Composition comprising glutamine source substance and use thereof

Abstract

The present invention provides a composition comprising a glutamine source substance and a vesicle, a preparation method therefor, and use thereof. The composition enables effective oral delivery of glutamine, has significant oral, gastric, and / or intestinal protection and repair functions, can be used for ameliorating bad conditions of the oral cavity, the stomach, and / or the intestinal tracts, is non-toxic, safe, and effective, and greatly improves patient compliance.

Description

Compositions containing glutamine sources and uses thereof Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composition comprising a glutamine source substance and vesicles, and a preparation method and application thereof. Background Art

[0002] Glutamine (Gln), whose chemical name is 2-amino-4-carbamoylbutyric acid, is a conditionally essential amino acid for the human body. It has the functions of maintaining the acid-base balance in the body, maintaining the normal structure and function of the small intestinal mucosa, maintaining the reserve of antioxidants in tissues and enhancing immune response.

[0003] Glutamine has low solubility and is unstable in aqueous solutions. It can generate toxic pyroglutamic acid and ammonia under heating and sterilization conditions. Alanyl-glutamine (Ala-Gln) and glycyl-glutamine (Gly-Gln) are important dipeptide derivatives of glutamine (Gln). Ala-Gln and Gly-Gln have solubilities of 586 g / L and 154 g / L, respectively, in water. They exhibit excellent thermal stability and can be stored at room temperature for two years.

[0004] Ala-Gln is primarily available as a drug called "Alanyl-Glutamine Injection." Its indications include parenteral nutrition for patients requiring glutamine supplementation, including those with catabolism and hypermetabolism. Ala-Gln and Gly-Gln, combined with other nutritional supplements, form a compound injection for parenteral nutrition. Summary of the Invention

[0005] The inventors unexpectedly discovered during their research that loading glutamine dipeptide into vesicles, such as extracellular vesicles, can achieve effective oral administration of glutamine, and can be used to protect the oral cavity, stomach and / or intestines, and improve adverse health conditions of the oral cavity, stomach and / or intestines, such as oral ulcers, gastric mucosal damage such as gastric ulcers, and ulcerative colitis.

[0006] The present invention provides a composition comprising a glutamine source material and vesicles, a preparation method thereof and an application thereof.

[0007] In a first aspect, the present invention provides a composition comprising a glutamine source and vesicles, wherein at least a portion of the glutamine source is loaded in the vesicles.

[0008] As used herein, the term "glutamine source" refers to a substance that can be broken down in the body to provide glutamine.

[0009] As used herein, the term "vesicle" refers to a small, enclosed, sac-like structure surrounded by at least one lipid membrane, which is used to store, digest, and transport substances. Examples of "vesicles" include, but are not limited to, liposomes, lipid particles, and extracellular vesicles.

[0010] As used herein, the term "loading" refers to the encapsulation of a glutamine source within the lumen of a vesicle, such as an extracellular vesicle.

[0011] As used herein, the terms "composition comprising a glutamine source and vesicles", "composition comprising a glutamine source and vesicles such as extracellular vesicles" and "composition in which vesicles are loaded with a glutamine source" are used interchangeably herein and all refer to a composition in which a glutamine source is loaded into a vesicle.

[0012] In one embodiment, the glutamine source is selected from glutamine polypeptide or modified glutamine.

[0013] The term "glutamine polypeptide" as used herein refers to a polypeptide derivative formed by glutamine and other amino acids. For example, the amino acids are the 20 amino acids that make up proteins in living organisms, including glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0014] The term "modified glutamine" as used herein refers to the addition of functional groups to glutamine, including but not limited to acetylation, alkylation, such as methylation, etc.

[0015] In one embodiment, the glutamine polypeptide is glutamine dipeptide.

[0016] The term "glutamine dipeptide" as used herein refers to a dipeptide formed by the dehydration condensation of glutamine and other amino acids. For example, alanyl-glutamine (Ala-Gln) is formed by the dehydration condensation of glutamine and alanine, and glycyl-glutamine (Gly-Gln) is formed by the dehydration condensation of glutamine and glycine. These dipeptides can be degraded by dipeptidases in the body and release glutamine.

[0017] In one embodiment, the glutamine dipeptide is selected from alanyl-glutamine or glycyl-glutamine; preferably, the concentration of alanyl-glutamine is 1 mg / mL-586 mg / mL, such as 1 mg / mL-100 mg / mL, 1 mg / mL-200 mg / mL, 1 mg / mL-300 mg / mL, 1 mg / mL-400 mg / mL, 1 mg / mL-500 mg / mL, preferably, for example, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL; the concentration of glycyl-glutamine is 1 mg / mL-154 mg / mL, such as 1 mg / mL-10 mg / mL, 1 mg / mL-20 mg / mL, 1 mg / mL-30 mg / mL, 1 mg / mL-40 mg / mL, 1 mg / mL-50mg / mL, 1mg / mL-60mg / mL, 1mg / mL-70mg / mL, 1mg / mL-80mg / mL, 1mg / mL-90mg / mL, 1mg / mL-100mg / mL, 1mg / mL-110mg / mL, 1mg / mL-120mg / mL, 1mg / mL-130mg / mL, 1mg / mL-140mg / mL, 1 mg / mL-150 mg / mL, preferably such as 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL.

[0018] In one embodiment, the vesicles are selected from extracellular vesicles.

[0019] The term "extracellular vesicles (EVs)" as used herein refers to double-membrane vesicles that are shed from the cell membrane or secreted by cells, or vesicles with the same structure, with diameters ranging from 40nm to 1000nm. The main forms are microvesicles (MVs) and exosomes (Exs). Extracellular vesicles are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, and breast milk). They carry a variety of cell-derived proteins, lipids, DNA, mRNA, miRNA, etc., and participate in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation.

[0020] In one embodiment, the extracellular vesicles are extracellular vesicles from animal milk.

[0021] The term "animal milk" used herein refers to the secretions of the mammary glands of female mammals, including but not limited to cow's milk, goat's milk, horse's milk, camel's milk, etc.

[0022] In one embodiment, the animal milk is selected from cow's milk or goat's milk.

[0023] In a second aspect, the present invention provides a method for preparing the composition comprising a glutamine source and vesicles, comprising:

[0024] Step 1, mixing a liquid containing vesicles with a glutamine source;

[0025] Step 2, incubating the mixture obtained in step 1; and

[0026] Step 3: Ultrasonicate the mixture obtained in step 2.

[0027] In one embodiment, the glutamine source material may be dissolved in a solution and then mixed with the vesicles, or the glutamine source material powder may be added to the vesicle liquid and mixed.

[0028] In one embodiment, the incubation in step 2 is incubating the mixture obtained in step 1 at 20° C. to 65° C. for 10 minutes to 5 hours.

[0029] In one embodiment, the sonication in step 3 is performed by sonicating the mixture obtained in step 2 at a frequency of 10 kHz to 60 kHz or a power of 5 W to 300 W at regular intervals. Ultrasound causes the extracellular vesicles to repeatedly rupture and reassemble, thereby encapsulating the glutamine source into the extracellular vesicles. The glutamine source is generally unable to penetrate the extracellular vesicle membrane and is loaded into the extracellular vesicles.

[0030] In a specific embodiment, step 2 and step 3 can be performed simultaneously. Specifically, the ultrasonication time is 1 to 60 seconds, with an interval of 1 to 30 minutes, until the incubation is completed to obtain a composition solution.

[0031] The composition solution prepared by incubation and ultrasonication can be diluted to a desired concentration for use.

[0032] In a third aspect, the present invention provides a method for improving poor oral, gastric and / or intestinal health conditions, comprising administering the composition comprising a glutamine source and vesicles or the composition prepared by the method to a subject in need thereof.

[0033] The term "ill health condition" as used herein refers to abnormal or damaged functions of the oral cavity, stomach or intestines, including, for example, oral ulcers, gastric mucosal damage (such as gastric ulcers) and ulcerative colitis.

[0034] The present invention also provides use of the composition or the composition prepared by the method in protecting the oral cavity, stomach and / or intestine.

[0035] In a fourth aspect, the present invention provides a composition comprising the composition comprising a glutamine source and vesicles according to the present invention or a composition prepared by the method.

[0036] In one embodiment, the composition comprising a glutamine source and vesicles such as extracellular vesicles of the present invention is in an oral form, such as an oral solution, a beverage, a syrup, a tablet, a soft capsule, a capsule, a powder, and the like.

[0037] The vesicle-loaded glutamine source composition prepared in the present invention achieves effective oral administration of the glutamine source and has significant oral, gastric and / or intestinal protective and repair functions. It can be used to improve adverse oral, gastric and / or intestinal health conditions, is non-toxic, safe and effective, and greatly improves patient compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0039] Figure 1 is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) diagram of milk extract protein; the extract contains whey protein, and the molecular weight of whey protein is approximately in the range of 10-100 kDa, among which the molecular weight of α-lactalbumin is 66 kDa, the molecular weight of β-lactoglobulin is 18 kDa, and the molecular weight of κ-lactoglobulin is 10 kDa. As can be seen from the figure, there are clear protein bands in the milk extract.

[0040] FIG2 is a particle size distribution diagram (DLS) of a solution of a milk extract-loaded alanyl-glutamine composition, wherein the particle size of the composition is measured using a dynamic light scattering instrument, and the average particle size is 155.2 nm, indicating nanoparticles.

[0041] Figure 3 shows the results of an animal test evaluating the auxiliary protective function of a milk-rich extracellular vesicle extract-loaded alanyl-glutamine composition in gastric mucosal injury, wherein Figure 3 (A) shows stomach photos of animals in different groups; and Figure 3 (B) shows bleeding point scores for acute alcohol injury in different groups.

[0042] Figure 4 shows the experimental results of a milk-rich extracellular vesicle extract-loaded alanyl-glutamine composition in the treatment of 3.5% DSS (dextran sulfate sodium) ulcerative colitis in mice, wherein Figure 4 (A) is a weight evaluation diagram of animals in different groups; Figure 4 (B) is a colon length evaluation diagram of mice in different groups; and Figure 4 (C) is a colon photograph of mice in different groups.

[0043] Figure 5 shows the results of an animal test evaluating the protective function of a goat milk-rich extracellular vesicle extract loaded with glycylglutamine composition in treating 4% DSS enteritis, wherein Figure 5 (A) is a graph evaluating the weight of animals in different groups; and Figure 5 (B) is a graph evaluating the colon length of mice in different groups.

[0044] FIG6 is a photograph of oral ulcers in rats in one embodiment, and photographs of the model group and the preparation group from day 1 to day 6.

[0045] FIG7 is a statistical diagram of the maximum area of ​​oral ulcers in rats in one embodiment, including comparisons of the model group, extract group, raw material group, and preparation group from day 1 to day 6 (n=8).

[0046] FIG8 is a graph showing the results of detecting the inflammatory factor TNF-a in the serum of different groups of rats with oral ulcers on the 7th day after orbital venous blood sampling.

[0047] FIG9 is a HE staining image of paraffin sections of oral mucosal tissues of different groups of rats with oral ulcers on the 7th day in one embodiment. DETAILED DESCRIPTION

[0048] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0049] The present invention discloses a method for preparing a composition comprising a glutamine source and vesicles. Those skilled in the art can refer to the contents herein and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to test and apply the technology of the present invention.

[0050] The present invention will be further described below in conjunction with the embodiments:

[0051] Example 1 Preparation of a milk extracellular vesicle extract-loaded alanyl-glutamine composition

[0052] See the literature Benmoussa, Abderrahim, et al. "Isolating Multiple Extracellular Vesicles Subsets, Including Exosomes and Membrane Vesicles, from Bovine Milk Using Sodium Citrate and Differential Ultracentrifugation." BIO-PROTOCOL 10.11 (2020), using the citric acid precipitation method (the dispersant is 2% histidine, and the added amount accounts for 6.7% of the total volume), and filtering with a 1 μm filter membrane to obtain a milk extract rich in extracellular vesicles, and using a tangential flow filtration hollow fiber column (pore size 500KD) to concentrate 3 times (900mL is concentrated to 300mL) as the solution to be loaded; weigh 60g of alanyl-glutamine, mix it thoroughly with the above-mentioned milk extract concentrate (solution to be loaded), and adjust the volume to 200mL, and the final alanyl-glutamine concentration is 300mg / mL; incubate at 37°C for 1 hour, use an ultrasonic power of 200W or an ultrasonic frequency of 35kHZ, an ultrasonic time of 20 seconds, and ultrasonicate once every 5 minutes until the incubation is completed.

[0053] A milk extract solution loaded with alanyl-glutamine (300 mg / mL) can be diluted with the milk extract solution as needed;

[0054] The protein SDS-PAGE of the milk extract is shown in FIG1 ; the particle size distribution of the composition loaded with alanyl glutamine milk extract was measured by dynamic light scattering (DLS) as shown in FIG2 . The solution is a nano solution with an average particle size of about 155 nm.

[0055] Example 2 Animal test to evaluate the auxiliary protective function against gastric mucosal injury

[0056] Animal experiments were conducted using an anhydrous ethanol-induced acute gastric mucosal injury model.

[0057] Healthy male SD rats, weighing 180-220 grams, were selected, with eight rats per group. The animals were randomly divided into three dose groups (low dose 500 mg / kg, medium dose 1000 mg / kg, and high dose 1500 mg / kg, calculated as alanyl-glutamine per animal body weight): a normal control group (blank control), a model group, an extract group (milk), a group receiving 500 mg / kg of the raw material prepared in purified water (alanyl-glutamine / animal body weight), a group receiving 1000 mg / kg of the raw material prepared in purified water (alanyl-glutamine / animal body weight), and a test sample (the alanyl-glutamine-loaded milk extract prepared in Example 1). Six days after oral administration of the sample to each dose group, all animals were strictly fasted for 24 hours (water was not allowed), and the test substance was also prohibited during this period. Except for the blank control, all animals in the experimental groups were given 1.0 ml of anhydrous ethanol. The animals were killed after 1 hour, the complete stomach was exposed, the pylorus was ligated, and an appropriate amount of 10% formaldehyde solution was perfused for fixation for 20 minutes. Then the stomach was cut open along the greater curvature, the stomach contents were washed, the gastric mucosa was unfolded, and the length and width of the bleeding point or bleeding band were measured with a vernier caliper under a stereoscopic dissection microscope or with the naked eye. Because the severity of the injury represented by the width is much greater than the length, double points are added. The scoring criteria are shown in the table below. This animal experiment and evaluation method refer to "Health Food Function Test and Evaluation Method (2023 Edition), Auxiliary Protection of Gastric Mucosa Test Method: 1.4.1 Anhydrous Ethanol Model of Acute Gastric Mucosal Injury p128-129". Table 1 Scoring criteria for naked eye observation of acute alcohol injury

[0058] The total score results of the above groups are shown in the table below. Table 2 Total score results of acute alcohol injury by naked eye observation in each group

[0059] Representative gastric dissections from each animal experiment group are shown in Figure 3(A), and the total score of acute alcohol damage in each experimental group as observed by naked eye is shown in Figure 3(B). The results show that at the same dose (500 mg / kg alanyl-glutamine), compared with the model group (total score of 157) and the raw material group (total score of 85), the low-dose group of the milk extract-loaded alanyl-glutamine composition (500 mg / kg) (total score of 0) showed excellent gastric mucosal protection. This indicates that milk extract-loaded alanyl-glutamine has a protective effect on the gastric mucosa that significantly exceeds that of free alanyl-glutamine, and can prevent gastric mucosal damage.

[0060] Example 3 Treatment of ulcerative colitis in DSS (dextran sulfate sodium) mice

[0061] Forty C57 male mice (9 weeks old) were ordered. Each mouse was labeled and weighed, and five mice were housed in each cage. Four mice were set up as a normal control group and received no treatment. The remaining 36 mice were treated with 3.5% DSS to establish an animal colitis model. Reference: Das S, Batra SK, Rachagani S. Mouse Model of Dextran Sodium Sulfate (DSS)-induced Colitis[J]. Bio-Protocol, 2017, 7: 2515. DOI: 10.21769 / BioProtoc.2515. The mice were weighed and recorded every day starting from day 0. The weight of the mice began to decrease on the fourth day. The weight of the mice was recorded and divided into 4 groups, including a model group fed with normal saline (normal saline group) (8 mice, 1 mouse was discarded because its weight did not meet the requirements), an extract group, a raw material group prepared with purified water (alanyl-glutamine 1000 mg / kg animal body weight), and a preparation group (milk extract prepared in Example 1 loaded with alanyl-glutamine, calculated as 1000 mg / kg animal body weight). The latter three groups each had 9 mice.

[0062] The mice were dosed daily and their weights were recorded until day 13. The results are shown in Figure 4(A). In the 3.5% DSS-treated mice with enteritis, one mouse died on day 12 in the model group. Compared to the model group, the milk extract group showed a slight increase in weight and no deaths, indicating improved survival. The 1000 mg / kg alanyl-glutamine group also improved the weight loss trend in mice, and the milk extract composition loaded with alanyl-glutamine significantly improved the weight loss trend in mice.

[0063] On the 13th day, all mice were killed, dissected and the colons were removed. The changes in colon length of mice in different groups were compared. The results are shown in the table below. Table 3 Statistical results of colon length of each group in the DSS mouse ulcerative colitis test

[0064] As can be seen from Figures 4(B), 4(C) and Table 3, compared with the average colon length of 4.7 cm in the normal saline group (model group), the average colon length of the mice in the milk extract group was slightly increased to 4.9 cm, the average colon length of the mice in the alanyl-glutamine raw material group (1000 mg / kg) was significantly increased to 5.4 cm, and the average colon length of the mice in the composition group loaded with alanyl-glutamine milk extract (1000 mg / kg) was 5.7 cm, the same as the average length of the normal control group, indicating that the preparation group had the most significant therapeutic effect on ulcerative colitis in mice.

[0065] Example 4 Preparation of a goat milk extracellular vesicle extract-loaded glycylglutamine composition

[0066] References Mukhopadhya, A., J. Santoro, and L. O'Driscoll. "Extracellular vesicle separation from milk and infant milk formula using acid precipitation and ultracentrifugation." STAR protocols 2.4(2021):100821, take acetic acid precipitate (dispersant is 2% histidine, and the added amount accounts for 6.7% of the total volume) to obtain a goat milk extract rich in extracellular vesicles, and use a tangential flow filtration hollow fiber column (pore size 500KD) to concentrate it 2 times (1000mL is concentrated to 500mL) as the loaded solution; weigh 45g of glycylglutamine, mix it thoroughly with the above-extracted goat milk extracellular vesicle extract concentrate and adjust the volume to 300mL, and the final glycylglutamine concentration is 150mg / mL; incubate at 37°C for a total of 1 hour, use an ultrasonic power of 80W, an ultrasonic time of 10 seconds, and ultrasonicate once every 10 minutes until the incubation is completed, or use an ultrasonic frequency of 35kHz, an ultrasonic time of 20 seconds, and ultrasonicate once every 5 minutes until the incubation is completed.

[0067] The goat milk extract solution loaded with glycylglutamine (150 mg / mL) can be diluted with the goat milk extract solution before use as needed.

[0068] Example 5 Treatment of ulcerative colitis in DSS (dextran sulfate sodium) mice

[0069] A total of 47 C57 male mice (8 weeks old) were used. Each mouse was labeled and weighed, and 5 mice were housed in each cage. Five mice served as a normal control group and were not treated. The remaining 42 mice were treated with 4% DSS to establish an animal colitis model. Reference: Das S, Batra SK, Rachagani S. Mouse Model of Dextran Sodium Sulfate (DSS)-induced Colitis[J]. Bio-Protocol, 2017, 7: 2515. DOI: 10.21769 / BioProtoc.2515. The mice were weighed and recorded every day starting from day 0. The weight of the mice began to decrease on the fourth day. The weight of the mice was recorded and divided equally into 7 groups, including a model group fed with normal saline, a control drug group (mesalazine enteric-coated tablets suspension 500 mg / kg), an extract group, a raw material group prepared with purified water (glycylglutamine 1000 mg / kg animal body weight), a low-dose group of the goat milk extract loaded with glycylglutamine prepared in Example 4 (measured at 250 mg / kg of glycylglutamine / animal body weight), a medium-dose group (measured at 500 mg / kg of glycylglutamine / animal body weight), and a high-dose group (measured at 1000 mg / kg of glycylglutamine / animal body weight).

[0070] The body weight of mice was recorded every day until the 12th day of drug administration. As shown in FIG5(A), in the 4% DSS mouse enteritis model experiment, one mouse died on the 8th day and one on the 10th day in the model group, for a total of two deaths; the graphical statistics only count up to the 9th day;

[0071] In the control group (mesalazine enteric-coated tablets suspension 500 mg / kg), two mice died consecutively on the 8th day, indicating that mesalazine enteric-coated tablets suspension has obvious gastrotoxicity; the statistical data in the graph only covers the 7th day;

[0072] In the extract group (goat milk rich in extracellular vesicle extract), one animal died on the 10th day, indicating that the modeling was more severe.

[0073] No mice died in the raw material group and the low-, medium-, and high-dose groups of the composition preparation, indicating that glycylglutamine and its goat milk extract composition are safe and non-toxic and can significantly improve the survival ability of mice;

[0074] Comparing on the 8th day, the body weight curves of the three groups of the combination preparation (low dose 250 mg / kg, medium dose 500 mg / kg, high dose 1000 mg / kg) were significantly higher than those of the raw material group (1000 mg / kg), indicating that the goat milk extract loaded with glycylglutamine can significantly improve the absorption of glycylglutamine in the intestine, improve the nutrition of mice and increase their weight.

[0075] On the 12th day, all mice were killed, dissected and the colons were removed. The changes in colon length of mice in different groups were compared. The results are shown in the table below. Table 4 Statistical results of colon length of each group in the DSS mouse ulcerative colitis test

[0076] As can be seen from Figure 5(B) and Table 4, compared with the average colon length of 4.30 cm in the normal saline group (model group) and the average colon length of 4.36 cm in the goat milk extract group, the average colon length of the mice in the glycylglutamine raw material group (1000 mg / kg) increased (4.73 cm), and all composition groups showed significant improvement, especially the average colon length of the medium-dose group (500 mg / kg) was 4.87 cm, and the average colon length of the high-dose group (1000 mg / kg) was 4.97 cm, which were significantly longer than the normal saline group, the extract group and the raw material group, indicating that the glycylglutamine and goat milk-enriched extracellular vesicles composition has a significant therapeutic effect on ulcerative colitis in mice. Judging from the colon length of mice in each group, the goat milk-enriched extracellular vesicle extract can increase the absorption of glycylglutamine in the intestine by nearly 100%.

[0077] Example 6: Milk extract-loaded alanyl-glutamine composition improves oral ulcers in rats induced by phenol (carbolic acid) burning method

[0078] The experiment used male SD rats weighing 180-220 g, which were provided by SPIEF (Beijing) Biotechnology Co., Ltd. with animal certificate number NO.110324251100578514. They were divided into 5 groups, with 8 rats in each group, namely normal group, model group, extract group, raw material group, and preparation group.

[0079] Phenol used in the rat oral ulcer model was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Cat. No.: P815401-500g, Lot#: C16547374). The extract group (milk-extracted extracellular vesicle solution), the raw material group prepared with purified water (100 mg / mL alanyl-glutamine solution), and the preparation group (milk-extracted extracellular vesicles loaded with alanyl-glutamine prepared according to Example 1 and diluted to 100 mg / mL) were used.

[0080] Model establishment: A 6-cm-long tube with a 3-mm diameter at the lower end is placed inside a small cotton ball, with the bottom of the ball level with the lower end of the tube. A 90% phenol solution is then dripped into the tube until the cotton ball is just soaked. The lower end of the glass tube is then placed flat on the buccal mucosa at the inside corner of the rat's mouth. After 30 seconds of cauterization, a white lesion approximately 3 mm in diameter appears on the mucosa in this area.

[0081] Experimental process: Rats with an oral ulcer model were given a topical application once a day, with a volume of 50 μL per rat each time. The animals were given the drug after anesthesia and kept in the drug-dosed state for 10 minutes. The animal signs were observed every day, the wound conditions were photographed and recorded once a day (photograph + ruler), and the ulcer diameter was counted. Rats in the normal group were also given a topical application once a day, with a volume of 50 μL per rat each time. The experimental period was 7 days. On the 7th day, blood was collected (orbital venous plexus), serum was separated, and the inflammatory factor TNF-a was detected using an ELISA kit (catalog number: YJ002859, Shanghai Enzyme-Linked Biotechnology Co., Ltd., batch number: 202410). After the 7th day, the rats were killed, and the ulcer tissue of one rat in each group was obtained. The mucosal tissue was fixed, sectioned, HE stained, and pathologically analyzed.

[0082] Results: When the rats in the oral ulcer model were dosed on the second day, it was found that except for the rats in the preparation group, which were more active in eating, the rats in other groups showed erect hair and a listless state. It was speculated that the rats' eating was affected by oral ulcers. When observed on Day 3, the rats in the preparation group showed a listless state, but the state was better than that of the rats in other groups, indicating that the preparation group (the composition of the milk-extracted extracellular vesicle solution loaded with 100 mg / mL alanyl-glutamine prepared in Example 1) can reduce the impact of oral ulcers on the growth state of rats.

[0083] Figure 6 shows comparative photographs of oral mucosal ulcers in rats from day 1 to day 6. From the comparative graph of oral ulcer areas in rats in Figure 7, the ulcer areas in the model group, extract group, and raw material group showed a slight healing trend from day 1 to day 6. On day 6, the oral ulcers in four rats in the preparation group had healed, and the ulcer surfaces in the remaining four rats were very small and close to healing, demonstrating that the preparation group (the composition of the milk-extracted extracellular vesicle solution loaded with 100 mg / mL alanyl-glutamine prepared in Example 1) can significantly shorten the recovery time of oral ulcers in rats.

[0084] Figure 8 shows a comparison of the test results of TNF-a, an inflammatory factor, in the serum of different groups of rats on the 7th day. From the results, it can be seen that the preparation group (a composition of milk-extracted extracellular vesicle solution loaded with 100 mg / mL alanyl-glutamine) has significantly better inhibition of inflammatory factors in serum than the raw material group, extract group and model group.

[0085] Figure 9 compares the pathological sections of the oral mucosa of rats in the normal group, model group, extract group, raw material group, and preparation group. It can be seen intuitively that compared with the model group, the extract group and the raw material group have the effect of improving ulcer healing, which is consistent with the ulcer area results; the pathological results of rats with oral ulcers using the preparation group (a composition of extracellular vesicle solution extracted from milk loaded with 100 mg / mL alanyl-glutamine) basically returned to those of the normal group, proving that the preparation group has the ability to treat oral ulcer healing.

Claims

1. A composition comprising a glutamine source and vesicles, wherein at least a portion of the glutamine source is loaded in the vesicles.

2. The composition according to claim 1, wherein the glutamine source is selected from glutamine polypeptide or modified glutamine.

3. The composition according to claim 1 or 2, wherein the glutamine polypeptide is glutamine dipeptide.

4. The composition according to any one of claims 1 to 3, wherein the glutamine dipeptide is selected from alanyl-glutamine or glycyl-glutamine.

5. The composition according to any one of claims 1 to 4, wherein the vesicles are selected from extracellular vesicles.

6. The composition according to any one of claims 1 to 5, wherein the extracellular vesicles are extracellular vesicles from animal milk.

7. The composition according to any one of claims 1 to 6, wherein the animal milk is selected from cow's milk or goat's milk.

8. A method for preparing the composition according to any one of claims 1 to 7, comprising: Step 1, mixing a liquid containing vesicles with a glutamine source; Step 2, incubating the mixture obtained in step 1; and Step 3: Ultrasonicate the mixture obtained in step 2.

9. The method according to claim 7, wherein: The incubation in step 2 is to incubate the mixture obtained in step 1 at 20° C. to 65° C. for 10 minutes to 5 hours.

10. The method according to claim 7 or 8, wherein The ultrasonication in step 3 is to ultrasonicate the mixture obtained in step 2 at a power of 5W to 300W.

11. The method according to claim 7 or 8, wherein: The ultrasonication in step 3 is to ultrasonicate the mixture obtained in step 2 at a frequency of 10 kHz to 60 kHz.

12. A method for improving poor oral, gastric and / or intestinal health conditions, comprising administering the composition of any one of claims 1 to 7 or the composition prepared by the method of any one of claims 8 to 11 to a subject in need thereof.

13. The method according to claim 12, wherein the oral, gastric and / or intestinal adverse health conditions are selected from oral ulcers, gastric mucosal damage and ulcerative colitis; preferably, the gastric mucosal damage is selected from gastric ulcers.

14. Use of the composition according to any one of claims 1 to 7 or the composition prepared by the method according to any one of claims 8 to 11 in protecting the oral cavity, stomach and / or intestines.

15. A composition comprising the composition according to any one of claims 1 to 7 or a composition prepared by the method according to any one of claims 8 to 11.

16. The composition according to any one of claims 1 to 7, the composition prepared by the method according to any one of claims 8 to 11 or the composition according to claim 15 is in an oral form.

17. The composition according to any one of claims 1 to 7, the composition prepared by the method according to any one of claims 8 to 11, or the composition according to claim 15 is an oral liquid, beverage, syrup, tablet, soft capsule, capsule, powder, etc.

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