral preparation as well as preparation method and medicine thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an oral preparation, a preparation method of yeast cell wall loaded insulin and a medicine. According to the oral preparation provided by the invention, the saccharomycetes cell wall is used as a carrier, the saccharomycetes cell wall is used for loading an active drug, the drug loading capacity is high, the encapsulation efficiency is high, the saccharomycetes cell wall is used as the carrier, drug encapsulation can be effectively carried out, the drug can be effectively protected from being damaged by a gastrointestinal fluid environment, and the bioavailability of the drug is improved; the saccharomycetes cell wall is natural polysaccharide, is high in biological safety, has a certain immunoregulation effect, can stabilize the blood sugar balance in the body, can enable the medicine to safely reach an absorption part in the body, and is beneficial to long-term treatment of the medicine on a diabetic patient. The invention further provides a preparation method of the oral preparation of the yeast cell wall loaded insulin, the method is simple, the reaction is mild, and the preparation method can be applied to industrialized production.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to an oral preparation, a preparation method of yeast cell wall-loaded insulin, and a medicine. Background Art

[0002] Diabetes is a metabolic disease characterized by increased blood sugar levels, which is manifested by defects in insulin secretion in the pancreas and decreased sensitivity of peripheral tissues to insulin, so that glucose in the blood cannot be converted into glycogen by tissues such as the liver and muscles. The disorder of material metabolism in diabetes can cause abnormal functions and structures of various tissues and organs throughout the body, and can be fatal in severe cases. Diabetes is also the main cause of diseases such as blindness, renal failure, heart disease, stroke and lower limb amputation. Insulin is the only protein hormone secreted by pancreatic islet cells that has the function of regulating blood sugar. It has a relative molecular mass of 5807.69 and is composed of 51 amino acids. It mainly includes two peptide chains, A chain and B chain. Among them, A chain contains 21 amino acids and B chain contains 30 amino acids. The A chain and the A and B chains are connected by disulfide bonds. Insulin is a biological macromolecule drug widely used in diabetic patients, but it has a short half-life, poor lipid solubility, and is not easy to penetrate biological membranes. Therefore, the only way to use insulin clinically is subcutaneous injection.

[0003] Although subcutaneous insulin has a rapid onset of action and high bioavailability, long-term subcutaneous insulin injection brings great pain to patients; the injection method is inconvenient to use, has poor compliance, and is often accompanied by injection complications such as allergies and skin damage. Therefore, in order to meet the needs of diabetic patients, prolong the action time of insulin in the body, and better treat diabetes, the development of non-injection routes of administration has become a research hotspot. The main non-injection routes of administration currently studied include pulmonary administration, nasal administration, transdermal administration, oral mucosal administration, and oral administration at the academic theoretical level. Although the above administration methods can improve the bioavailability of insulin to a certain extent, long-term administration is prone to complications such as pulmonary edema, skin inflammation, hematoma, etc. Among all non-injection routes of administration, oral administration is the safest and most convenient, and patient compliance is also the best. However, insulin drugs are usually injected, and there is no oral insulin preparation. Summary of the invention

[0004] The purpose of the present invention is to provide an oral preparation of yeast cell wall loaded insulin and a preparation method thereof, so as to improve the bioavailability of insulin.

[0005] In order to solve the above technical problems, the following technical solutions are proposed:

[0006] The invention provides an oral preparation, comprising a carrier and an active drug loaded in the carrier; the active drug comprises insulin; and the carrier comprises a yeast cell wall.

[0007] Preferably, the mass content of insulin in the oral preparation is 13% to 16%.

[0008] Preferably, the particle size of the oral preparation is 1 to 3 μm; the dosage form of the oral preparation includes oral enteric-coated capsules.

[0009] Preferably, the oral preparation further comprises a functional excipient; the functional excipient comprises a gastrointestinal absorption enhancer.

[0010] The present invention provides a method for preparing the oral preparation described in the above technical solution, comprising:

[0011] Insulin is mixed with a hydrochloric acid aqueous solution to obtain a mixture, and yeast cell walls are added into the mixture to carry out a self-assembly reaction to obtain a yeast cell wall-loaded insulin oral preparation.

[0012] Preferably, the method for preparing the yeast cell wall comprises:

[0013] The yeast and the first organic solvent are mixed and then subjected to ultra-high pressure treatment to obtain yeast cells;

[0014] The yeast cells are sequentially subjected to alkali treatment, acid treatment and washing with a second organic solvent to obtain yeast cell walls;

[0015] The ultra-high pressure temperature is 25-30° C., the pressure is 50-300 MPa, the ultra-high pressure treatment time is 5-30 minutes, and the ultra-high pressure treatment times are 1-6 times.

[0016] Preferably, the first organic solvent includes anhydrous ethanol; and the second organic solvent includes acetone.

[0017] Preferably, the mass ratio of the yeast cell wall to insulin is 9:(1-4).

[0018] Preferably, the self-assembly method includes water bath stirring; the water bath stirring temperature is 15 to 40° C., and the water bath stirring time is 30 min to 6 h.

[0019] The present invention provides a medicine, which comprises a pharmaceutical active ingredient and a pharmaceutical excipient, wherein the pharmaceutical active ingredient comprises the oral preparation described in any one of the above technical solutions or the oral preparation prepared by the preparation method described in any one of the above technical solutions.

[0020] Beneficial effects of the present invention: The present invention provides an oral preparation, comprising a carrier and an active drug loaded in the carrier; the active drug comprises insulin; and the carrier comprises a yeast cell wall.

[0021] The present invention uses yeast cell wall as a carrier, and yeast cell wall has strong biocompatibility and safety. As a carrier, it can effectively carry out drug encapsulation, and can also effectively protect drugs from being destroyed by the gastrointestinal fluid environment and inactivated, which is beneficial to the transportation of drugs to the small intestine, and the bioavailability of drugs is improved; and the yeast cell wall is a natural polysaccharide, which has high biological safety and certain immunomodulatory effects, and can stabilize the blood sugar balance in the body. And the oral preparation of the present invention also avoids complications at the site of administration. After oral administration of insulin, it can directly participate in the metabolism of sugar by the liver after entering the body circulation through the portal vein, simulate the effect of endogenous insulin secreted by the pancreas, reduce the fluctuation of blood sugar in the body, thereby reducing damage to blood vessels, improve the bioavailability of insulin, and enable the drug to safely reach the absorption site in the body, which is beneficial to the long-term treatment of diabetic patients with drugs.

[0022] The present invention also provides a method for preparing an oral preparation of yeast cell wall loaded insulin. The present invention uses a self-assembly loading method in a hydrochloric acid aqueous solution of yeast cell wall and insulin to achieve loading of active drugs on yeast cell wall. The yeast cell wall loaded insulin oral preparation is obtained after centrifugal freeze drying after self-assembly reaction. The drug loading amount and encapsulation rate are high. The preparation method provided by the present invention is simple, the reaction is mild, the materials and solvents used in the preparation are non-toxic, and it has good environmental benefits and can be applied to factory production.

[0023] The present invention adds functional excipients, such as gastrointestinal absorption enhancers, to the provided oral preparation of insulin loaded on yeast cell walls, and can enhance drug absorption by improving drug solubility and oral delivery efficiency. At the same time, it can also reduce drug metabolism in the body, improve the tight connection of the digestive tract, enhance the fluidity of cell membranes, promote intestinal transport of drugs, and enhance the combination of drugs with epithelial cells, thereby improving the drug's penetration efficiency in the digestive tract and oral bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0025] Figure 1 TEM images of yeast, yeast cell wall, and yeast cell wall loaded with insulin;

[0026] Figure 2 SEM images of yeast cell wall, insulin and yeast cell wall loaded with insulin; from left to right: a, b, c;

[0027] Figure 3 FTIR images of insulin, yeast cell wall, yeast cell wall loaded with insulin and mixture;

[0028] Figure 4 XRD diagrams of insulin, yeast cell wall, yeast cell wall loaded with insulin and mixture;

[0029] Figure 5 The DSC diagrams of insulin, yeast cell wall, yeast cell wall loaded with insulin and the mixture;

[0030] Figure 6 TG curves of insulin, yeast cell wall, yeast cell wall loaded with insulin and mixture;

[0031] Figure 7 The in vitro dissolution diagrams of insulin group and yeast cell wall loaded insulin group in simulated gastrointestinal fluid environment; a is artificial gastric fluid, b is artificial intestinal fluid;

[0032] Figure 8 The graphs show the hypoglycemic effects of insulin group, yeast cell wall loaded insulin group, yeast cell wall loaded insulin + gastrointestinal absorption enhancer group and insulin injection group in male rats;

[0033] Fig. 9 The graphs show the hypoglycemic effects of the insulin group, the yeast cell wall loaded insulin group, the yeast cell wall loaded insulin + gastrointestinal absorption enhancer group and the insulin injection group in female rats;

[0034] Fig.10 This is the insulin blood concentration graph of rats in the insulin group, yeast cell wall loaded insulin group, yeast cell wall loaded insulin + gastrointestinal absorption enhancer group and insulin injection group within 36 hours;

[0035] Fig.11 These are H&E staining images of sections of major organs of mice in the blank group, insulin group, and yeast cell wall-loaded insulin group. DETAILED DESCRIPTION

[0036] The invention provides an oral preparation, comprising a carrier and an active drug loaded in the carrier; the active drug comprises insulin; and the carrier comprises a yeast cell wall.

[0037] In the present invention, unless otherwise specified, insulin is a commercially available product well known to those skilled in the art.

[0038] In the present invention, the method for preparing the yeast cell wall preferably comprises:

[0039] The yeast and the first organic solvent are mixed and then subjected to ultra-high pressure treatment to obtain yeast cells;

[0040] The yeast cells are sequentially subjected to alkali treatment, acid treatment and washing with a second organic solvent to obtain yeast cell walls.

[0041] The temperature of the ultrahigh pressure of the present invention is preferably 25-30°C, more preferably 26-28°C; the pressure is preferably 50-300MPa, more preferably 50-150MPa; in the embodiment of the present invention, the ultrahigh pressure can be 50MPa, 100MPa, 150MPa, 200MPa or 300MPa. The time of ultrahigh pressure treatment is preferably 5-30min, more preferably 5-15min, more preferably 5min; in the embodiment of the present invention, the time of ultrahigh pressure treatment can be 5min, 10min, 15min, 20min or 30min. The number of ultrahigh pressure treatments is preferably 1-6 times, more preferably 1-3 times, more preferably 1 time; in the embodiment of the present invention, the number of ultrahigh pressure treatments can be 1, 2, 3, 4 or 6 times. The ultrahigh pressure treatment can completely release the material components in the yeast body to form a cavity carrier. The first organic solvent preferably includes anhydrous ethanol; the mass volume ratio of the yeast and the first organic solvent is preferably (10-20g): 1L. The ultra-high pressure combined with the first organic solvent treatment of the present invention is to mix the yeast with the first organic solvent and then perform the ultra-high pressure treatment.

[0042] After ultrahigh pressure and the first organic solvent treatment, the present invention preferably stirs and then centrifuges to obtain yeast cells. The stirring time is preferably 30 min to 120 min, more preferably 60 min; in an embodiment of the present invention, the stirring time may be 30 min, 60 min or 120 min. The centrifugal speed is preferably 3000 to 5000 rpm, more preferably 4000 rpm; in an embodiment of the present invention, the centrifugal speed may be 3000, 4000 or 5000 rpm. The centrifugal time is preferably 8 to 15 min, more preferably 10 min; in an embodiment of the present invention, the centrifugal time may be 8, 10 or 15 min.

[0043] The yeast cell is obtained. The present invention preferably sequentially treats the yeast cell with alkali, acid and washes it with a second organic solvent to obtain a yeast cell wall. In the present invention, the reagent used for the alkali treatment preferably includes a NaOH solution; the molar concentration of the NaOH solution is preferably 0.5-1.5 mol / L, more preferably 1 mol / L; in an embodiment of the present invention, the molar concentration of the NaOH solution may be 0.5, 1 or 1.5 mol / L. The temperature of the alkali treatment is preferably 60-100°C, more preferably 85°C, and in an embodiment of the present invention, the temperature of the alkali treatment may be 60, 70, 80, 85 or 100°C. The time of the alkali treatment is preferably 30-120 min, more preferably 60 min; in an embodiment of the present invention, the time of the alkali treatment may be 30, 60 or 120 min. The alkali treatment method of the present invention preferably includes water bath stirring; the present invention washes the cell by the alkali treatment to remove the protein in the cell; when washing the cell, the mass of the cell and the volume ratio of the alkali treatment reagent are preferably (10-20 g): 1 L. The present invention preferably centrifuges the alkali-treated product to obtain a precipitate, washes the precipitate, and then performs an acid treatment; the centrifugal speed is preferably 3000-5000 rpm, more preferably 4000 rpm; in an embodiment of the present invention, the centrifugal speed may be 3000, 4000 or 5000 rpm. The centrifugal time is preferably 8-15 min, more preferably 10 min; in an embodiment of the present invention, the centrifugal time may be 8, 10 or 15 min. The reagent used for the acid treatment preferably includes an HCl solution; the molar concentration of the HCl solution is preferably 0.5-1.5 mol / L, more preferably 1 mol / L; in an embodiment of the present invention, the molar concentration of the HCl solution may be 0.5 or 1 mol / L; the pH value of the HCl solution treatment is preferably 4.5-5.0; in an embodiment of the present invention, the pH value of the HCl solution treatment may be 4.5, 4.8 or 5.0. The temperature of the acid treatment is preferably 50-80°C, more preferably 65°C; in an embodiment of the present invention, the temperature of the acid treatment may be 50, 65 or 80°C. The acid treatment time is preferably 30 min to 120 min, more preferably 60 min; in an embodiment of the present invention, the acid treatment time can be 30, 60 or 120 min. The acid treatment method of the present invention preferably includes water bath stirring; the acid treatment is used to wash the cells and remove proteins in the cells. When washing the cells, the mass of the cells and the volume ratio of the acid treatment reagent are preferably (10 to 20 g): 1 L.

[0044] The present invention preferably centrifuges the acid-treated product to obtain a precipitate, washes the precipitate, and then rinses it with a second organic solvent. In the present invention, the centrifugal speed is preferably 3000-5000rpm, more preferably 4000rpm; in an embodiment of the present invention, the centrifugal speed may be 3000, 4000 or 5000rpm. The centrifugal time of the present invention is preferably 8-15min, more preferably 10min; in an embodiment of the present invention, the centrifugal time may be 8, 10 or 15min. The second organic solvent of the present invention preferably includes acetone. The number of acetone rinses is preferably 1-2 times, more preferably 1 time; the mass volume ratio of the bacterial precipitate to the second organic solvent is preferably (10-20g): 200mL.

[0045] The present invention preferably dries the product obtained by washing with the second organic solvent to obtain the yeast cell wall carrier; the drying method is preferably natural drying at 25-30° C. indoors; the natural drying time is preferably 5-15 hours, more preferably 12 hours.

[0046] In the yeast cell wall preparation process of the present invention, the yeast is treated with ultra-high pressure and organic reagents, washed with alkaline solution and acid solution, and then washed with organic reagents, which are all helpful to remove the protein and other active ingredients inside the yeast, ensuring that the empty yeast cell wall is obtained after centrifugal drying.

[0047] The mass content of insulin in the oral preparation is preferably 10% to 20%, more preferably 12% to 18%, and more preferably 13% to 16%. The particle size of the oral preparation is preferably 1 to 3 μm, more preferably 2 to 3 μm. The dosage form of the oral preparation preferably includes oral enteric-coated capsules.

[0048] The oral preparation of the present invention also includes functional excipients; the functional excipients include gastrointestinal absorption promoters; the gastrointestinal absorption promoters include one or more of ethylenediaminetetraacetic acid (EDTA), diethylenetriammonium pentaacetic acid (DTPA), 8-(2-hydroxybenzamido) sodium caprylate (SNAC), chitosan and its derivatives, carbomer, alkyl glycosides and sodium taurocholate.

[0049] However, insulin drugs are usually injected, and there is no oral insulin preparation. The present invention uses yeast cell wall as a carrier of oral preparation, so that insulin can avoid being damaged by the complex gastrointestinal environment, effectively protect insulin from being safely transported to the mucus layer of the small intestine through the gastrointestinal environment for absorption, and improve oral bioavailability, which is beneficial to the long-term treatment of diabetic patients. The yeast cell wall-loaded insulin oral preparation is obtained by centrifugal freeze-drying through a self-assembly reaction.

[0050] The present invention selects natural macromolecular polysaccharide yeast cell wall with immunomodulatory effect, washes it with alkaline solution, acid solution and organic reagent to obtain a carrier for loading insulin.

[0051] The present invention provides a method for preparing the oral preparation described in the above technical solution, comprising:

[0052] Insulin is mixed with a hydrochloric acid aqueous solution to obtain a mixture, and yeast cell walls are added into the mixture to carry out a self-assembly reaction to obtain a yeast cell wall-loaded insulin oral preparation.

[0053] The present invention preferably mixes the insulin with an aqueous hydrochloric acid solution to obtain a mixture. The concentration of the aqueous hydrochloric acid solution of the present invention is not required. The pH value of the aqueous hydrochloric acid solution of the present invention is preferably 1.2 to 3.0. In a specific embodiment of the present invention, the concentration of the aqueous hydrochloric acid solution may be 1.2, 2.0, 2.8 or 3.0. The concentration of insulin in the mixture of the present invention is preferably 0.5 to 3.0 mg / mL, more preferably 1.2 to 1.6 mg / mL. After obtaining the mixture, the present invention preferably self-assembles the mixture with yeast cell walls to obtain a yeast cell wall-loaded insulin oral preparation. The mass ratio of the yeast cell wall to insulin is preferably 9:(1 to 4), more preferably 9:(1 to 2). The pH value of the solution after the yeast cell wall and the mixture are mixed is preferably 1.2 to 3.0, more preferably 2.8 to 3.0. The self-assembly method preferably includes a water bath stirring method; the present invention achieves encapsulation of yeast cell wall-loaded insulin by water bath stirring. In the present invention, the temperature of the water bath stirring is preferably 15-40°C, more preferably 25-35°C; in a specific embodiment of the present invention, the temperature of the water bath stirring can be 15, 25, 30, 35 or 40°C; the time of the water bath stirring is preferably 0.5h-6h, more preferably 2h; in a specific embodiment of the present invention, the time of the water bath stirring can be 0.5h, 2h, 3h, 4h or 6h.

[0054] After the self-assembly, a drug-loaded solution is obtained. The present invention preferably further comprises centrifuging the drug-loaded solution and discarding the supernatant to obtain a precipitate; freeze-drying the precipitate to obtain the yeast cell wall-loaded insulin oral preparation. The rotation speed of the centrifugal separation of the present invention is preferably 5000-8000rpm, more preferably 6000-7000rpm; the time of the centrifugal separation is preferably 8-15min, more preferably 10min. The freeze-drying temperature of the present invention is preferably 0-5°C, more preferably 0°C; the freeze-drying vacuum pressure is preferably 1-50Pa, more preferably 10Pa; the freeze-drying time is preferably 24-72h, more preferably 48h.

[0055] The yeast cell wall-loaded insulin oral preparation prepared by the present invention has a drug loading amount of preferably 13-16% and an encapsulation rate of preferably 90-100%.

[0056] The preparation method provided by the invention is simple, has mild reaction, and the materials and solvents used in the preparation are non-toxic, has good environmental benefits, and can be applied to factory production.

[0057] The present invention provides a medicine, which comprises a pharmaceutical active ingredient and a pharmaceutical excipient, wherein the pharmaceutical active ingredient is the yeast cell wall-loaded insulin oral preparation described in the above technical scheme or the yeast cell wall-loaded insulin oral preparation prepared by the preparation method described in the above technical scheme.

[0058] In the present invention, the pharmaceutical excipient is a pharmaceutically acceptable excipient. The present invention has no particular limitation on the type of the pharmaceutically acceptable excipient, and any excipient well known to those skilled in the art can be used.

[0059] In the present invention, the dosage form of the drug is preferably an oral preparation; the oral preparation is preferably an oral enteric-coated capsule.

[0060] The yeast cell wall of the present invention is a natural green biomacromolecule carrier, which has the advantages of high biocompatibility, small toxic side effects and low cost. Selecting a suitable carrier yeast cell wall for the treatment of specific diabetes can efficiently utilize the carrier and the drug, which is beneficial to the absorption of the drug and the treatment of the disease. The present invention selects the natural green macromolecular carrier material yeast cell wall to prepare an oral preparation loaded with insulin to improve the stability and bioavailability of insulin in the gastrointestinal tract. The oral preparation prepared by the present invention has the characteristics of small particles, large specific surface area, strong adsorption capacity, etc., which can not only enhance the convenience and compliance of patients' medication, but also reflect the physiological advantages of oral administration, and has important scientific research value and clinical significance.

[0061] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0062] Unless otherwise specified, all raw and auxiliary materials, reagents and instruments used in the present invention are well known in the art.

[0063] Example 1

[0064] 1. Take 10g of yeast and disperse it in 1L of anhydrous ethanol, stir it evenly and then perform ultra-high pressure treatment. The pressure of ultra-high pressure treatment is 100MPa, the time of one ultra-high pressure treatment is 10min, and the number of ultra-high pressure treatment is 6 times; then continue stirring for 30min to obtain a stirring liquid.

[0065] 2. After centrifugation of the stirred liquid, the precipitate is obtained, which is the yeast cell. The cell is mixed with NaOH solution, the molar concentration of the NaOH solution is 0.5 moL / L, and the volume is 1 L. After mixing, stir in a water bath at 60°C for 30 minutes to allow for sufficient reaction, and then centrifuge at 3000 rpm for 8 minutes to obtain the cell precipitate.

[0066] 3. The bacterial precipitate obtained in step 2 is placed in a 0.5 mol / L HCl solution, the pH is adjusted to 4.5 until stable, stirred in a water bath at 50°C for 30 min, and then centrifuged at 3000 rpm for 8 min to obtain a bacterial precipitate, and then the obtained precipitate is rinsed with 200 mL of acetone, the rinsing number is 1; after rinsing, it is placed at room temperature to dry naturally to obtain a yeast cell wall carrier.

[0067] 4. Dissolve insulin in a hydrochloric acid aqueous solution, the pH value of the hydrochloric acid aqueous solution is 1.2, and the concentration of insulin in the hydrochloric acid aqueous solution is 0.5 mg / mL. Slowly add the yeast cell wall obtained by drying in step 3 to the insulin hydrochloric acid aqueous solution, the mass ratio of insulin to yeast cell wall is 1:9, and then use water bath stirring to load insulin on the yeast cell wall, the water bath stirring drug loading time is 30 minutes, and the water bath stirring temperature is 15°C to obtain a self-assembled drug loading solution.

[0068] 5. The self-assembled drug-loaded solution was centrifuged at 5000 rpm for 8 min, the supernatant was discarded, and the precipitate was vacuum freeze-dried to obtain a yeast cell wall-loaded insulin oral preparation.

[0069] The content of insulin in the oral preparation of yeast cell wall loaded insulin was detected by HPLC, and the drug loading and encapsulation efficiency of insulin were calculated. The drug loading and encapsulation efficiency were 9.77% and 85.31%, respectively.

[0070] Example 2

[0071] 1. Take 15g of yeast and disperse it in 1L of anhydrous ethanol, stir it evenly and then perform ultra-high pressure treatment. The pressure of ultra-high pressure treatment is 150MPa, the time of one ultra-high pressure treatment is 15min, the number of ultra-high pressure treatment is 4 times, and then continue stirring for 2h to obtain a stirring liquid.

[0072] 2. After centrifugation of the stirred liquid, the precipitate, i.e., yeast cells, was obtained. The cells were mixed with a NaOH solution having a molar concentration of 1.5 moL / L and a volume of 1 L. After mixing, the mixture was stirred in a water bath at 70°C for 2 h to allow for sufficient reaction, and then centrifuged at 5000 rpm for 15 min to obtain a cell precipitate.

[0073] 3. The bacterial precipitate obtained in step 2 was placed in a 1 mol / L HCl solution, and the pH was adjusted to 5.0 until stable, and then stirred in a water bath at 80°C for 2 hours. Then centrifuged at 5000 rpm for 15 minutes to obtain a precipitate, and then the precipitate was rinsed with 200 mL of acetone for 2 times. After rinsing, the precipitate was placed at room temperature for natural drying to obtain a yeast cell wall carrier.

[0074] 4. Dissolve insulin in a hydrochloric acid aqueous solution, the pH value of the hydrochloric acid water is 2.0, and the insulin concentration in the hydrochloric acid aqueous solution is 3.0 mg / mL. Slowly add the dried yeast cell wall to the insulin hydrochloric acid aqueous solution, the mass ratio of insulin to yeast cell wall is 4:9, and then use a water bath to stir the yeast cell wall to load insulin. The water bath stirring drug loading time is 3 hours, and the water bath stirring temperature is 25°C to obtain a self-assembled drug loading solution.

[0075] 5. The self-assembled drug-loaded solution obtained in step 4 was centrifuged at 8000 rpm for 12 min, the supernatant was discarded, and the precipitate was freeze-dried to obtain a yeast cell wall-loaded insulin oral preparation.

[0076] The content of insulin in yeast cell wall-loaded insulin was detected by HPLC, and the drug loading and encapsulation efficiency of insulin were calculated. The drug loading and encapsulation efficiency were 11.12% and 88.56%, respectively.

[0077] Example 3

[0078] 1. Take 15g of yeast and disperse it in 1L of anhydrous ethanol, stir it evenly and then perform ultra-high pressure treatment. The pressure of ultra-high pressure treatment is 200MPa, the time of ultra-high pressure treatment is 20min, the number of ultra-high pressure treatment is 3 times, and then continue stirring for 2h to obtain a stirring liquid.

[0079] 2. After centrifugation of the stirred liquid, the precipitate, i.e., yeast cells, is obtained. The cells are then mixed with a NaOH solution having a molar concentration of 1 moL / L and a volume of 1 L. After mixing, the mixture is stirred in a water bath at 80°C for 1 hour to allow for sufficient reaction, and then centrifuged at 4000 rpm for 10 minutes to obtain a precipitate.

[0080] 3. The precipitate obtained in step 2 is placed in 1 mol / L HCl solution, the pH is adjusted to 4.8 until stable, and then stirred in a water bath at 65°C for 1 hour to react fully; the reaction solution is then centrifuged at 3000 rpm for 10 minutes to obtain a precipitate, and then the precipitate is rinsed with 200 mL of acetone for 2 times. After rinsing, the precipitate is placed at room temperature for natural drying to obtain a yeast cell wall carrier.

[0081] 4. Dissolve insulin in a hydrochloric acid aqueous solution, the pH value of the hydrochloric acid water is 2.8, and the insulin concentration in the hydrochloric acid aqueous solution is 1 mg / mL. Slowly add the yeast cell wall obtained by drying in step 3 to the insulin hydrochloric acid aqueous solution, the mass ratio of insulin to yeast cell wall is 2:9, and then use water bath stirring to load insulin on the yeast cell wall, the water bath stirring drug loading time is 4h, and the water bath stirring temperature is 35°C to obtain a self-assembled drug loading solution.

[0082] 5. The self-assembled drug-loaded solution obtained in step 4 was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the precipitate was freeze-dried to obtain a yeast cell wall-loaded insulin oral preparation.

[0083] The content of insulin in yeast cell wall-loaded insulin was detected by HPLC, and the drug loading and encapsulation efficiency of insulin were calculated. The drug loading and encapsulation efficiency were 12.34% and 91.37%, respectively.

[0084] Example 4

[0085] 1. Take 20g of yeast and disperse it in 1L of anhydrous ethanol, stir it evenly and then perform ultra-high pressure treatment. The pressure of ultra-high pressure treatment is 300MPa, the time of ultra-high pressure treatment is 30min, the number of ultra-high pressure treatment is 2 times, and then continue stirring for 2h to obtain a stirring liquid.

[0086] 2. After centrifugation of the stirred liquid, the precipitate, i.e., yeast cells, was obtained. The cells were mixed with a NaOH solution having a molar concentration of 1.5 moL / L and a volume of 1 L. After mixing, the mixture was stirred in a water bath at 100°C for 1 hour to allow for sufficient reaction, and then centrifuged at 4000 rpm for 10 minutes to obtain a precipitate.

[0087] 3. The precipitate obtained in step 2 is placed in 1 mol / L HCl solution, the pH is adjusted to 4.5 until stable, and then stirred in a water bath at 65°C for 1 hour to react fully; the reaction solution is centrifuged at 3000 rpm for 10 minutes to obtain a precipitate, and then the precipitate is rinsed with 200 mL of acetone, the rinse number is 1, and the precipitate is placed at room temperature for natural drying after rinsing to obtain a yeast cell wall carrier.

[0088] 4. Dissolve insulin in a hydrochloric acid aqueous solution, the pH value of the hydrochloric acid water is 3.0, and the insulin concentration in the hydrochloric acid aqueous solution is 2 mg / mL. Slowly add the dried yeast cell wall to the insulin hydrochloric acid aqueous solution, the mass ratio of insulin to yeast cell wall is 3:9, and then use a water bath to stir the yeast cell wall to load insulin. The water bath stirring drug loading time is 6 hours, and the water bath stirring temperature is 40°C to obtain a self-assembled drug loading solution.

[0089] 5. The self-assembled drug-loaded solution obtained in step 4 was centrifuged at 7000 rpm for 10 min, the supernatant was discarded, and the precipitate was freeze-dried to obtain a yeast cell wall-loaded insulin oral preparation.

[0090] The content of insulin in yeast cell wall-loaded insulin was detected by HPLC, and the drug loading and encapsulation efficiency of insulin were calculated. The drug loading and encapsulation efficiency were 14.15% and 94.22%, respectively.

[0091] Example 5

[0092] 1. Take 10g of yeast and disperse it in 1L of anhydrous ethanol, stir it evenly and then perform ultra-high pressure treatment. The pressure of ultra-high pressure treatment is 50MPa, the time of ultra-high pressure treatment is 5min, the number of ultra-high pressure treatment is 1, and then continue stirring for 1h to obtain a stirring liquid.

[0093] 2. After centrifugation of the stirred liquid, a precipitate, i.e., yeast cells, was obtained; the cells were mixed with a NaOH solution having a molar concentration of 1 moL / L and a volume of 1 L; after mixing, the mixture was stirred in a water bath at 85°C for 1 hour to allow for sufficient reaction, and then centrifuged at 4000 rpm for 10 minutes to obtain a precipitate;

[0094] 3. The precipitate obtained in step 2 is placed in 1 mol / L HCl solution, the pH is adjusted to 4.5 until stable, and then stirred in a water bath at 65°C for 1 hour to react; the reaction solution is centrifuged at 4000 rpm for 10 minutes to obtain a precipitate, and then the precipitate is rinsed with 200 mL of acetone, the rinse number is 1, and the precipitate is naturally dried at room temperature after rinsing to obtain a yeast cell wall carrier (i.e., yeast cell wall, referred to as Yps).

[0095] 4. Dissolve insulin in a hydrochloric acid aqueous solution, the pH value of the hydrochloric acid water is 3.0, and the insulin concentration in the hydrochloric acid aqueous solution is 1.5 mg / mL. Slowly add the dried yeast cell wall to the insulin hydrochloric acid aqueous solution, the mass ratio of insulin to yeast cell wall is 1.5:9, and then use a water bath to stir the yeast cell wall to load insulin. The water bath stirring drug loading time is 2 hours, and the water bath stirring temperature is 30°C to obtain a self-assembled drug loading solution.

[0096] 5. The self-assembled drug-loaded solution obtained in step 4 was centrifuged at 6500 rpm for 10 min, the supernatant was discarded, and the precipitate was freeze-dried to obtain a yeast cell wall-loaded insulin oral preparation, namely, yeast cell wall-loaded insulin, namely, Yps-Ins.

[0097] The content of insulin in yeast cell wall-loaded insulin was detected by HPLC, and the drug loading and encapsulation efficiency of insulin were calculated. The drug loading and encapsulation efficiency were 15.41% and 96.72%, respectively.

[0098] The following test examples are described by taking the yeast cell wall-loaded insulin oral preparation prepared in Example 5 and the analysis of yeast cell wall as examples:

[0099] Test Example 1

[0100] Transmission electron microscopy and scanning electron microscopy were used to characterize the properties of insulin, yeast cell wall-loaded insulin oral preparations, yeast cell walls, and untreated yeast. Figure 1 and Figure 2 As shown:

[0101] Figure 1 Figure a is a transmission electron microscope image of the yeast used in Example 5; Figure 1 b is a transmission electron micrograph of the yeast cell wall prepared in Example 5; Figure 1 Figure c is a transmission electron micrograph of the yeast cell wall loaded with insulin prepared in Example 5; it can be seen that a is a solid sphere for the untreated yeast. b is the hollow porous structure of the yeast cell wall, indicating that after treatment, all substances in the yeast cell are removed. The hollow structure of the yeast cell is a necessary condition for the adsorption of insulin, which is conducive to achieving a higher insulin load. c is the yeast cell wall loaded with insulin, with obvious black particles in the center, and it can be seen that insulin is successfully loaded into the yeast cell wall.

[0102] Figure 2 Figure a is a scanning electron microscope image of the yeast cell wall in Example 5; Figure 2 b is a scanning electron microscope image of insulin in Example 5; Figure 2 Figure c is a scanning electron micrograph of the yeast cell wall loaded with insulin prepared in Example 5. It can be seen that the surface of the yeast cell wall shrinks significantly after treatment, and the internal substances of the yeast cells have been successfully separated. After loading with insulin, the surface shrinkage of the yeast cell wall decreases, the volume increases significantly, and the diameter is close to 3 μm, indicating that insulin has been successfully loaded into the yeast cell wall.

[0103] Test Example 2

[0104] The preparation method of the mixture of insulin and yeast cell walls is as follows: equal amounts of insulin and yeast cell walls are weighed and placed in a container, and mixed thoroughly and evenly with a vortex mixer to obtain the mixture of insulin and yeast cell walls.

[0105] The performance of yeast cell wall loaded insulin (denoted as Yps-Ins), yeast cell wall (denoted as Yps), insulin and yeast cell wall mixture (referred to as physical mixture, physical mixture), and insulin (denoted as Ins) were characterized. The specific results are as follows Figure 3 , Figure 4 and Figure 5 shown.

[0106] Figure 3 The FTIR images of Yps-Ins, Yps, Ins and the mixture in Example 5 are shown in Table 1. -1 The peak at 1513 cm is caused by the stretching vibration peak of C=O. -1 The peaks are caused by the bending vibration of NH and the stretching vibration of CN. In the spectrum of yeast cell wall loaded insulin, since insulin enters the carrier cavity and is tightly wrapped by the carrier, no corresponding characteristic peaks of insulin appear. After loading insulin, its structural characteristics are not changed. -1 The intermolecular hydrogen bonding force was not weakened. In the physical mixing state, the characteristic peaks were similar to those of INS, but the peak shape was weakened, and the characteristic peaks of insulin enteric-coated capsule carriers did not appear, indicating that the carrier successfully loaded INS without changing its own structure.

[0107] Figure 4 The XRD patterns of Yps-Ins, Yps, Ins and the mixture in Example 5 are shown in Table 1. It can be seen that the diffraction peaks in the XRD pattern of insulin are relatively broad. The XRD spectra of yeast cell wall and yeast cell wall loaded insulin are basically the same, and there are no strong diffraction peaks. This indicates that insulin is in an amorphous state after being loaded by yeast cell wall.

[0108] Figure 5 The DSC graphs of Yps-Ins, Yps, Ins and the mixture prepared in Example 5 show that insulin shows an exothermic peak at 280°C and a weak endothermic peak at 302°C, which is the melting point of insulin. The peaks of the mixture state and insulin are similar. The DSC curves of yeast cell wall and yeast cell wall loaded insulin are similar, and the exothermic and endothermic peaks of insulin disappear, indicating that insulin has been transformed from crystalline to amorphous and successfully encapsulated in the yeast cell wall, which is consistent with the XRD results.

[0109] Figure 6 TG curves of Yps-Ins, Yps, Ins and the mixture prepared in Example 5. It can be seen that the yeast cell wall starts to lose weight at 278°C, insulin starts to lose weight significantly at 225°C, the mixture starts to lose weight at 265°C, and the yeast cell wall loaded insulin starts to lose weight at 260°C. This shows that after insulin is loaded by the yeast cell wall, the retention rate of insulin is significantly improved and the thermal stability is improved.

[0110] Test Example 3

[0111] The performance test of the oral insulin preparation loaded with yeast cell wall prepared in Example 5 was carried out as follows:

[0112] The environment in the body is extremely complex. In order to investigate the effects of different environments in the digestive system on the release of insulin from yeast cell wall-loaded insulin, dissolution experiments were conducted on insulin and yeast cell wall-loaded insulin in artificial gastric juice and artificial intestinal juice. The speed and cumulative release amount of free drugs released by the drug through the dialysis bag were tested. The specific steps of the dissolution test in artificial gastric juice and artificial intestinal juice are as follows:

[0113] Treatment 1 (yeast-loaded insulin group): Prepare 2 portions of 20 mL artificial gastric juice and 2 portions of 20 mL artificial intestinal juice, and place them in beakers respectively; place 10 mg insulin loaded with yeast cell walls in a small amount of distilled water, pour into a cellulose dialysis bag with a molecular weight cutoff of 50,000 Da, and place them in artificial gastric juice and artificial intestinal juice respectively. Place 4 beakers in a 37°C water bath at 100 rpm. Take out 1 mL of the dissolution solution at 0.083 h, 0.167 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, respectively, and add 1 mL of blank artificial intestinal juice or artificial gastric juice to the beakers after taking them out.

[0114] Treatment 2 (insulin group): Same as treatment 1, the only difference being that the yeast cell wall-loaded insulin (insulin 10 mg) was replaced with only 10 mg insulin.

[0115] Insulin and yeast cell wall-loaded insulin were taken out from artificial gastric juice and artificial intestinal juice at different time points in treatment 1 and treatment 2, and the insulin content was detected by HPLC after passing through a 0.22 μm filter membrane. The cumulative release percentage (%) was calculated and the drug release curve was drawn. Figure 7 .

[0116] Figure 7 Figure a shows the dissolution curves of insulin and yeast cell wall-loaded insulin in artificial gastric juice. Figure 7 Figure b shows the dissolution curves of insulin and yeast cell wall-loaded insulin in artificial intestinal fluid. The results show that the cumulative release rate of insulin in artificial gastrointestinal fluid is higher than that of yeast cell wall-loaded insulin, indicating that the yeast cell wall protects insulin from being released in the stomach environment, plays a protective and sustained-release role, prevents insulin from being released prematurely, and safely reaches the small intestine and is effectively released. This behavior is conducive to the application of oral drugs. The reason for the sustained release is mainly because insulin is wrapped inside by the yeast cell wall. Therefore, the yeast cell wall is a good carrier for oral drugs, which increases the dissolution of the original drug and reduces the side effects of the drug. At this time, the yeast cell wall-loaded insulin oral preparation overcomes the biochemical barrier of the gastrointestinal tract and enters the mucus layer of the small intestine, where it is released and transported to the blood circulation.

[0117] Test Example 4

[0118] The in vivo functional test of the oral insulin preparation loaded with yeast cell wall prepared in Example 5 was carried out as follows:

[0119] The specific steps of the in vivo glucose-lowering test in diabetic rats are as follows:

[0120] Preparation method of sodium citrate buffer: weigh 2.1g citric acid and 2.94g sodium citrate into two beakers, add 100mL deionized water to each beaker, and sonicate until completely dissolved. Mix the citric acid solution and the sodium citrate solution in a volume ratio of 1:1 to obtain a sodium citrate buffer solution (pH = 4.5), and store it at 4°C away from light.

[0121] Streptozotocin (STZ) powder was dissolved in the sodium citrate buffer prepared as above to prepare a STZ solution with a concentration of 10 g / L, and the solution was kept in an ice bath and protected from light.

[0122] Twelve male and female rats were selected and, after fasting for 12 hours, injected with the prepared STZ solution at a dose of 60 mg / kg. One day later, each rat was injected with STZ solution again in the same manner and at the same dose.

[0123] 72 hours after the second injection of STZ solution, the fasting blood glucose level of the rats was measured. If the fasting blood glucose level was ≥11.6mmol / L, it indicated that the type 1 diabetic rat model was successfully established and it could be determined to be a diabetic animal model.

[0124] Seven days after the diabetic model was established in male and female rats, two parallel groups were set up for the male and female rats with successful modeling (fasting blood glucose concentration greater than 11.6mmol / L), that is, the female rats were randomly divided into 4 groups, namely, group A1, group B1, group C1 and group D1, with 3 rats in each group; the male rats were randomly divided into 4 groups, namely, group A2, group B2, group C2 and group D2, with 3 rats in each group. Group A1 and A2 were insulin groups (50IU / kg); Group B1 and B2 were yeast cell wall-loaded insulin groups (insulin dosage 50IU / kg); Group C1 and C2 were yeast cell wall-loaded insulin + gastrointestinal absorption enhancer (ethylenediaminetetraacetic acid) groups (insulin dosage 50IU / kg), and the application dosage of ethylenediaminetetraacetic acid was 16mg / kg; Group D1 and D2 were tail vein injection insulin groups (5IU / kg).

[0125] Rats in group A1, A2, B1, B2, C1, C2, D1 and D2 were fasted for 12 h before administration and had free access to water during this period. The administration was by gavage. Rats in group A1, A2, B1, B2, C1, C2, D1 and D2 were administered at the same time. Blood was collected from the medial angular venous plexus of rats at each time point (0.5, 1, 2, 3, 4, 6, 8, 12, 24 and 36 h), and the blood glucose level of rats within 36 h after administration was measured using a blood glucose meter.

[0126] Results Figure 8 and Fig. 9 , Figure 8 This is a graph showing the blood sugar lowering effects of the insulin group, yeast cell wall-loaded insulin group, yeast cell wall-loaded insulin + gastrointestinal absorption enhancer (ethylenediaminetetraacetic acid) group and insulin injection group on diabetic male rats within 36 hours. Fig. 9 The following is a graph showing the hypoglycemic effect of insulin group, yeast cell wall loaded insulin group, yeast cell wall loaded insulin + gastrointestinal absorption enhancer group (ethylenediaminetetraacetic acid) group and insulin injection group on diabetic female rats. The original insulin group lost its drug activity due to degradation caused by the gastrointestinal environment in the body, and had no obvious hypoglycemic effect. Due to the differences between male and female individuals, male rats absorb and metabolize faster than female rats. The blood sugar levels of the tail vein injection insulin group in male and female rats dropped rapidly within 30min and 2h, and the blood sugar levels dropped to 4.3 and 4.9mmoL / L as low as 4.3 and 4.9mmoL / L, but then the blood sugar levels began to rise rapidly, indicating that although subcutaneous injection can quickly restore blood sugar levels to normal levels, it cannot maintain long-term stability of blood sugar in the body. This is mainly due to the short half-life of injected insulin in the body. In addition, subcutaneous injection of insulin may cause drastic fluctuations in blood sugar levels, causing potential harm to the human body, and cannot meet the needs of diabetic patients for long-term stable blood sugar levels. However, for the yeast cell wall loaded insulin group, insulin can overcome multiple barriers in the body, achieve a slow release effect and be effectively absorbed by the body, and the blood glucose levels of male and female mice dropped to 8.0 and 6.7mmoL / L at 24h and 36h, respectively, which are close to the normal range. In the yeast cell wall loaded insulin + gastrointestinal absorption enhancer (ethylenediaminetetraacetic acid) group, the application amount of ethylenediaminetetraacetic acid was 16mg / kg, and the blood glucose levels of male and female mice at 24h and 36h also dropped to 7.8 and 4.9mmoL / L, respectively, with a significant hypoglycemic effect. This shows that in the complex environment of the gastrointestinal tract, most of the insulin is protected by the yeast cell wall, which has a certain resistance to gastrointestinal proteases, helps to overcome the obstruction of the intestinal mucosa, prolongs the residence time in the intestine, and thus promotes the absorption of intestinal epithelial cells, achieving the purpose of slow release in the intestine and efficient absorption by the blood, which is consistent with the in vitro dissolution effect, indicating that yeast cell wall loaded insulin has potential value in the oral insulin delivery system for diabetic patients.

[0127] Test Example 5

[0128] The yeast cell wall-loaded insulin oral preparation prepared in Example 5 was subjected to in vivo functional test and bioavailability test. The specific steps are as follows:

[0129] Male diabetic rats with an average weight of 180-200 g were fasted overnight before the experiment and had free access to water. Twelve male rats were randomly divided into 4 groups, 3 rats in each group. The 4 groups were:

[0130] Insulin group: Rats were intragastrically administered with insulin at a dose of 50 IU / kg.

[0131] Yeast cell wall loaded insulin group: Rats were gavaged with insulin at a dose of 50 IU / kg; the drug administered was yeast cell wall loaded insulin.

[0132] Yeast cell wall loaded insulin + gastrointestinal absorption enhancer group: rats were gavaged at a dose of 50 IU / kg of insulin; the drug for gavage was yeast cell wall loaded insulin + gastrointestinal absorption enhancer; the gavage amount of EDTA was 16 mg / kg.

[0133] Insulin injection group: Insulin was injected into the tail vein of rats at a dose of 5 IU / kg.

[0134] For the above four groups, blood (600-800 μL) was collected from the anesthetized rats at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h and 36 h after administration. The blood samples were centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant (serum) was transferred to a new 1.5 mL centrifuge tube for quantitative determination of insulin content in plasma. Fig.10 .

[0135] Fig.10 The insulin blood concentration diagram of the insulin group, yeast cell wall loaded insulin group, yeast cell wall loaded insulin + gastrointestinal absorption enhancer (ethylenediaminetetraacetic acid) group and insulin injection group in rats. After calculation, the AUC of oral administration of insulin original drug (0-36h) =41.862μIU / mL*h, AUC of yeast cell wall loaded insulin (0-36h) =409.022μIU / mL*h, AUC of yeast cell wall loaded insulin + gastrointestinal absorption enhancer (ethylenediaminetetraacetic acid) (0-36h) =218.699μIU / mL*h, AUC of injected insulin (0-36h)=91.749μIU / mL*h, considering that the oral dose is 50IU / kg and the injection dose is 5IU / kg, the absolute bioavailability is calculated by the formula: Fabs=(AUCT*Div) / (AUCiv*DT)×100%, where AUC represents the area under the drug-time curve, Div and DT represent the doses of intravenous injection and test preparation, respectively. The absolute bioavailabilities of oral original insulin, yeast cell wall-loaded insulin and yeast cell wall-loaded insulin + gastrointestinal absorption enhancer (ethylenediaminetetraacetic acid) are 4.56%, 44.6% and 23.9%, respectively. The oral bioavailability of yeast cell wall-loaded insulin is significantly improved compared with original insulin.

[0136] Test Example 6

[0137] The in vivo functional test of the oral insulin preparation loaded with yeast cell wall prepared in Example 5 was carried out as follows:

[0138] In vivo safety evaluation, the specific steps are as follows:

[0139] Insulin group (INS): Insulin was administered continuously for 15 days, and the main organs of mice were collected for histopathological examination.

[0140] Yeast cell wall-loaded insulin oral preparation group (YPS-INS): Yeast cell wall-loaded insulin oral preparation was administered continuously for 15 days, and the main organs of mice were collected for histopathological examination to evaluate the in vivo biosafety of yeast cell wall-loaded insulin oral preparation.

[0141] Blank group (control): The mice were orally gavaged with normal saline for 15 consecutive days, and the main organs of the mice were collected for histopathological examination as the normal control group.

[0142] The main organs of mice were collected for histopathological examination, and the operation process was specifically divided into two parts: paraffin embedding and H&E staining.

[0143] Paraffin embedding: (1) Tissue fixation: The tissue specimens were fully fixed with 10% formaldehyde. (2) Flowing water rinsing: The fixed tissue specimens were rinsed in flowing water for 30 minutes to remove excess fixative. (3) Block trimming: The tissues were cut into blocks of appropriate size. (4) Dehydration: The tissues were immersed in 70%, 80%, 90% and 100% ethanol for 1 hour each to gradually dehydrate the tissues. (5) Transparentization: The tissues were then immersed in xylene: ethanol (v / v = 1:1) and pure xylene for 20 minutes each to make the tissues transparent. (6) Wax immersion: The transparent tissues were placed in the melted paraffin (I), paraffin (II) and pure paraffin for 1.5 hours each. Paraffin (I) consisted of xylene and paraffin, and the volume ratio of xylene to paraffin was 2:1; paraffin (II) consisted of xylene and paraffin, and the volume ratio of xylene to paraffin was 1:1. (7) Embedding: The immersed tissue is embedded into blocks in sequence for subsequent sectioning.

[0144] H&E staining: (1) Sectioning: Cut the conventional paraffin-embedded tissue into sections with a thickness of 6 μm. (2) Processing: The sections are subjected to xylene twice for 5 min each, anhydrous ethanol twice for 5 min each, 90%, 80%, and 70% ethanol for 5 min each, and distilled water for 5 min. (3) Staining: Stain with hematoxylin for 5 min, differentiate in hydrochloric acid ethanol solution for 20 seconds, and eosin staining for 5 min. (4) Cleaning: Soak in distilled water for 5 min, 70% and 80% alcohol for 5 min each, anhydrous ethanol twice for 5 min each, and xylene twice for 5 min each. (5) Sealing: Finally, seal the sections in neutral gum and take photos under a microscope at 200 times.

[0145] Fig.11 The following are H&E staining images of sections of the main organs (heart, liver, spleen, intestine and kidney) of mice in the blank group, insulin group and yeast cell wall loaded insulin group. Compared with the blank group, the insulin group and yeast cell wall loaded insulin showed a clear boundary between the cytoplasm and the nucleus of all organs, and the cells were arranged compactly and regularly. No obvious organ damage was found, and there was no significant difference compared with the blank group. This further verified that the yeast cell wall loaded insulin oral enteric-coated capsule is a relatively safe and non-toxic drug delivery system, which has no toxic side effects on tissues in the body and can also improve the hypoglycemic effect, providing a greater possibility for future clinical use.

[0146] In summary, the yeast cell wall-loaded insulin proposed in the present invention has high drug loading capacity and encapsulation efficiency, thereby improving the bioavailability of insulin.

[0147] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An oral preparation, characterized in that The invention comprises a carrier and an active drug loaded in the carrier; the active drug comprises insulin; and the carrier comprises a yeast cell wall.

2. The oral preparation according to claim 1, characterized in that The mass content of insulin in the oral preparation is 13% to 16%.

3. The oral preparation according to claim 1 or 2, characterized in that The particle size of the oral preparation is 1 to 3 μm; the dosage form of the oral preparation includes oral enteric-coated capsules.

4. The oral preparation according to claim 1, characterized in that The oral preparation further comprises functional excipients; the functional excipients comprise gastrointestinal absorption enhancers.

5. The method for preparing the oral preparation according to any one of claims 1 to 4, characterized in that: include: Insulin is mixed with a hydrochloric acid aqueous solution to obtain a mixture, and yeast cell walls are added into the mixture to carry out a self-assembly reaction to obtain a yeast cell wall-loaded insulin oral preparation.

6. The preparation method according to claim 5, characterized in that: The preparation method of the yeast cell wall comprises: The yeast and the first organic solvent are mixed and then subjected to ultra-high pressure treatment to obtain yeast cells; The yeast cells are sequentially subjected to alkali treatment, acid treatment and washing with a second organic solvent to obtain yeast cell walls; The ultra-high pressure temperature is 25-30° C., the pressure is 50-300 MPa, the ultra-high pressure treatment time is 5-30 minutes, and the ultra-high pressure treatment times are 1-6 times.

7. The preparation method according to claim 6, characterized in that: The first organic solvent includes anhydrous ethanol; the second organic solvent includes acetone.

8. The preparation method according to claim 5, characterized in that: The mass ratio of the yeast cell wall to insulin is 9:(1-4).

9. The preparation method according to claim 5, characterized in that: The self-assembly method includes a water bath stirring method; the water bath stirring temperature is 15 to 40° C., and the water bath stirring time is 30 minutes to 6 hours.

10. A drug, characterized in that The drug comprises active pharmaceutical ingredients and pharmaceutical excipients, and the active pharmaceutical ingredients comprise the oral preparation according to any one of claims 1 to 4 or the oral preparation prepared by the preparation method according to any one of claims 5 to 9.

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