Supramolecular extraction method of carnosine, efficacy-superposed uric acid-reducing product and application of efficacy-superposed uric acid-reducing product

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a supramolecular extraction method of ancarnosine, an efficacy-superposed uric acid-reducing product and application. The supramolecular extraction method of the ancarnosine comprises the following steps: firstly, adding water into tuna muscles, and crushing to obtain homogenate; adding alkaline protease and cyclodextrin into the homogenate, then adding special enzyme for aquatic products and flavourzyme, carrying out enzymolysis, adding a catalyst in batches, and carrying out purification, so as to obtain the product. According to the invention, the synergistic effect among the alkaline protease, the special enzyme for aquatic products and the flavourzyme is catalyzed by the catalyst, supramolecular extraction of the carnosine with high bioavailability and high extraction rate is carried out, and the carnosine and other effective components are mixed by using an embedding technology to obtain the embedding product for reducing uric acid. Animal model tests show that the uric acid reducing effect of the traditional Chinese medicine composition is better than that of western medicines including benzbromarone and allopurinol, and the traditional Chinese medicine composition has the advantages of being simple in formula, obvious in curative effect and free of toxic and side effects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical preparations, and specifically relates to a supramolecular extraction method of anserine and a uric acid-lowering product with performance superposition and application. BACKGROUND

[0002] Hyperuricemia refers to a serum urate concentration exceeding the solubility limit (about 6.8 mg / L), and is also a common biochemical abnormality reflecting extracellular fluid supersaturation. It is mainly caused by excessive uric acid secretion or insufficient renal uric acid excretion, and both excessive uric acid secretion and insufficient excretion can disrupt serum uric acid homeostasis. Modern medicine believes that hyperuricemia is mainly caused by a large amount of high-purine, high-protein or high-fructose diet, which leads to purine metabolism disorder, resulting in imbalance of purine metabolism end products (uric acid) and accumulation in the blood, and a series of adverse reactions and multiple organ damage such as kidney are caused by the increase of the content.

[0003] At present, the drugs for treating hyperuricemia mainly include the following types: (1) drugs for inhibiting uric acid generation, the most commonly used is allopurinol, which can inhibit the key enzyme of uric acid synthesis, thereby reducing the production of uric acid. (2) drugs for promoting uric acid excretion, such as benzbromarone and probenecid, which can reduce blood uric acid level by increasing the excretion of uric acid from the kidney. (3) uricase drugs, such as rasburicase and pegloticase, which are suitable for patients with severe hyperuricemia or refractory gout, and can directly degrade uric acid in the blood. However, most of the uric acid-lowering drugs have side effects, and long-term use cannot be tolerated by patients, so there is an urgent need for effective and less toxic treatment in the clinical treatment of hyperuricemia.

[0004] For example, Chinese invention patent publication No. CN118903390A discloses a uric acid-lowering functional peptide composition and product thereof, which is composed of the following components in parts by mass: Portulaca oleracea polypeptide extract 41 parts, anserine 25 parts, elastin peptide 5 parts, hyaluronic acid sodium 5 parts, Bifidobacterium adolescentis ATCC15703 2 parts, Lactobacillus paracasei ET-22 3 parts, Lactobacillus rhamnosus CRL1505 2 parts, astaxanthin 5 parts, vitamin C 2 parts, soybean fiber 5 parts, microcrystalline cellulose 2 parts, propolis 2 parts and lemon powder 1 part. The functional peptide composition has the effects of significantly reducing the content of uric acid and xanthine oxidase in blood, and can reduce the content of creatinine and urea nitrogen in blood, improve kidney health, and promote the excretion of excess uric acid. However, the uric acid-lowering functional peptide composition of the present application has complex components, contains microorganisms, has high cost, and the preparation process involves enzymolysis, which is complicated and not suitable for large-scale production.

[0005] Another Chinese invention patent publication No. CN108498783A discloses a composition for treating hyperuricemia and a preparation method thereof. The active ingredients of the composition include sunflower disc extract, corn silk extract, sour cherry extract, cactus fruit extract, plantain seed extract, red radish extract, goose peptide and minerals. The composition has the effect of reducing hyperuricemia and protecting the kidneys, and can synergistically increase the efficacy and reduce the toxicity of clinical uric acid-lowering drugs. However, the composition of the invention has poor effect on reducing uric acid.

[0006] Goose peptide, also known as amyl peptide, aminodi-peptide, and beta-alanyl-1-methyl L-histidine, is a highly stable water-soluble dipeptide. Related studies have found that goose peptide can quickly promote acid metabolism, open the path of acid and energy conversion, avoid acid accumulation, eliminate human fatigue and soreness, and ensure energy supply to the human body. Goose peptide has the advantages of stable acid-lowering effect, rapid acid-lowering speed, and no toxic side effects and dependence after long-term use. Especially for patients with kidney damage caused by long-term medication, goose peptide is an ideal choice. Its clear targeting ensures high efficiency of acid reduction, but the extraction rate of goose peptide in the prior art is low.

[0007] Therefore, there is an urgent need in the art to provide an extraction method for improving the extraction rate of amyl peptide, and a uric acid-lowering drug composition that can quickly reduce the concentration of uric acid in serum, has simple composition, obvious curative effect and no toxic side effects. SUMMARY

[0008] The present application provides a supermolecular extraction method of amyl peptide and a uric acid-lowering product with efficiency stacking and application.

[0009] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows: A supermolecular extraction method of amyl peptide, comprising the following steps: (1) adding water to tuna muscle, crushing to obtain homogenate; (2) adding alkaline protease and cyclodextrin to the homogenate first, then adding special enzymes for aquatic products and flavor protease, and performing enzymolysis while adding catalysts in batches, and purifying to obtain the product.

[0010] Preferably, in step (1), the mass ratio of tuna muscle to water is 1:3-6; the crushing is treated by colloid mill and sieved through a 50-100 mesh sieve.

[0011] Preferably, in step (2), the mass ratio of alkaline protease, special enzymes for aquatic products and flavor protease is 4-6:2-4:1-3.

[0012] More preferably, the mass ratio of alkaline protease, special enzymes for aquatic products and flavor protease is 5:3:2.

[0013] Preferably, the mass ratio of the homogenate to the sum of alkaline protease, aqua-specific enzyme and flavor protease is 150-300:0.5-4.5.

[0014] More preferably, the mass ratio of the homogenate to the sum of alkaline protease, aqua-specific enzyme and flavor protease is 250-300:1.5-3.5.

[0015] Further preferably, the mass ratio of the homogenate to the sum of alkaline protease, aqua-specific enzyme and flavor protease is 300:2.5-3.5.

[0016] Preferably, the cyclodextrin in step (2) is hydroxypropyl-β-cyclodextrin or β-cyclodextrin.

[0017] Preferably, the mass ratio of the cyclodextrin to the homogenate in step (2) is 1:25-50.

[0018] More preferably, the mass ratio of the cyclodextrin to the homogenate is 1:30-50.

[0019] Further preferably, the mass ratio of the cyclodextrin to the homogenate is 1:50.

[0020] Preferably, the enzymolysis condition in step (2) is intermittent ultrasonic enzymolysis at 40-60℃, 200-300 W for 0.5-5 h.

[0021] More preferably, the enzymolysis condition in step (2) is intermittent ultrasonic enzymolysis at 50-60℃, 250-300 W for 1-5 h.

[0022] Preferably, the catalyst in step (2) is calcium ion, which is from calcium chloride, calcium lactate or calcium citrate, and the final concentration of the calcium ion in the homogenate is 1-10 mM.

[0023] More preferably, the final concentration of the calcium ion in the homogenate is 1-5 mM.

[0024] Further preferably, the final concentration of the calcium ion in the homogenate is 2.5 mM.

[0025] Preferably, the process of adding the catalyst in batches in step (2) includes adding 50-60% of the total calcium first, and then adding 40-50% of the total calcium.

[0026] Preferably, the purification process in step (2) comprises: centrifuging at a speed of 1000-1500 rpm for 20-40 min to obtain an enzyme hydrolysis supernatant, then filtering the enzyme hydrolysis supernatant through a 3000-5000 Da ultrafiltration membrane, and finally loading the filtrate onto a 724 cation exchange resin and eluting with 0.1-0.3 mol / L NH4Cl solution to collect the eluate and dry it to obtain the product.

[0027] The present application also provides a uric acid-lowering composition comprising the following components: the ampelopsin, pyrroloquinoline quinone disodium salt, Ampelopsis megalophylla extract, Gardenia extract, Noni fruit extract, Portulaca oleracea extract, Eucommia ulmoides leaf extract, Cichorium intybus extract and Apium graveolens seed extract obtained by the above-mentioned supramolecular extraction method.

[0028] In the uric acid-lowering composition of the present application, in addition to the ampelopsin, the other components provide natural flavonoids, natural polyphenols, natural coenzymes, natural coumarins, natural phenolic acids and iridoid glycosides, etc. active factors, thereby playing a synergistic effect.

[0029] The present application also provides a uric acid-lowering embedded preparation comprising an embedding agent and the above-mentioned uric acid-lowering composition.

[0030] Preferably, the embedding agent is selected from one or more of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, whey protein, microcrystalline cellulose, sodium alginate and maltodextrin.

[0031] More preferably, the embedding agent is β-cyclodextrin, hydroxypropyl-β-cyclodextrin, whey protein or sodium alginate.

[0032] Preferably, the dosage form of the preparation is tablets, capsules, powders, granules or pills.

[0033] The present application also provides a preparation method of the above-mentioned embedded preparation, comprising the following steps: (1) dissolving the embedding agent in water, then adding the Ampelopsis megalophylla extract, Gardenia extract and pyrroloquinoline quinone disodium salt, homogenizing or ultrasonicating to obtain a homogenate; (2) adding the ampelopsin, Noni fruit extract, Portulaca oleracea extract, Eucommia ulmoides leaf extract, Cichorium intybus extract and Apium graveolens seed extract to the homogenate, mixing evenly, and drying to obtain the product.

[0034] Preferably, the mass ratio of the embedding agent, water, Ampelopsis megalophylla extract, Gardenia extract, pyrroloquinoline quinone disodium salt, ampelopsin, Noni fruit extract, Portulaca oleracea extract, Eucommia ulmoides leaf extract, Cichorium intybus extract and Apium graveolens seed extract is 1-5:30-90:1-5:0.4-2:0.02-1:1-3:2-5:1-3:2-4:1-3:2-4.

[0035] More preferably, the mass ratio of the embedding agent, water, Ampelopsis japonica leaves extract, Gardenia extract, pyrroloquinoline quinone disodium salt, anserine, Noni fruit extract, Portulaca oleracea extract, Eucommia ulmoides leaf extract, Cichorium intybus extract and celery seed extract is 2-5:30-80:1-3:1-2:0.02-0.3:1-3:2-4:1-3:2-4:1-3:2-4.

[0036] Further preferably, the mass ratio of the embedding agent, water, Ampelopsis japonica leaves extract, Gardenia extract, pyrroloquinoline quinone disodium salt, anserine, Noni fruit extract, Portulaca oleracea extract, Eucommia ulmoides leaf extract, Cichorium intybus extract and celery seed extract is 3:80:2:1.5:0.1:2:3:2:3:1.5:2.5.

[0037] Preferably, the homogenization conditions in step (1) include: 2-6 cycles of high pressure homogenization treatment at 25-50℃ and 50-100 MPa.

[0038] Preferably, the ultrasonic conditions in step (1) include: ultrasonic treatment at 40-60℃ and 200-300 W for 0.5-3 h.

[0039] The present application also provides a product with superimposed efficacy for reducing uric acid, comprising the above-mentioned composition for reducing uric acid.

[0040] The present application also provides the use of the above-mentioned composition for reducing uric acid or the above-mentioned embedding preparation in the preparation of a medicine for reducing uric acid.

[0041] Compared with the prior art, the present application has the following beneficial effects: (1) The present application extracts anserine with high bioavailability and high extraction rate through the synergistic effect of calcium ion catalyzed alkaline protease, special enzyme for aquatic products and flavor protease.

[0042] (2) The present application uses embedding technology to combine anserine with other effective components to obtain an embedding preparation for reducing uric acid. The preparation can quickly reduce the concentration of uric acid in serum, accelerate the excretion of uric acid, shorten the recovery time of kidney function, and has better effect on reducing uric acid than western medicines such as benzbromarone and allopurinol, with the advantages of simple prescription, obvious curative effect and no toxic and side effects.

[0043] (3) The anserine and the composition for reducing uric acid provided by the present application do not harm the liver and kidney, and have a repairing effect on damaged kidneys. DETAILED DESCRIPTION

[0044] It is worth mentioning that the raw materials used in the present application are all ordinary commercially available products. Among them, the extract of Ampelopsis acontifolia contains 98%; the extract of Gardenia contains 50% of gardenoside; the extract of Noni contains 90%; the extract of Portulaca contains 80%; the extract of Eucommia ulmoides contains 90%; the extract of Chichorium intybus contains 90%; the extract of Apium graveolens contains 90%; the enzyme for aquatic products is Papain 2709 from Nanning Dongheng Huadao Biological Technology Co., Ltd.; and the whey protein contains 80%.

[0045] Example 1 A preparation for reducing uric acid is prepared as follows: (1) 3 parts of hydroxypropyl-β-cyclodextrin are dissolved in 80 parts of water, and then 2 parts of Ampelopsis acontifolia extract, 1.5 parts of Gardenia extract and 0.1 part of pyrroloquinoline quinone disodium salt are added. The mixture is treated by 80 MPa high pressure homogenization for 4 cycles at 30°C to obtain a homogenate; (2) 2 parts of anmusipeptide, 3 parts of Noni extract, 2 parts of Portulaca extract, 3 parts of Eucommia ulmoides extract, 1.5 parts of Chichorium intybus extract and 2.5 parts of Apium graveolens extract are added to the homogenate and mixed uniformly, and then dried to obtain the preparation.

[0046] The extraction process of anmusipeptide is as follows: (1) Water is added to tuna muscle (the mass ratio of tuna muscle to water is 1:4), and the mixture is treated by colloid mill and then passed through a 50-mesh sieve to obtain a homogenate; (2) Part of calcium chloride (60% of the total calcium amount) is added to the homogenate, mixed for 0.5 h, and then β-cyclodextrin and alkaline protease are added. The mixture is subjected to intermittent ultrasonic enzymolysis at 50°C and 250 W for 1.5 h (intermittent for 20 min, and then ultrasonic for 10 min). Finally, the remaining calcium chloride (40% of the total calcium amount), the enzyme for aquatic products and flavor protease are added, and the mixture is subjected to intermittent ultrasonic enzymolysis under the same conditions for 2.5 h. The mass ratio of alkaline protease, the enzyme for aquatic products and flavor protease is 5:3:2 (i.e. compound enzyme), and the mass ratio of homogenate to compound enzyme is 100:1. The mass ratio of β-cyclodextrin to homogenate is 1:50. The final concentration of calcium chloride in the homogenate is 2.5 mM.

[0047] (3) After the enzymolysis is completed, the enzymolysis solution is centrifuged at a speed of 1500 rpm for 30 min to obtain an enzymolysis supernatant. The enzymolysis supernatant is filtered through a 3000 Da ultrafiltration membrane, and the filtrate is loaded onto a 724 cation exchange resin. The resin is eluted with 0.2 mol / L NH4Cl solution, and the eluate is collected and freeze-dried to obtain anmusipeptide. The extraction rate is 92.8%.

[0048] Example 2 A uric acid-lowering embedding preparation is prepared by the following method: (1) 2 parts of β-cyclodextrin are dissolved in 35 parts of water, then 2.5 parts of Ampelopsis acontifolia leaf extract, 1 part of Gardenia extract and 0.2 parts of pyrroloquinoline quinone disodium salt are added, and the mixture is treated by 60 MPa high-pressure homogenization for 4 cycles at 40°C to obtain a homogenate; (2) 2.5 parts of anmusipeptide, 3.5 parts of Noni fruit extract, 1.5 parts of Purslane extract, 4 parts of Eucommia ulmoides leaf extract, 2 parts of Chicory extract and 4 parts of Celery seed extract are added to the homogenate, mixed and dried to obtain the embedding preparation.

[0049] The extraction process of anmusipeptide is as follows: (1) Water is added to tuna muscle (the mass ratio of tuna muscle to water is 1:5), and the mixture is treated by colloid mill and then passed through a 100-mesh sieve to obtain a homogenate; (2) Part of calcium chloride (55% of the total calcium amount) is added to the homogenate, mixed for 0.5 h, then β-cyclodextrin and alkaline protease are added, and the mixture is subjected to intermittent ultrasonic enzymolysis at 50°C and 250 W for 1.5 h (intermittent for 20 min, then ultrasonic for 10 min), and finally the remaining calcium chloride (45% of the total calcium amount), special enzyme for aquatic products and flavor protease are added, and the mixture is subjected to intermittent ultrasonic enzymolysis under the same conditions for 2.5 h. The mass ratio of alkaline protease, special enzyme for aquatic products and flavor protease is 5:2:3 (i.e. compound enzyme), and the mass ratio of homogenate to compound enzyme is 100:1; the mass ratio of β-cyclodextrin to homogenate is 1:30; and the final concentration of calcium chloride in the homogenate is 3 mM.

[0050] (3) After the enzymolysis is completed, the enzymolysis solution is centrifuged at a speed of 1200 rpm for 35 min to obtain an enzymolysis supernatant, and then the enzymolysis supernatant is filtered through a 3000 Da ultrafiltration membrane, and finally the filtrate is loaded onto a 724 cation exchange resin, eluted with 0.2 mol / L NH4Cl solution, and the eluate is collected and freeze-dried to obtain anmusipeptide, and the extraction rate is 91.5%.

[0051] Example 3 A uric acid-lowering embedding preparation is prepared by the following method: (1) 2 parts of β-cyclodextrin are dissolved in 35 parts of water, then 2.5 parts of Ampelopsis acontifolia leaf extract, 1 part of Gardenia extract and 0.2 parts of pyrroloquinoline quinone disodium salt are added, and the mixture is treated by 60 MPa high-pressure homogenization for 4 cycles at 40°C to obtain a homogenate; (2) 2.5 parts of anmusipeptide, 3.5 parts of Noni fruit extract, 1.5 parts of Purslane extract, 4 parts of Eucommia ulmoides leaf extract, 2 parts of Chicory extract and 4 parts of Celery seed extract are added to the homogenate, mixed and dried to obtain the embedding preparation.

[0052] The extraction process of the anmusin is as follows: (1) First, water is added to the tuna muscle (the mass ratio of tuna muscle to water is 1:3), and a colloidal mill is used for treatment, and then a 50-mesh sieve is used to obtain a homogenate liquid; (2) Then, part of calcium chloride (58% of the total amount of calcium) is added to the homogenate liquid, mixed for 0.5 h, and then β-cyclodextrin and alkaline protease are added, and intermittent ultrasonic enzymolysis is carried out at 55°C and 200 W for 1 h (first intermittent for 20 min, and then ultrasonic for 10 min), and finally the remaining calcium chloride (42% of the total amount of calcium), special enzymes for aquatic products and flavor protease are added, and intermittent ultrasonic enzymolysis is continued under the same conditions for 2 h. The mass ratio of alkaline protease, special enzymes for aquatic products and flavor protease is 4:4:3 (i.e. compound enzyme), and the mass ratio of homogenate liquid to compound enzyme is 300:0.5; the mass ratio of β-cyclodextrin to homogenate liquid is 1:30; and the final concentration of calcium chloride in the homogenate liquid is 2 mM.

[0053] (3) After the enzymolysis is completed, the enzymolysis liquid is first centrifuged at a speed of 1000 rpm for 40 min to obtain an enzymolysis clear liquid, and then the enzymolysis clear liquid is filtered through a 5000 Da ultrafiltration membrane, and finally the filtrate is loaded onto a 724 cation exchange resin, eluted with 0.1 mol / L NH4Cl liquid, and the eluate is collected and freeze-dried to obtain the anmusin, and the extraction rate reaches 90.2%.

[0054] Example 4 A preparation for reducing uric acid is prepared by the following method: (1) First, 5 parts of microcrystalline cellulose are dissolved in 80 parts of water, and then 3 parts of Ampelopsis aconitifolia leaf extract, 1.5 parts of gardenia extract and 0.3 parts of pyrroloquinoline quinone disodium salt are added, and the mixture is treated by 100 MPa high-pressure homogenization for 2 cycles at 50°C to obtain a homogenate liquid; (2) 3 parts of anmusin, 4 parts of noni fruit extract, 1 part of purslane extract, 4 parts of eucommia leaf extract, 3 parts of chicory extract and 2 parts of celery seed extract are added to the homogenate liquid, and then mixed and dried to obtain the preparation.

[0055] The extraction process of the anmusin is as follows: (1) First, water is added to the tuna muscle (the mass ratio of tuna muscle to water is 1:3), and a colloidal mill is used for treatment, and then a 50-mesh sieve is used to obtain a homogenate liquid; (2) Then add part of calcium chloride (60% of the total calcium amount) to the homogenate, mix for 0.5 h, then add β-cyclodextrin and alkaline protease, and perform intermittent ultrasonic enzymolysis at 60°C and 300 W for 2 h (intermittent for 20 min first, then ultrasonic for 10 min), finally add the remaining calcium chloride (40% of the total calcium amount), special enzyme for aquatic products and flavor protease, and continue to perform intermittent ultrasonic enzymolysis under the same conditions for 3 h. The mass ratio of alkaline protease, special enzyme for aquatic products and flavor protease is 6:2:1 (i.e. compound enzyme), and the mass ratio of homogenate to compound enzyme is 150:5; the mass ratio of β-cyclodextrin to homogenate is 1:25; and the final concentration of calcium chloride in the homogenate is 10 mM.

[0056] (3) After the enzymolysis is completed, centrifuge the enzymolysis solution at a speed of 1000 rpm for 40 min to obtain an enzymolysis clear solution, then pass the enzymolysis clear solution through a 5000 Da ultrafiltration membrane, and finally load the filtrate onto a 724 cation exchange resin, elute with 0.3 mol / L NH4Cl solution, collect the eluate, and freeze-dry to obtain anserine, with an extraction rate of 91.0%.

[0057] Comparative Example 1 Compared with Example 1, the only difference is that the compound enzyme in the anserine extraction process is alkaline protease and special enzyme for aquatic products with a mass ratio of 5:3, and anserine with an extraction rate of 78.4% is obtained. Further, a uric acid-lowering embedding preparation is prepared.

[0058] Comparative Example 2 Compared with Example 1, the only difference is that the compound enzyme in the anserine extraction process is alkaline protease and flavor protease with a mass ratio of 5:2, and anserine with an extraction rate of 61.3% is obtained. Further, a uric acid-lowering embedding preparation is prepared.

[0059] Comparative Example 3 Compared with Example 1, the only difference is that the compound enzyme in the anserine extraction process is special enzyme for aquatic products and flavor protease with a mass ratio of 3:2, and anserine with an extraction rate of 75.2% is obtained. Further, a uric acid-lowering embedding preparation is prepared.

[0060] Comparative Example 4 Compared with Example 1, the only difference is that the calcium chloride in the anserine extraction process is replaced by zinc chloride, and anserine with an extraction rate of 68.7% is obtained. Further, a uric acid-lowering embedding preparation is prepared.

[0061] Comparative Example 5 Compared with Example 1, the only difference is that the β-cyclodextrin in the anserine extraction process is replaced by malt dextrin, and anserine with an extraction rate of 52.4% is obtained. Further, a uric acid-lowering embedding preparation is prepared.

[0062] Comparative Example 6 Compared with Example 1, the only difference is that hydroxypropyl-β-cyclodextrin is replaced by chitosan to prepare the uric acid-lowering embedding preparation.

[0063] Test Example 1 The uric acid-lowering embedding preparation of Example 1 was subjected to a mouse uric acid-lowering function test, as follows: 1. Sample to be tested The uric acid-lowering embedding preparation prepared in Example 1.

[0064] 2. Experimental animals ICR male mice, 4-6 weeks old, 60 mice, mice were adapted for 2 weeks, and free feeding and drinking water during the adaptation period.

[0065] 3. Experimental method After the adaptation period, the experiment was officially started for 14 days, and the mice were randomly divided into 6 groups, namely the blank control group, the model group, the positive control group and Example 1 (low-dose group, medium-dose group and high-dose group), 10 mice in each group.

[0066] During the entire experiment (Day 1-14), at 9:00 am every day, the blank control group and the model group were given 0.5% carboxymethylcellulose sodium (CMC-Na) solution by gavage; the positive control group was given 150 mg / kg benzbromarone by gavage; the low, medium and high dose groups of Example 1 were given 20 mg / kg, 100 mg / kg and 500 mg / kg of the uric acid-lowering embedding preparation by gavage, respectively. From the fourth day (Day 4), at 8:00 am every day (i.e. about 1 hour before receiving gavage), except for the blank control group of mice injected with normal saline, the other groups of mice were injected with the same volume of modeling solution (250 mg / kg oxonate and 150 mg / kg hypoxanthine) intraperitoneally. During this period, the mice were free to eat and drink.

[0067] On the day of the end of the experiment (Day 14), at 10:00, the whole blood of each mouse was collected separately, 30-50 μL, and stored in an anticoagulant tube.

[0068] 4. Index detection The whole blood was placed in an anticoagulant tube and centrifuged at 3000 rpm and 4°C for 15 min. The supernatant (plasma) was mixed with methanol at a volume ratio of 1:4, shaken and mixed, then centrifuged at 10000 rpm for 10-15 min. An appropriate amount of supernatant was centrifuged at 12000 rpm for 10 min, and then subjected to HPLC detection. After drawing a standard curve using different concentrations of uric acid standard solution, the peak area of the sample to be tested was quantitatively converted to obtain the blood uric acid concentration.

[0069] The HPLC detection conditions are as follows: C18 chromatographic column; mobile phase A: 0.52 mM sodium pentane sulfonate and 0.2 M aqueous potassium dihydrogen phosphate, mobile phase B: acetonitrile; detection wavelength: 254 nm.

[0070] 5. Experimental results The results are shown in Table 1. As can be seen from Table 1, compared with the blank control group, the modeling treatment caused the blood uric acid concentration of the mice to increase by 28 times (P < 0.001), as high as 324.4 ± 64.2 μM, indicating that the hyperuricemia modeling was successful. The different doses of the uric acid-lowering embedded preparation of Example 1 can promote the relief of the phenotype of the hyperuricemia mice, and with the increase of the dose, the blood uric acid content gradually decreases, showing a dose effect. p <0.001), as high as 324.4 ± 64.2 μM, indicating that the hyperuricemia modeling was successful. The different doses of the uric acid-lowering embedded preparation of Example 1 can promote the relief of the phenotype of the hyperuricemia mice, and with the increase of the dose, the blood uric acid content gradually decreases, showing a dose effect.

[0071] The blood uric acid concentration of the dose group in Example 1 is not much different from that of the positive control group, indicating that the dose of the uric acid-lowering embedded preparation in the application can achieve an effect comparable to that of benzbromarone, wherein the high-dose group of Example 1 can reduce the blood uric acid concentration to 172.4 ± 20.4 μM, which is 53.1% of the content of the model group.

[0072] Table 1 Blood uric acid concentration of mice

[0073] Note: compared with the control group, ### indicates p <0.001; compared with the model group, @@ indicates p <0.01, @@@ indicates p <0.001.

[0074] Test Example 2 The uric acid-lowering embedded preparations prepared in Examples 1-4 and Comparative Examples 1-6 were respectively subjected to zebrafish uric acid-lowering function tests, specifically as follows: 1. Test sample The uric acid-lowering embedded preparations prepared in Examples 1-4 and Comparative Examples 1-6.

[0075] 2. Experimental animals 5 dpf wild-type AB zebrafish larvae.

[0076] 3. Experimental method 3.1 Acute toxicity experiment of the uric acid-lowering embedded preparation of Example 1 600 AB zebrafish larvae were selected and treated with different concentrations of the uric acid-lowering embedding preparation of Example 1, and 3 parallels were set for each group (n = 10 per group). The uric acid-lowering embedding preparation treatment groups were divided into I, II, III, and IV, a total of 4 groups, and each uric acid-lowering embedding preparation was set up at five concentration gradients: 500 μg / mL, 250 μg / mL, 125 μg / mL, 60 μg / mL, and 30 μg / mL. After incubation in a constant temperature incubator at 28.5°C for 48 h, the mortality of each group was recorded.

[0077] 3.2 Uric acid-lowering effect of the uric acid-lowering embedding preparation of the application on zebrafish with hyperuricemia 390 AB zebrafish were selected and divided into 13 groups, and the experiment was divided into a blank control group, a model group, a positive control group, and uric acid-lowering embedding preparation treatment groups prepared from Examples 1-4 and Comparative Examples 1-6, with 3 parallels set for each group (n = 10 per group). The zebrafish were placed in a six-well plate, 10 zebrafish per well, and the volume of each well was 4 mL. The plates were labeled with the group and treatment information and placed in an incubator at 28.5°C for incubation.

[0078] From the fifth day of incubation (Day 5), the rest of the groups were given 600 μM potassium oxonate in the six-well plate for 24 h, except for the blank control group, and on the seventh day (Day 7), the treatment of 30 μM sodium xanthate was continued for 24 h, thereby constructing a zebrafish hyperuricemia model.

[0079] On the ninth day (Day 9), the positive control group was given 2000 μM allopurinol (APL), and the Examples 1-4 and Comparative Examples 1-6 groups were given 500 μg / mL of the corresponding uric acid-lowering embedding preparation, except for the blank control group and the model group, and incubated in the six-well plate for 24 h. After the experiment ended on the tenth day (Day 10), the experimental animals were treated, and the uric acid levels of different experimental groups were detected to determine the uric acid-lowering effect of the uric acid-lowering embedding preparation of the application.

[0080] 4. Detection of uric acid content After the drug treatment of the zebrafish larvae was completed, the zebrafish were collected in a 1.5 mL EP tube, and the liquid in the EP tube was sucked out. 40 μL of PBS was added to each EP tube, and the collected zebrafish were homogenized and broken up on ice using a homogenizer until there were no obvious tissue fragments. Centrifugation was performed at 15000 x g and 4°C for 15 min. The supernatant was transferred to a new tube, and the protein concentration and uric acid concentration were determined using a BCA kit and high-performance liquid chromatography. The remaining was stored in a -80°C refrigerator for standby use.

[0081] High-performance liquid chromatography and double-flow gradient were used to appropriately detect and separate all target analytes. The high-performance liquid chromatography acquisition conditions were as follows: Chromatographic column: Microsorb-MV 100-5 C18 (150x4.6 mm); mobile phase A: ultrapure water containing 0.52 mmol / L of sodium 1-pentanesulfonate and 0.20 mmol / L of potassium dihydrogen phosphate, pH=4.0; mobile phase B: 10% acetonitrile solution; elution condition: isocratic elution, A phase:B phase=85:15 (V / V); flow rate set to 1.0 mL / min; column temperature 25°C; detection wavelength 254 nm; injection volume 10 μL; running time 15 min.

[0082] After equilibrating the chromatographic column with the mobile phase for at least 30 min, start injecting samples, and the sampling sequence is from low concentration to high concentration samples, and a needle of methanol needs to be run before each group of samples is injected. Among them, the mobile phase A is prepared as 10x stock solution in advance, stored at 4°C, and diluted to 1x working solution with ultrapure water when used, and the pH is adjusted to 4.0 with 75% phosphoric acid, and then filtered and ultrasonicated for standby.

[0083] According to the protein standard and the number of zebrafish samples, prepare an appropriate amount of BCA working solution according to the instructions of the Biyun Tian BCA protein concentration determination kit, mix thoroughly, and store at room temperature for 24 h. Add 20 μL of protein standard / zebrafish sample to the sample well of the 96-well plate, add 200 μL of BCA working solution to each well, incubate at 37°C in the dark for 30 min, and use the enzyme label instrument to determine the absorbance value at 562 nm. According to the protein standard curve, the protein concentration of the zebrafish sample is calculated.

[0084] The uric acid concentration of zebrafish is determined by high performance liquid chromatography, and based on the uric acid standard curve and the protein standard curve, the relative content of uric acid in the protein of the zebrafish sample is calculated.

[0085] 5. Experimental results 5.1 Acute toxicity experiment results As shown in Table 2, during the acute toxicity experiment period, the zebrafish treated with the uric acid-lowering embedded preparation of Example 1 at different concentrations survived well within 48 h, with normal behavior, and no obvious lethal effect was observed.

[0086] Table 2 Lethal rate of the uric acid-lowering embedded preparation of Example 1 at different concentrations

[0087] Note: "-" indicates that the lethal rate is 0.

[0088] 5.2 Uric acid-lowering experiment results of the uric acid-lowering embedded preparation of the application As shown in Table 3, after the treatment of potassium oxonate and sodium xanthine salt modeling, the uric acid level in zebrafish was increased by more than 3 times, reaching 29.5±5.3 μmol / g; under the treatment condition of the uric acid-lowering embedded preparation with a concentration of 500 μg / mL, the treatment groups of Examples 1-4 all had a significant effect on reducing the uric acid level of zebrafish. Among them, compared with the model group, the positive drug control group (allopurinol) could reduce the uric acid level of zebrafish by about 27.1%, Example 1 could reduce the uric acid level of zebrafish by about 54.2%, Example 2 could reduce the uric acid level of zebrafish by about 49.8%, Example 3 could reduce the uric acid level of zebrafish by about 48.8%, and Example 4 could reduce the uric acid level of zebrafish by about 43.7%.

[0089] In addition, the uric acid-lowering level of the treatment groups of Examples 1-4 of the present application was significantly better than that of the positive control group, indicating that the uric acid-lowering effect of the uric acid-lowering embedded preparation of the present application was better than that of the western medicine allopurinol, which could quickly reduce the uric acid concentration in serum, accelerate the excretion of uric acid, shorten the recovery time of kidney function, and had the advantages of simple prescription, obvious curative effect and no toxic side effects.

[0090] Table 3 Effect of the uric acid-lowering embedded preparation of the present application on the uric acid-lowering level of zebrafish

[0091] Note: compared with the model group, # indicates p <0.05, ## indicates p <0.01, ### indicates p <0.001.

[0092] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A method for extracting an ultramolecular peptide of anisomelic peptide, characterized by, It comprises the following steps: (1) adding water to tuna muscle, crushing to obtain homogenate liquid; (2) adding alkaline protease and cyclodextrin to the homogenate liquid first, then adding aquaculture special enzyme and flavor protease, carrying out enzymolysis, adding catalyst in batches, purifying, and obtaining the product.

2. The supramolecular extraction method according to claim 1, characterized in that, In step (1), the mass ratio of tuna muscle to water is 1:3-6; the crushing is carried out by colloid mill treatment and passing through a 50-100 mesh screen.

3. The supramolecular extraction method of claim 1, wherein, In step (2), the mass ratio of alkaline protease, aquaculture special enzyme and flavor protease is 4-6:2-4:1-3.

4. The supramolecular extraction method according to claim 3, characterized in that, The mass ratio of the sum of homogenate liquid, alkaline protease, aquaculture special enzyme and flavor protease is 150-300:0.5-4.

5.

5. The supramolecular extraction method of claim 1, wherein, In step (2), the cyclodextrin is hydroxypropyl-β-cyclodextrin or β-cyclodextrin; the mass ratio of cyclodextrin to homogenate liquid is 1:25-50; the enzymolysis condition is intermittent ultrasonic enzymolysis at 40-60℃, 200-300 W for 0.5-5 h.

6. The supramolecular extraction method of claim 1, wherein, In step (2), the catalyst is calcium ion, which is from calcium chloride, calcium lactate or calcium citrate, and the final concentration of calcium ion in the homogenate liquid is 1-10 mM.

7. The supramolecular extraction method according to claim 6, characterized in that, In step (2), the process of adding catalyst in batches comprises adding 50-60% of the total calcium first, and then adding 40-50% of the total calcium.

8. The supramolecular extraction method of claim 1, wherein, In step (2), the process of purification comprises centrifuging at 1000-1500 rpm for 20-40 min to obtain enzymolysis supernatant, then passing the enzymolysis supernatant through a 3000-5000 Da ultrafiltration membrane, and finally loading the filtrate onto a 724 cation exchange resin, eluting with 0.1-0.3 mol / L NH4Cl solution, collecting the eluate, and drying to obtain the product.

9. A composition for reducing uric acid, comprising, It comprises the following components: anjin peptide, pyrroloquinoline quinone disodium salt, ampelopsis japonica leaf extract, gardenia extract, noni fruit extract, portulaca oleracea extract, eucommia ulmoides leaf extract, chicory extract and celery seed extract obtained by the supramolecular extraction method according to any one of claims 1-8.

10. A uric acid-lowering embedded formulation, characterized in that, It comprises embedding agent and the uric acid-lowering composition of claim 9.

11. The embedding formulation of claim 10, wherein, The embedding agent is selected from one or more of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, whey protein, microcrystalline cellulose, sodium alginate and malt dextrin, and the dosage form of the preparation is tablet, capsule, powder, granule or pill.

12. A method for preparing an embedding formulation as claimed in claim 10 or 11, characterized in that It comprises the following steps: (1) dissolving the embedding agent in water, then adding ampelopsis japonica leaf extract, gardenia extract and pyrroloquinoline quinone disodium salt, homogenizing or ultrasonic, to obtain homogenate liquid; (2) adding anjin peptide, noni fruit extract, portulaca oleracea extract, eucommia ulmoides leaf extract, chicory extract and celery seed extract to the homogenate liquid, mixing evenly, and drying to obtain the product.

13. The method of claim 12, wherein, The mass ratio of embedding agent, water, ampelopsis japonica leaf extract, gardenia extract, pyrroloquinoline quinone disodium salt, anjin peptide, noni fruit extract, portulaca oleracea extract, eucommia ulmoides leaf extract, chicory extract and celery seed extract is 1-5:30-90:1-5:0.4-2:0.02-1:1-3:2-5:1-3:2-4:1-3:2-4.

14. The method of claim 12, wherein, In step (1), the homogenization conditions include: 2-6 cycles of high pressure homogenization at 25-50℃, 50-100 MPa; the ultrasonic conditions include: 0.5-3 h of ultrasonic treatment at 40-60℃, 200-300 W.

15. A performance stack hypouricemic product, characterized in that, The composition for reducing uric acid of claim 9.

16. Use of the composition for reducing uric acid of claim 9 or the embedding preparation of claim 10 or 11 in the preparation of a product for preventing and / or treating hyperuricemia and acute gout.

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