Diarginine salt and composition

Abstract

Disclosed in the present invention are a physiologically acceptable salt of diarginine and a preparation method therefor. Further disclosed is a composition containing the diarginine and salts thereof, and arginine and salts thereof. The preparation method of the present invention can be used for efficient and stable production of the salt of the diarginine. The composition of the present invention does not require the separation of a small amount of arginine from the diarginine, and thus has great cost advantage and commercial potential regarding both synthesis and purification.

Description

Arginine dipeptide salts and compositions Technical Field

[0001] The present invention belongs to the field of biochemical engineering and specifically relates to a physiologically acceptable salt of arginine dipeptide and a preparation method thereof. It also relates to an arginine dipeptide composition comprising arginine dipeptide and its salt and arginine and its salt. Background Art

[0002] Arginine participates in the ornithine cycle in the human body, promoting the formation of urea. This process converts ammonia produced in the body into non-toxic urea, which is excreted in the urine, thereby reducing blood ammonia concentrations. A high concentration of hydrogen ions helps correct the acid-base balance in hepatic encephalopathy. Arginine has numerous physiological activities and numerous health benefits. However, its bitter, astringent, and fishy odor is difficult to mask simply through flavoring or seasoning. This significantly affects the flavor and taste of arginine-based products, reduces consumer enjoyment, and limits their application.

[0003] A compound formed by the condensation of two amino acid molecules is called a dipeptide. Dipeptides are typically hydrolyzed in the body by dipeptidases into two free amino acids. Arginine dipeptide is a dipeptide formed by a peptide bond between two arginine amino acids. Currently, research and reports on arginine dipeptides are limited. How to make arginine dipeptides and their suitable forms, such as compositions, widely applicable in dietary supplements, foods, nutritional supplements, and health products remains a key area of ​​research for those skilled in the art. Summary of the Invention

[0004] The present invention can prepare a stable physiologically acceptable salt of arginine dipeptide, which has the advantages of low energy consumption and material consumption, and the arginine dipeptide salt of the present invention can be better applied in the fields of dietary supplements, food and the like.

[0005] In one aspect, the present invention provides a physiologically acceptable salt of arginine dipeptide, wherein the salt is selected from one or more of sodium salt, potassium salt, calcium salt, and magnesium salt.

[0006] In some embodiments, the molar ratio of arginine dipeptide to metal is 1: 1 to 2: 1. In some embodiments, the molar ratio of arginine dipeptide to metal is 1: 1, 2:1.

[0007] In some embodiments, the salt is in a crystalline form, an amorphous form, or a mixture thereof. In some embodiments, the salt is in an amorphous form.

[0008] In some embodiments, the salt is sodium arginine dipeptide, which has an X-ray diffraction pattern substantially as shown in Figure 1 A. In some embodiments, sodium arginine dipeptide has an infrared spectrum substantially as shown in Figure 2A.

[0009] In some embodiments, the salt is potassium arginine dipeptide, which has an X-ray diffraction pattern substantially as shown in Figure 1 B. In some embodiments, potassium arginine dipeptide has an infrared spectrum substantially as shown in Figure 2B.

[0010] In some embodiments, the salt is arginine dipeptide calcium, which has an X-ray diffraction pattern substantially as shown in Figure 1 C. In some embodiments, arginine dipeptide calcium has an infrared spectrum substantially as shown in Figure 2C.

[0011] In some embodiments, the salt is magnesium arginine dipeptide, which has an X-ray diffraction pattern substantially as shown in Figure ID. In some embodiments, magnesium arginine dipeptide has an infrared spectrum substantially as shown in Figure 2D.

[0012] In another aspect, the present invention provides a method for preparing the salt as described above, comprising: reacting arginine dipeptide with an alkaline metal compound; concentrating, adding a solvent, heating, and dissolving; cooling to precipitate a solid, and drying to obtain the salt.

[0013] In some embodiments, the basic metal compound is selected from one or more of the following: oxides, peroxides, hydrides, hydroxides, and organometallic bases of sodium, potassium, calcium, and magnesium. In some embodiments, the organometallic base includes methoxides, ethoxides, and the like. In some embodiments, the basic metal compound may also be carbonates and bicarbonates.

[0014] In some embodiments, the solvent is selected from one or more of the following: methanol, ethanol, propanol, isopropanol, butanol, acetonitrile, acetone, methyl ethyl ketone, ethyl acetate, butyl acetate, isopropyl acetate, and water.

[0015] In some embodiments, the temperature is raised to 40-80°C.

[0016] In some embodiments, the salt is used in the preparation of a food, beverage, supplement, or nutraceutical.

[0017] In another aspect, the present invention provides a composition comprising the salt as described above, and arginine dipeptide.

[0018] In some embodiments, the proportion of the physiologically acceptable salt of arginine dipeptide is greater than 55%.

[0019] In some embodiments, the composition further comprises a physiologically acceptable carrier.

[0020] In some embodiments, the composition is used to prepare a food, a beverage, a supplement, or a nutraceutical.

[0021] In one aspect, the present invention provides a composition comprising arginine dipeptide and a salt thereof; and arginine and a salt thereof.

[0022] In some embodiments, the content of arginine and its salts in the composition is no more than 10wt%, 9wt%, 8wt%, 7wt%, or 6wt%. In some embodiments, the content of arginine and its salts in the composition is no more than 5wt%. Further, the content of arginine and its salts in the composition is less than 4.8wt%, 4.5wt%, 4wt%, 3.8wt%, 3.5wt%, 3wt%, 2.8wt%, 2.5wt%, 2wt%, 1.5wt%, 1wt%, 0.5wt%, 0.3wt%, 0.2wt%, or 0.1wt%.

[0023] In some embodiments, the composition is a solid formulation or a liquid formulation.

[0024] In some embodiments, the salt includes metal salts, such as hydrochloride, acetate, sulfate, citrate, malate, succinate, and metal salts include sodium, potassium, calcium, and magnesium salts.

[0025] In some embodiments, the salt comprises a physiologically acceptable salt of arginine dipeptide as described above. In some embodiments, the metal salt comprises sodium arginine dipeptide, potassium arginine dipeptide, calcium arginine dipeptide and / or magnesium arginine dipeptide as described above.

[0026] In some embodiments, the composition of the present invention is used to prepare a product for improving muscle function and / or enhancing athletic performance.

[0027] In some embodiments, the composition can be in the form of a suppository, tablet, pill, granule, powder, film, capsule, beverage, aerosol, elixir, tincture, tonic, liquid suspension, or syrup.

[0028] In another aspect, the present invention provides a nutritional supplement, food, beverage, or animal feed comprising the composition, wherein the composition comprises 0.01 to 90.0% (w / w) of the nutritional supplement, food, beverage, or animal feed. For example, 0.005% to 90%, 0.01% to 85%, 0.05% to 80%, 0.05% to 80%, 0.5% to 75%, 1% to 70%, 2% to 50%, 0.005% to 45%, 0.01% to 40%, 0.02% to 35%, 0.05% to 30%, 0.1% to 25%, 0.2% to 20%, 0.5% to 15%, 1% to 10%, 0.1% to 25%, 0.2% to 10%, 0.5% to 9%, 1% to 7%, 2% to 7% (w / w).

[0029] In another aspect, the present invention provides a method of using the composition as described above, comprising administering the composition to a subject in need thereof in an amount of 1 to 2000 mg per day. In some embodiments, the composition is administered to a subject in need thereof in an amount of 0.01 to 6000 mg, 1 to 3000 mg, 5 to 1500 mg, 10 to 1200 mg, 15 to 1000 mg, or 30 to 800 mg per day.

[0030] Compared with the existing technology, the present invention can efficiently and stably produce the salt of arginine dipeptide; and the arginine dipeptide salt of the present invention has excellent properties such as good stability, and can have a wider application prospect in the fields of dietary supplements, food and so on. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 1A to 1D are X-ray diffraction patterns of arginine dipeptide sodium, arginine dipeptide potassium, arginine dipeptide calcium, and arginine dipeptide magnesium, respectively.

[0032] 2A to 2D are infrared spectra of arginine dipeptide sodium, arginine dipeptide potassium, arginine dipeptide calcium, and arginine dipeptide magnesium, respectively.

[0033] Figure 3 shows the area under the curve of NO in each group.

[0034] FIG4 shows the area under the curve of cGMP in each group.

[0035] Figure 5 shows the weighted swimming time of each group.

[0036] Figure 6 shows the running distance of each group.

[0037] Figure 7 shows the blood urea nitrogen content in each group.

[0038] Figure 8 shows the blood lactate levels in each group.

[0039] Figure 9 shows the liver glycogen content in each group. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention will now be described in detail with reference to the embodiments thereof. Although the present invention will be described in conjunction with the preferred embodiments, it should be understood that they are not intended to limit the present invention to these embodiments. On the contrary, the present invention is intended to cover substitutions, modifications and equivalents, which may be included within the spirit and scope of the present invention as defined in the claims.

[0041] As used herein, the term "or" is intended to include "and" and "or." In other words, the term "or" can also be replaced with "and / or."

[0042] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0043] As used herein, the term "comprises" or "includes" or variations thereof refers to instances where the term is used in its non-limiting sense, meaning that items following the term are included, but items not specifically mentioned are not excluded. It also includes the more restrictive verbs 'consisting essentially of' and 'consisting of.'

[0044] As used herein, the terms "about" and "approximately" provide flexibility in numerical values ​​by providing that a given value may be "slightly above" or "less than" an endpoint. The flexibility of this term can be determined by the particular variable and is within the knowledge of those skilled in the art to determine based on experience and the relevant description herein.

[0045] As used herein, the terms "subject" or "individual" are used interchangeably to refer to any subject to which the compositions and methods of the present disclosure can be applied or administered. The subject may have a disease or condition, but the subject does not need to be sick to benefit from the methods and compositions of the present disclosure. The subject may need to improve his or her overall health, but the subject may also have a generally healthy condition and wish to maintain or further improve his or her overall health. Therefore, any subject can be administered with the disclosed compositions or become a recipient of the disclosed methods. Herein, the term "subject" refers to an animal (e.g., birds, reptiles, and mammals). In certain embodiments, the subject can be a mammal including non-primates (e.g., camels, donkeys, zebras, cows, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys, chimpanzees, humans). In certain embodiments, the subject can be a non-human mammal. In other embodiments, the subject can be a human.

[0046] As used herein, the term "administer" refers to the process of delivering the disclosed compositions or active ingredients to a subject. The compositions of the present invention are preferably administered orally, intravenously, intramuscularly, intraperitoneally, subcutaneously, topically, or sublingually, but may also be administered by other conventional routes to achieve the desired effect.

[0047] As used herein, the terms "physiologically acceptable," "dietarily or pharmaceutically acceptable" refer to pharmaceutically, physiologically, dietary and / or nutritionally acceptable compositions or agents, materials or combinations of compositions and / or dosage forms thereof that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals, compatible with the other ingredients of the composition, without excessive toxicity, irritation, allergic response or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0048] In some embodiments, the salts of the present invention can be prepared as compositions together with dietary or pharmaceutically acceptable carriers. In the present invention, the administration form of the composition is provided and involves liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the composition and is harmless to the subject, i.e., suitable for consumption or nutritionally acceptable.

[0049] In some embodiments, the salts of the present invention may be administered with other supplements, such as vitamins, minerals, nootropics, and other supplements known in the art.

[0050] The compositions of the present invention will be formulated into nutritional supplements or dietary supplements, (medical) foods, animal feeds in liquid or solid form, and optionally contain a dietary or pharmaceutically acceptable carrier. For example, when the composition is in solid form, the composition can be formulated into snack bars, yogurts, lozenges, tablets or capsules, or applied to cereal products, contained in baked goods. On the other hand, when the supplement is in liquid form, the composition can be formulated into tinctures, soft gel capsules, liquid capsules, syrups, carbonated beverages, brewed beverages (such as coffee or tea), juices, energy drinks, sports drinks or flavored waters. Although specifically contemplated for human use, it should be understood that the compositions of the present invention and their formulations can also be used for veterinary purposes (e.g., animal feed for domestic companion animals ("pets") or animal feed for farm animals. In addition, the amount of the composition to be added can vary depending on the type and form of the nutritional supplement or dietary supplement, (medical) food, animal feed. For oral administration, the compositions of the present invention can be in any suitable form, including solutions, tablets, gel capsules, capsules or alternative nutritional foods or nutritional supplements.

[0051] The composition of the present invention is simple and convenient to prepare. During the preparation of the arginine dipeptide, a mixture of the two can be obtained by suitable separation and purification methods. During the preparation of the composition, the components can be added appropriately using mixing methods well known in the art, depending on the properties of the raw materials. The composition of the present invention can be formulated using appropriate content selections as needed.

[0052] The polymorphic state of solid pharmaceuticals is an important aspect of studying the state of drug existence. For most chemical drugs, polymorphism generally exists. Because these different crystalline substances affect the physical and chemical properties and biological activity of drugs, the existence of drug crystalline forms should be considered when studying drug raw materials, formulations, etc. Amorphous is a form of material polymorphism and a special crystalline state. Like crystalline substances, the amorphous state of solid pharmaceuticals can also exist in different forms. This phenomenon is called the polymorphism of solid substances without amorphous forms, also known as amorphous polymorphism.

[0053] The following examples are illustrative of selected embodiments of the present invention and are not intended to limit the scope of the invention.

[0054] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0055] Example 1. Preparation of Sodium Arginine Dipeptide

[0056] Arginine dipeptide (50 g, 1.0 eq) and sodium hydroxide (6.1 g, 1.0 eq) were placed in 250 mL of purified water and heated to 20-30°C for 2 h to dissolve completely. The solution was concentrated to remove water, and 100 mL of isopropanol was added and heated to 60°C to dissolve the solution. The solution was then slowly cooled to crystallize, filtered, and dried to obtain 41.1 g of a white solid with a yield of 77.0%.

[0057] Example 2. Preparation of potassium arginine dipeptide

[0058] Preparation 1: Arginine dipeptide (50 g, 1.0 eq) and potassium hydroxide (8.5 g, 1.0 eq) were placed in 250 mL of purified water and heated to 20-30°C for 4 h to dissolve the mixture. The mixture was concentrated to remove the water, and 100 mL of isopropanol was added and heated to 60°C to dissolve the mixture. The mixture was slowly cooled to crystallize, filtered, and dried to obtain 39.8 g of a white solid (71.4% yield).

[0059] Preparation 2: Arginine dipeptide (50 g, 1.0 eq) and potassium peroxide (10.8 g, 1.0 eq) were placed in 250 mL of purified water and heated to 20-30°C for 4 h to dissolve completely. The solution was concentrated to remove the water, and 100 mL of ethanol was added and heated to 50°C to dissolve the solution. The solution was slowly cooled to crystallize, filtered, and dried to obtain 38.5 g of a white solid (yield 69.1%).

[0060] Example 3. Preparation of arginine dipeptide calcium

[0061] Preparation 1: Arginine dipeptide (50 g, 1.0 eq) and calcium hydroxide (5.6 g, 0.5 eq) were placed in 250 mL of purified water and heated to 30-40°C for 4 h to dissolve completely. The mixture was concentrated to remove the water, and 100 mL of isopropanol was added and heated to 60°C to dissolve the clear solution. The mixture was slowly cooled to crystallize, filtered, and dried to obtain 27.9 g of a white solid (52.9% yield).

[0062] Preparation 2: Arginine dipeptide (50 g, 1.0 eq) and calcium oxide (4.2 g, 0.5 eq) were placed in 250 mL of purified water and heated to 30-40°C for 4 h to dissolve completely. The solution was concentrated to remove water, and 100 mL of water was added and heated to 70°C to dissolve the solution. The solution was slowly cooled to crystallize, filtered, and dried to obtain 25.8 g of a white solid (48.8% yield).

[0063] Example 4. Preparation of magnesium arginine dipeptide

[0064] Preparation 1: Arginine dipeptide (50 g, 1.0 eq) and magnesium hydroxide (4.4 g, 0.5 eq) were placed in 250 mL of purified water and heated to 60-70°C for 6 h to dissolve completely. The mixture was concentrated to remove the water, and 100 mL of isopropanol was added and heated to 60°C to dissolve the clear solution. The mixture was slowly cooled to crystallize, filtered, and dried to obtain 23.1 g of a white solid (44.7% yield).

[0065] Preparation 2: Arginine dipeptide (50 g, 1.0 eq) and magnesium methoxide (6.5 g, 0.5 eq) were placed in 250 mL of purified water and heated to 60-70°C for 6 h to dissolve the residue. The mixture was concentrated to remove the water, and 100 mL of methanol was added and heated to 60°C to dissolve the residue. The mixture was slowly cooled to crystallize, filtered, and dried to obtain 21.4 g of a white solid (41.3% yield).

[0066] X-ray diffraction

[0067] X-ray powder diffraction patterns were obtained using a SmartLab 3KW X-ray powder diffractometer under the following conditions: diffraction line: Cu_K-beta (40 kV, 40 mA), scan rate: 20.00 deg / min, scan range: 5° to 40°. The X-ray diffraction patterns of the arginine dipeptide salts of Examples 1 to 4 are shown in Figures 1A, 1B, 1C, and 1D, respectively.

[0068] Infrared spectroscopy analysis

[0069] The arginine dipeptide salts of each example were analyzed by infrared spectroscopy using a Shimadzu Fourier transform attenuated total reflection infrared spectrometer. The infrared spectra of the arginine dipeptide salts of Examples 1 to 4 are shown in Figures 2A, 2B, 2C, and 2D, respectively.

[0070] Elemental analysis

[0071] The arginine dipeptide salts of each example were subjected to elemental analysis using an elemental analyzer, and the elemental analysis results of the salts were substantially consistent with those of the examples.

[0072] Quantitative NMR

[0073] The arginine dipeptide salt of each example was recorded in a spectrometer. 1 H NMR spectrum. Arginine dipeptide sodium of Example 1: 1 H NMR (400 MHz, D2O): δ 4.12-4.15 (m, 1H), 3.77-3.80 (m, 1H), 3.14-3.18 (m, 4H), 1.56-1.82 (m, 8H). Potassium arginine dipeptide of Example 2: 1 H NMR (400 MHz, D2O): δ 4.12-4.15 (m, 1H), 3.77-3.80 (m, 1H), 3.14-3.18 (m, 4H), 1.56-1.82 (m, 8H). Arginine dipeptide calcium of Example 3: 1 H NMR (400 MHz, D2O): δ 4.08-4.11 (m, 1H), 3.33-3.36 (m, 1H), 3.06-3.10; 2.48-2.53 (m, 4H), 1.47-1.77 (m, 8H). Magnesium arginine dipeptide of Example 4: 1 H NMR (400MHz, D2O): δ4.08-4.11(m,1H), 3.33-3.36(m,1H), 3.06-3.10; 2.48-2.53(m,4H), 1.47-1.77(m,8H).

[0074] The HPLC-MS analysis of the sodium arginine dipeptide in Example 1 showed that its content was as high as 98.7%. The HPLC-MS results of other arginine dipeptide salts of the present invention were basically consistent with those of Example 1.

[0075] The solubility of arginine, arginine dipeptide, and the arginine dipeptide salts of Examples 1 to 4 was measured. The data are shown in Table 1 below:

[0076] Table 1

[0077] Taste test

[0078] 5.4 g of each of the arginine dipeptide salts and arginine dipeptide from Examples 1 to 4 were weighed and dissolved in 240 mL of deionized water. The samples were numbered and 10 randomly selected subjects were asked to rate the taste of the sample powders and solutions (fishy, ​​bitter, salty, and bitter aftertaste). The evaluations were conducted in a separate evaluation room with a white wall and white paper background under white fluorescent light. Only one subject performed the evaluations at a time, and the results were recorded.

[0079] Taste evaluation included fishy, ​​bitter, salty, and aftertaste. Participants were instructed to taste approximately 15 mL of the sample in an evaluation room and then rate the fishy (strong fishy: 0, strong fishy: 1, fishy: 2, moderate fishy: 3, slightly fishy: 4, no fishy: 5), bitterness (strong bitterness: 0, strong bitterness: 1, moderate bitterness: 2, weak bitterness: 3, weak bitterness: 4, no bitterness: 5), saltiness (strong saltiness: 0, strong saltiness: 1, moderate saltiness: 2, weak saltiness: 3, weak saltiness: 4, no saltiness: 5), and aftertaste (strong aftertaste: 0, strong aftertaste: 1, moderate aftertaste: 2, weak aftertaste: 3, weak aftertaste: 4, no aftertaste: 5). Higher scores indicate fewer unpleasant tastes. The taste evaluation results are shown in Table 2.

[0080] Table 2

[0081] According to the above evaluation results, the taste of several arginine dipeptide salts is better than arginine dipeptide, with higher overall scores and higher acceptance among subjects.

[0082] The present invention can efficiently and stably produce salts of arginine dipeptide; and the arginine dipeptide salt of the present invention exists in a specific amorphous state, that is, a special crystalline state, and has excellent properties such as improved taste and good stability, and has high solubility, and can have a wider application prospect in the fields of dietary supplements, food, etc.

[0083] Composition preparation

[0084] The compositions of samples 1 to 9 were prepared according to Table 3 below.

[0085] Table 3

[0086] The composition of the present invention can obtain a mixture of the two by a suitable separation and purification method during the preparation of arginine dipeptide. For example, in the preparation of arginine dipeptide, peptide bond connection is completed under certain conditions by a specific enzyme, and a mixture of the two is obtained by a suitable separation and purification method. This method has a simple preparation process and a simple process purification, which greatly reduces the process cost. The preparation method can be: dissolving arginine in water, adding amino acid ligase, adding auxiliary materials, keeping the reaction at 30-35°C for a period of time, HPLC control, arginine ≤5.0% in the reaction liquid, stopping the reaction, filtering to remove insoluble matter, and purifying the aqueous solution with a cationic resin to obtain an arginine dipeptide / arginine mixture. Detection confirms that arginine accounts for about 4.2% and arginine dipeptide 95.8%. Nutritional supplements, food, beverages or animal feed.

[0087] The composition of arginine dipeptide salt and arginine was prepared as shown in Table 3.

[0088] Any of the compositions of Samples 1-9 is prepared into a nutritional supplement, food, beverage or animal feed, wherein the weight of the composition accounts for 0.01 to 90.0% (w / w) of the nutritional supplement, food, beverage or animal feed.

[0089] Smell and taste testing

[0090] According to the formula shown in Table 3, samples 4-7 were prepared, and 5.4 g of arginine and an equal amount of sample were dissolved in 240 mL of deionized water. The sample powders were paired with the resulting solutions one by one and numbered. Ten subjects were randomly recruited to rate the smell (fishy smell, rancid smell) and taste (fishy smell, bitterness, saltiness, and bitter aftertaste) of the sample powders and solutions, and a total score was finally given. The evaluation was carried out in a separate evaluation room, in front of a white wall and white paper background, under a white fluorescent light, and only one subject evaluated and recorded the results.

[0091] Odor evaluation included both fishy and rancid odors. Participants were instructed to sniff the sample for 3 seconds in an evaluation room and then rate the fishy odor (0 for strong fishy odor, 1 for relatively strong fishy odor, 2 for fishy odor, 3 for moderate fishy odor, 4 for slightly fishy odor, and 5 for no fishy odor) and rancid odor (0 for strong rancid odor, 1 for relatively strong rancid odor, 2 for rancid odor, 3 for moderate rancid odor, 4 for slightly rancid odor, and 5 for no rancid odor). Higher scores indicate less objectionable odor. The odor evaluation results are shown in Table 4.

[0092] Table 4

[0093] According to the evaluation results in Table 4, the fishy and rancid smell scores of samples 4-7 were higher than that of arginine, and the sample odor evaluation was higher than that of arginine.

[0094] Taste evaluation included fishy, ​​bitter, salty, and aftertaste. In the evaluation room, participants were instructed to taste approximately 15 mL of the sample and then rate the fishy (strong fishy: 0, strong fishy: 1, moderate fishy: 2, moderate fishy: 3, slightly fishy: 4, no fishy: 5), bitterness (strong bitterness: 0, strong bitterness: 1, moderate bitterness: 2, weak bitterness: 3, weak bitterness: 4, no bitterness: 5), saltiness (strong saltiness: 0, strong saltiness: 1, moderate saltiness: 2, weak saltiness: 3, weak saltiness: 4, no saltiness: 5), and aftertaste (strong aftertaste: 0, strong aftertaste: 1, moderate aftertaste: 2, weak aftertaste: 3, weak aftertaste: 4, no aftertaste: 5). Higher scores indicate fewer unpleasant tastes. The taste evaluation results are shown in Table 5.

[0095] Table 5

[0096] According to the results in Table 5, the taste of the solutions of samples 4-7 is better than that of arginine in terms of fishy taste, bitterness, saltiness, and aftertaste, and the overall taste score of sample 4-7 is higher.

[0097] Table 6 is the overall evaluation of each sample by the subjects (0-5 points)

[0098] According to Table 6, the total scores of samples 4-7 were higher than that of arginine, indicating that samples 4-7 were superior to arginine in taste and smell and were more easily accepted by the subjects.

[0099] Sports performance and fatigue resistance testing

[0100] 40 6-8 week old SD rats were randomly divided into 5 groups, including a control group, an arginine group, sample group 1, sample group 2, and sample group 3 in Table 3. The dosage of each composition was 500 mg / kg. After fasting for 16 hours one day in advance, blood was collected from the orbit before gavage as a baseline. After gavage (control group gavage with water), blood was collected at 15, 30, 60, 120, and 180 minutes, and serum was separated and obtained for detection of NO and cGMP. Serum detection was performed according to the kit instructions, and the area under the curve values ​​of NO and cGMP within 3 hours were obtained.

[0101] As shown in the area under the NO curve in Figure 1, each sample group was superior to arginine in promoting NO. Samples 1, 2, and 3 were 22%, 26%, and 24% better than arginine alone in increasing NO, respectively. As shown in the area under the cGMP curve in Figure 2, each sample group was superior to arginine in promoting cGMP. Samples 1, 2, and 3 were 9%, 12%, and 10% better than arginine alone in increasing cGMP, respectively.

[0102] Forty-eight ICR mice were randomly divided into a normal control group (blank, Group 1), an exercise group (exercise, Group 2), an exercise + arginine group (700 mg / kg, Group 3), an exercise + sample 1 group (350 mg / kg, Group 4), an exercise + sample 2 group (350 mg / kg, Group 5), and an exercise + sample 3 group (350 mg / kg, Group 6), with eight mice in each group. Animals were gavaged continuously for 32 days. On the 28th day, a 5% weight-bearing swimming test was performed, and the swimming exhaustion time was recorded. A treadmill exhaustion test was performed on the 30th day of administration, and the exhaustion distance of the mice was recorded. On the 32nd day of administration, the mice were subjected to a 90-minute weight-bearing swimming test. Blood and tissue samples were collected immediately after the swim. After standing at room temperature for 4 hours, the blood was centrifuged at 1500 x g for 10 minutes at 4°C, and all serum was transferred to cryogenic tubes and stored at -80°C. All animals underwent exercise testing 30 minutes after receiving the gavage.

[0103] Weighted swimming test: 30 minutes after administration, immediately wrap a lead wire around the tail. The weight of the wire is 5% of the animal's body weight. Use a stopwatch to record the time from when the animal's head is completely submerged in water for 10 seconds until it cannot surface. The swimming time for each mouse is recorded.

[0104] Treadmill endurance test: Muscle endurance test was performed on the 30th day of administration for each group. Muscle endurance was performed on a motorized treadmill with a speed range of 0 to 50 cm / s and an incline of 10 degrees. Uphill running involves concentric muscle contraction, which increases muscle work and leads to faster fatigue compared to running on flat ground. The belt speed started at approximately 15 cm / s and increased by 5 cm / s every 2 minutes until a speed of 50 cm / s was reached. The animals performed the treadmill test until exhaustion. The exhaustion point was defined as the time point at which the animal fell into the shock zone. The movement distance (m) was measured as a marker of exercise performance.

[0105] After 32 days of administration, serum was collected after 90 minutes of weight-free swimming for analysis of clinical biochemical parameters, including lactate (LA) and urea nitrogen (BUN). In addition, part of the liver was removed for determination of liver glycogen (LG) content.

[0106] Figures 3 and 4 show the weighted swimming time and running distance of each group, respectively. The results confirm the performance-enhancing effect of the composition. In terms of swimming time, the duration of the swim was extended by 48%, 57%, and 53% for Samples 1, 2, and 3, respectively, compared to arginine alone. In terms of running distance, the distances run by Samples 1, 2, and 3 were increased by 17%, 21%, and 19%, respectively, compared to arginine alone.

[0107] Figures 5, 6, and 7 show the blood urea nitrogen, blood lactate, and liver glycogen levels of each group, respectively. As shown in Figure 5, Samples 1, 2, and 3 were superior to arginine alone in inhibiting urea nitrogen accumulation, with results exceeding arginine by 7%, 15%, and 13%, respectively. As shown in Figure 6, Samples 1, 2, and 3 were superior to arginine alone in inhibiting lactic acid accumulation, with results exceeding arginine alone by 4.6%, 7.2%, and 5.9%, respectively. As shown in Figure 7, Samples 1, 2, and 3 were superior to arginine alone in reducing glycogen depletion, with results exceeding arginine alone by 11.9%, 19.7%, and 15.5%, respectively. This demonstrates that the combination has a superior anti-fatigue effect.

[0108] When synthesizing arginine dipeptide using arginine as raw material, if you want to convert it completely, you need a long reaction time and add more auxiliary materials, which greatly increases the cost. The subsequent arginine and arginine dipeptide separation process is cumbersome, and the batch yield is reduced, which is not conducive to industrialization. The composition of the present invention does not need to separate arginine dipeptide and a small amount of arginine. The arginine dipeptide / arginine composition has great cost advantages and commercial potential in synthesis and purification. While retaining the good efficacy of arginine dipeptide, the process is simple and easy to implement, greatly reducing costs.

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations to these embodiments without departing from the principles and purpose of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A physiologically acceptable salt of arginine dipeptide, characterized in that The salt is selected from one or more of sodium salt, potassium salt, calcium salt and magnesium salt.

2. The salt according to claim 1, characterized in that The molar ratio of the arginine dipeptide to the metal is 1:1 to 2:

1.

3. The salt according to claim 1 or 2, characterized in that The salt is in a crystalline form, an amorphous form or a mixture thereof.

4. The salt according to any one of claims 1 to 3, characterized in that The salt is sodium arginine dipeptide, which has an X-ray diffraction pattern substantially as shown in FIG. 1A .

5. The salt according to any one of claims 1 to 3, characterized in that The salt is potassium arginine dipeptide, which has an X-ray diffraction pattern substantially as shown in FIG. 1B .

6. The salt according to any one of claims 1 to 3, characterized in that The salt is calcium arginine dipeptide, which has an X-ray diffraction pattern substantially as shown in FIG1C .

7. The salt according to any one of claims 1 to 3, characterized in that The salt is magnesium arginine dipeptide, which has an X-ray diffraction pattern substantially as shown in Figure ID.

8. A method for preparing the salt according to any one of claims 1 to 3, characterized in that: The method comprises: reacting arginine dipeptide with an alkaline metal compound; concentrating, adding a solvent, heating, dissolving; cooling to precipitate a solid, and drying to obtain the salt.

9. The preparation method according to claim 4, characterized in that The alkaline metal compound is selected from one or more of the following: oxides, peroxides, hydrides, hydroxides, and organic metal bases of sodium, potassium, calcium, and magnesium.

10. The preparation method according to claim 8 or 9, characterized in that: The solvent is selected from one or more of the following: methanol, ethanol, propanol, isopropanol, butanol, acetonitrile, acetone, methyl ethyl ketone, ethyl acetate, butyl acetate, isopropanol acetate, and water.

11. The preparation method according to any one of claims 8 to 10, characterized in that Raise the temperature to 40-80℃.

12. The salt according to any one of claims 8 to 11, characterized in that The salt is used to prepare food, beverages, supplements, and nutritional products.

13. A composition, characterized in that The composition comprises the salt according to any one of claims 1 to 7, and arginine dipeptide.

14. The composition according to claim 13, characterized in that The proportion of the physiologically acceptable salt of the arginine dipeptide is greater than 55%.

15. The composition according to claim 13 or 14, characterized in that The composition also includes a physiologically acceptable carrier.

16. The composition according to any one of claims 13 to 15, characterized in that The composition is used for preparing food, beverage, supplement and nutrition product.

17. A composition, characterized in that Contains arginine dipeptide and its salts; and arginine and its salts.

18. The composition according to claim 17, characterized in that The content of the arginine and its salts in the composition is no more than 10 wt%.

19. The composition according to claim 17 or 18, characterized in that The content of the arginine and its salts in the composition is no more than 5 wt%.

20. The composition according to any one of claims 17 to 19, characterized in that The content of arginine and its salt in the composition is less than 2 wt %.

21. The composition according to any one of claims 17 to 20, characterized in that The composition is a solid preparation or a liquid preparation.

22. The composition according to any one of claims 17 to 21, characterized in that The salts include metal salts, hydrochlorides, acetates, sulfates, citrates, malates, and succinates.

23. The composition according to any one of claims 17 to 22, characterized in that The salt is the salt according to any one of claims 1 to 7.

24. The composition according to any one of claims 17 to 23, characterized in that The composition is used in preparing a product for improving muscle function and / or enhancing sports performance.

25. The composition according to any one of claims 17 to 24, characterized in that The composition is in the form of suppositories, tablets, pills, granules, powders, films, capsules, beverages, aerosols, spirits, tinctures, tonics, liquid suspensions, or syrups.

26. A nutritional supplement, food, beverage or animal feed, characterized in that The nutritional supplement, food, beverage, or animal feed comprises the composition according to any one of claims 17 to 25.

27. The nutritional supplement, food, beverage or animal feed according to claim 26, wherein The composition comprises 0.01 to 90.0% (w / w) of the nutritional supplement, food, beverage or animal feed.

28. A method of using the composition according to any one of claims 17 to 25, characterized in that: The method of use comprises administering the composition to a subject in need thereof in an amount of 1 to 2000 mg per day.