CN117482126B - Anserine composition for inhibiting absorption of food-borne purine and application thereof - Google Patents
Anserine composition for inhibiting absorption of food-borne purine and application thereof Download PDFInfo
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- CN117482126B CN117482126B CN202311839657.2A CN202311839657A CN117482126B CN 117482126 B CN117482126 B CN 117482126B CN 202311839657 A CN202311839657 A CN 202311839657A CN 117482126 B CN117482126 B CN 117482126B
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- anserine
- composition
- chicory
- extract
- purine
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention belongs to the technical field of biological medicines, and discloses a anserine composition for inhibiting absorption of food-borne purine and application thereof. The goose carnosine composition is prepared by mixing goose carnosine, chicory extract and euglena extract according to the weight ratio of 75-85:10-20:2-12. Proved by verification, the anserine composition provided by the invention has the beneficial effects of obviously inhibiting the absorption of purine compounds, reducing the expression of CNT2 transport proteins, promoting the metabolism of the purine compounds into allantoin, regulating intestinal flora and the like. The anserine composition provided by the invention has the advantages of remarkable effect, mildness, safety and no side effect, and provides a feasible scheme for the research and development of medical foods, health-care products, pharmaceutical compositions and the like for patients with gout or hyperuricemia.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a anserine composition for inhibiting absorption of food-borne purine and application thereof.
Background
Hyperuricemia is a metabolic disease caused by purine metabolic disorders and/or uric acid excretion disorders, which is clinically defined as: the fasting serum uric acid content of men is more than 420 mu mol/L, and the fasting serum content of women is more than 357 mu mol/L. With the improvement of living standard and the change of dietary structure, the incidence of hyperuricemia is increasing. Hyperuricemia has become the second most metabolic disease next to diabetes and presents a trend of younger men than women, coastal areas than inland areas.
The causative factors of hyperuricemia are diverse, and purine metabolic disorder and uric acid excretion disorder are the main intrinsic factors inducing hyperuricemia, and food-borne purine malabsorption is the direct extrinsic factor inducing hyperuricemia. With the improvement of living standard, the excessive intake of high-purine foods such as meat, seafood, beer and the like aggravates the risk of hyperuricemia. At present, clinical medicines aiming at inhibiting purine absorption or preventing hyperuricemia are very deficient, and only purine intake can be reduced by controlling daily diet, so that the life quality of patients is seriously affected. Therefore, the development of a safe drug or health-care product capable of inhibiting purine absorption would be of great significance.
The goose carnosine is histidine dipeptide naturally existing in vertebrate muscles and brains, is abundant in chicken, beef and marine fish muscles, and has a remarkable uric acid reducing function. However, the molecular mechanism of reducing uric acid by anserine is not known at present, and whether the anserine has an effect of inhibiting the absorption of purine compounds is not reported. Compared with other existing approaches, if the anserine can inhibit the absorption of purine, the problems of diet limitation, life quality reduction and the like of gout patients or hyperuricemia patients can be radically solved.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a anserine composition, and the technical scheme of the invention is as follows.
The invention provides a goose carnosine composition, which comprises the active components of goose carnosine, chicory extract and euglena extract, wherein the mass ratio of the goose carnosine to the chicory extract to the euglena extract is 75-85:10-20:2-12, and the goose carnosine composition is used for inhibiting purine compounds from absorbing.
The preparation method of the chicory extract comprises the following steps: crushing the roots of the chicory, sieving the crushed roots of the chicory with a 100-mesh sieve to obtain chicory powder, extracting the chicory powder with water at 80-90 ℃ for 4-5 times, each time for 1-2 hours, combining the filtrates, concentrating under reduced pressure, carrying out alcohol precipitation on the water extract of the chicory powder with 40% -50% ethanol for 20-40 hours, filtering, and freeze-drying the alcohol precipitate to obtain the chicory extract.
The preparation method of the euglena extract comprises the following steps: sieving Euglena dry powder with 80 mesh sieve, soaking in acetic acid aqueous solution, heating at 40deg.C under stirring for 6 hr, adding equal volume of sodium hydroxide aqueous solution, heating under stirring for 4 hr, standing, filtering to obtain filtrate, adding absolute ethanol for alcohol precipitation, standing, filtering to collect alcohol precipitate, and freeze drying to obtain Euglena extract.
Further, in the above composition, the anserine is prepared by chemical synthesis or extraction, and the anserine extraction preparation method comprises the following steps: mincing fish meat with water, homogenizing, performing enzymolysis according to the addition amount of papain 500U/g and flavourzyme 200U/g, inactivating enzyme, centrifuging at 4deg.C and 3500×g for 15min, deoiling, decolorizing, separating, purifying, and lyophilizing to obtain anserine dry powder.
The invention provides an application of the anserine composition in preparing a medicament for preventing and/or treating gout or hyperuricemia.
In one aspect, in the above application, the anserine composition has the effect of reducing the expression of CNT2 transporter and thus inhibiting the absorption of purine compounds.
In another aspect, in the above application, the anserine composition has the effect of promoting the metabolism of purine compounds into allantoin.
Also, in the above application, the anserine composition has an effect of regulating intestinal flora. At the phylum level, the anserine composition has the effect of increasing the relative abundance of microorganisms of the phylum firmicutes of the intestinal tract; at the genus level, the anserine composition has the effect of increasing the relative abundance of microorganisms of the genus lactobacillus.
The invention also claims a medicine for preventing and/or treating gout or hyperuricemia, and the active ingredients of the medicine contain the anserine composition.
The invention also claims a health-care product or medical food for preventing and/or treating gout or hyperuricemia, and the active ingredients of the health-care product or medical food contain the anserine composition.
Compared with the prior art, the anserine composition for inhibiting the absorption of the food-borne purine and the application thereof have the following beneficial effects:
The anserine composition provided by the invention has the effect of inhibiting absorption of food-borne purine. CNT2 transporter is a concentration-dependent protein that exists in the small intestine and, upon ingestion of foods containing a large amount of purine compounds, the expression level of CNT2 transporter is increased accordingly to promote absorption of purine compounds. According to the research, the anserine composition can reduce the expression quantity of the CNT2 transporter in the small intestine, and the high-dose group has the best inhibition effect on the expression of the CNT2 transporter. The goose carnosine composition can inhibit the absorption of purine compounds in food by the small intestine by reducing the expression level of the CNT2 transporter.
The anserine composition provided by the invention has the effect of promoting food-borne purine metabolism. Compared with the model group, the anserine composition can significantly increase the content of allantoin in the intestinal contents of rats, and the allantoin is one of the end products of purine metabolism, which indicates that the anserine composition can promote the catabolism of purine compounds to generate the allantoin.
The anserine composition provided by the invention has the effect of regulating intestinal flora. The anserine composition is capable of significantly increasing beneficial microorganisms (e.g., lactobacillus) in the intestine and decreasing harmful microorganisms (e.g., proteus, actinomycetes) in the intestine, as compared to the model group, and this change in intestinal colonial structure indicates that the anserine composition can help restore the disorder of intestinal microbiota.
In summary, the invention provides a goose carnosine composition for gout patients or hyperuricemia patients, which has the beneficial effects of inhibiting purine compound absorption, reducing expression of CNT2 transport protein, promoting purine compound metabolism, regulating intestinal flora and the like, has the potential of preparing health care products, medical foods or pharmaceutical compositions, and provides a selection way for treating gout or hyperuricemia.
Drawings
FIG. 1 shows the effect of anserine composition on the purine content of rat blood.
FIG. 2 is a graph showing the effect of a anserine composition on rat blood uric acid levels.
FIG. 3 is a graph showing the effect of anserine compositions on expression of the rat intestinal CNT2 transporter.
FIG. 4 is a graph showing the effect of a anserine composition on the microbial composition of the rat gut at the portal level.
FIG. 5 is a graph showing the effect of anserine composition on the relative abundance of Lactobacillus intestinal in rats.
In the drawings of the present invention, "composition-low" refers to a low dose group of a composition, "composition-medium" refers to a medium dose group of a composition, "composition-high" refers to a high dose group of a composition, "chicory, euglena binary extract" and "binary extract control" each refer to a chicory, euglena binary extract group.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The anserine of the present invention may be prepared using the procedure recommended in example 1, or using commercially available anserine products. The technical object of the present invention can be achieved by the anserine prepared in example 1, or by using the anserine commercially available.
Example 1
This example provides methods of extraction of the components and preparation of the anserine composition.
1. Separation and extraction of anserine
Taking bonito with flat rudder, mincing, adding distilled water 900mL into every 100g of fish meat, homogenizing, and performing enzymolysis on every gram of fish meat for 3 hours under the condition of pH=6.5 according to the addition amount of 500U/g papain and 200U/g flavourzyme, and centrifuging the enzymolysis product for 15 minutes at 3500×g at 4 ℃ after enzyme deactivation in water bath. The supernatant obtained by centrifugation is deoiled by a tube centrifuge, and then 3% perlite is added for decolorization. Performing primary filtration by using a ceramic membrane (the pressure is 2 multiplied by 10 5 Pa, the time is 360 min), performing primary separation and low molecular peptide enrichment on the primary filtrate by using a 5000Da ultrafiltration membrane and a 1000Da ultrafiltration membrane, and then further separating and purifying the low molecular peptide by using gel chromatography and a C18 reversed phase chromatographic column under the chromatographic conditions: ZORBAX SB-Phenyl (250 mm. Times.4.6 mm), 20mmo/L dipotassium hydrogen phosphate (pH=7.80): methanol=85:15; flow rate: 0.6mL/min; the column temperature is 25 ℃, the detection wavelength is 210nm, the sample injection amount is 10 mu L, and the purified sample is collected for freeze drying under the following conditions: pre-freezing at-40deg.C under 10Pa, and cold-trap at-70deg.C to obtain anserine dry powder.
2. Preparation of chicory extract
Selecting chicory root, cleaning, airing, crushing, and sieving with a 100-mesh sieve to obtain chicory powder. Adding 10 times of purified water into the chicory powder, extracting for 4-5 times at 80-90 ℃ for 1-2 hours each time, merging filtrate, concentrating under reduced pressure, carrying out alcohol precipitation on the water extract by 40% -50% ethanol for 20-40 hours, filtering and recovering ethanol, and freeze-drying the alcohol precipitate, wherein the conditions are as follows: pre-freezing at-40deg.C under 10Pa, and cooling in cold trap at-70deg.C to obtain herba Cichorii extract.
3. Preparation of Euglena extract
The euglena dry powder is sieved by a 80-mesh sieve, soaked by 10 times of acetic acid aqueous solution (pH=6.5) and heated and stirred for 6 hours in a water bath kettle at 40 ℃, then sodium hydroxide aqueous solution (pH=7.5) with the same volume as the acetic acid aqueous solution is added for continuous heating and stirring for 4 hours, and the mixture is stood for 8 hours and filtered to obtain filtrate. Adding absolute ethyl alcohol into the filtrate for alcohol precipitation, standing for 8 hours, filtering and collecting alcohol precipitate, and freeze-drying under the following conditions: pre-freezing at-40deg.C under 10Pa, and cooling to-70deg.C to obtain Euglena extract.
4. Combined proportion of anserine composition
And uniformly mixing the prepared anserine dry powder, the chicory extract and the euglena extract to prepare the anserine composition. The goose carnosine, namely chicory extract, namely euglena extract=75-85:10-20:2-12 in terms of mass ratio. Further preferably, the anserine is chicory extract to euglena extract=75:15:10 in mass ratio. Further preferably, the goose carnosine: chicory extract: euglena extract=80:12:8 by mass ratio. Further preferably, the anserine is chicory extract to euglena extract=85:10:5 in mass ratio. Further preferably, the anserine is chicory extract to euglena extract=76:20:2 in mass ratio.
Example 2
This example provides the inhibitory effect of anserine compositions on the intestinal absorption of food-borne purine compounds in rats. The anserine composition used in this example comprises, in mass ratio, anserine from chicory extract to euglena extract=80:12:8.
1. Grouping and management of laboratory animals
84 Male rats with the weight of 200+/-20 g are subjected to free feeding with basic feed and distilled water for 7 days, and the feeding conditions are as follows: 24+/-2 ℃ and 55% -60% of relative humidity, and 12 hours of light/dark circulation. All rats were then randomly divided into 7 groups of 12, the specific groupings and management are shown in table 1.
The feeding period of the rats was 35d, the rats were sacrificed on day 35 by capillary tube Cong Caixie at the orbital veins of the rats, fasted and free drinking water was given 12 hours before the death of the rats, the anesthetized rats were intraperitoneally injected with sodium pentobarbital (dose 30mg/kg, concentration 3 mg/mL), the rats were sacrificed by breaking the cervical vertebrae after blood collection of the eyeballs, and the small intestine and intestinal contents were collected.
TABLE 1 animal experiment design
Note that: mg/kgBW.d represents mg per kg body weight per day, and 15 g/kgBW.d represents 15g per kg body weight per day.
2. Index measurement
1) HPLC (high Performance liquid chromatography) detection of purine content of yeast powder and rat serum
Sample pretreatment: after 2h of gastric lavage of the yeast powder, the orbital vein Cong Caixie 0.5.5 mL of the rat, and the blood sample of the rat was centrifuged at 3000r/min for 10min to obtain serum. Weighing yeast powder, adding distilled water for dissolution, and preparing a 1000 mug/mL solution. Adding 3 times of methanol into the rat serum and yeast powder solution respectively, standing at 4deg.C for 12 hr, centrifuging at 12000r/min for 20min, and collecting supernatant.
Chromatographic conditions: mobile phase: 50mmol/L ammonium acetate acetonitrile; flow rate: 1.0mL/min; ph=4.7; sample loading amount: 10L (100 [ mu ] g/mL); gradient conditions: 0-6 min:98 percent of 2 percent; 6-8 min:98% -95%; 8-30 min:95 percent of 5 percent; ultraviolet detection wavelength 254nm; column temperature was 30 ℃.
Preparing a standard solution: and accurately weighing 10.0mg of guanylic acid (GMP), 5' -sodium Inosinate (IMP), adenylic Acid (AMP), hypoxanthine, xanthine, guanine, adenine, inosine, guanosine and adenosine respectively, using a small amount of 1.0mol/L NaOH solution for assisting dissolution, adding distilled water to a volume of 10.0mL, and preparing a standard substance solution of 1.0 mg/mL.
Drawing a standard curve: preparing standard substance solutions with serial gradients of 1, 5, 10, 20, 40, 80 and 100 mug/mL, performing high performance liquid chromatography analysis by using a determined optimal chromatography method, and drawing standard curves of 10 purine compounds according to peak areas and standard substance concentrations.
The results show that 10 purine compounds are detected in the yeast powder, the content of the purine compounds is shown in table 2, the total content of the purine compounds accounts for 17.79% of the mass of the yeast powder, the variety is rich, and the yeast powder meets the condition that the purine sources in daily diet are complex and various, and is a high-quality hyperuricemia inducer.
TABLE 2.1000 content of purine Compounds in mu g/mL Yeast powder
7 Purine compounds can be detected in the serum of the rats, and the total amount of the purine compounds of the rats in different treatment groups is changed along with the feeding time as shown in figure 1. Compared with the blank group, the total amount of purine compounds of the yeast powder fed rats is obviously increased. Compared with a model group, the total amount of purine compounds in a low-dose, medium-dose and high-dose group of the anserine composition, a anserine control group, a chicory and naked-algae binary extract group is obviously reduced, the inhibition effect of the anserine composition group on purine absorption is superior to that of the anserine composition group, the chicory and naked-algae binary extract group, the high-dose group of the anserine composition has the best effect, and the total amount of serum purine compounds is reduced to 12.45 mug/mL.
2) HPLC (high Performance liquid chromatography) detection of serum uric acid content of rats
The treatment method and the detection method of the rat blood sample are the same.
As a result, as shown in fig. 2, serum uric acid levels of rats in the blank group increased significantly with the prolongation of the feeding time. Compared with the blank group, uric acid levels of rats in the model group gradually rise with the prolongation of the feeding time, and reach hyperuricemia levels on day 14. Compared with a model group, the blood uric acid content of rats in the anserine composition group is firstly increased and then decreased, and the uric acid level of rats in the anserine composition group is lower than that of rats in the model group at each blood sampling time point, which shows that the anserine composition effectively inhibits abnormal increase of uric acid of rats induced by yeast powder, and the effect is superior to that of a anserine control group and a chicory and naked algae binary extract group, and the blood uric acid content of rats in the anserine composition group with low, medium and high doses is respectively decreased to 78.88, 82.16 and 67.94 mug/mL.
3) Allantoin detection
0.10G of the intestinal contents of the rats are weighed, 5.0mL of distilled water is added for uniform mixing, magnetic beads are added for grinding for 2min at 90Hz in a tissue grinder, and then the mixture is centrifuged for 20min at 12000r/min, and the supernatant is taken. HPLC detection conditions: the mobile phase is water and methanol (83:17); the flow rate is 1.0mL/min; the ultraviolet detection wavelength is 210nm; the loading amount is 10 mu L (standard substance: 100 mu g/mL), and the experimental results are shown in Table 3.
TABLE 3 allantoin content of the intestinal contents of rats
The results show (Table 3) that the allantoin content was significantly increased in rats in the model group compared to the blank group, because the yeast powder provided abundant purine compounds, which were decomposed in the intestinal tract to form allantoin. Compared with the model group, the anserine composition group (low dose group, medium dose group and high dose group), the anserine control group and the chicory and naked alga binary extract group are used for remarkably increasing the allantoin content of the intestinal contents of rats after intervention, which indicates that the anserine composition can promote the catabolism of purine compounds and promote the generation of allantoin.
4) Determination of intestinal CNT2 transporter
Sample treatment: 0.10g of small intestine sample was weighed, washed 3 times with cold PBS solution to remove dirt, 10 times of tissue lysate (main ingredients are 50mM tromethamine, 150mM sodium chloride, 1% triton X-100 and 1% deoxycholate sodium) containing SPMF was added, and after grinding in a tissue grinder for 2min, the sample was cracked on ice for 30min (during which time it was repeatedly blown by a pipette), and then centrifuged at 12000r/min for 15min to collect a supernatant protein sample.
Protein concentration determination: and taking a proper amount of protein sample, and adding distilled water according to the ratio of 1:5 for dilution. And (3) detecting the protein concentration by a BCA method, adding a buffer solution into a 96-well plate according to the operation steps of a kit instruction, respectively adding 20 mu L of standard protein and 20 mu L of diluted samples, incubating for 20min at a constant temperature of 37 ℃, detecting a light absorption value by an enzyme-labeled instrument 595nm, substituting the light absorption value of the detected sample protein into a standard curve equation, and calculating the value of X to obtain the protein concentration of the sample.
SDS-PAGE electrophoresis: taking a proper amount of sample, adding distilled water to dilute the sample to 6 mug/mL, adding 5X SDS loading buffer, and carrying out boiling water bath for 5min to denature protein. The electrophoresis gel is fixed on an electrophoresis frame and is placed in an electrophoresis tank, tris-buffer solution is added into the electrophoresis tank, 7 mu L Marker and 10 mu L diluted denatured sample are added into a sample hole, and constant 80V voltage is used for electrophoresis for 30min. Then the voltage is increased to 120V, and the electrophoresis is finished until the bromophenol blue dye reaches the bottom of the electrophoresis gel.
Western blot: after electrophoresis, the gel is taken down, and a target strip is cut off by using a gel cutting plate according to the position of a Marker. And (3) flattening the cathode plate of the transfer printing device, sequentially stacking sponge, filter paper, gel strips and PVDF film, covering the anode plate, and clamping. And placing the transfer printing device into an electric transfer tank, adding pre-cooling transfer film liquid and an ice bag, and carrying out electric transfer for 50min at a constant current of 160 mA. The PVDF membrane was transferred to an incubator pre-filled with a rapid blocking solution and the shaker was blocked for 10min.
Incubation resistance: primary antibody (1:1000) was added and incubated at 4℃for 12h in a refrigerator.
Secondary antibody incubation: the primary antibody is recovered, washed 3 times with washing liquid for 15min each time, and then secondary antibody (1:2000) is added for 2h of shaking table incubation. Finally, the immunoreactive bands were observed using chemiluminescence and quantified using image J1.8 software.
The results show that the intestinal CNT2 transporter expression is significantly elevated in the model group compared to the blank group. The CNT2 transporter expression was significantly reduced in rats of the anserine composition group, anserine control group, chicory, and the euglena binary extract group compared to the model group (see fig. 3). The CNT2 transporter expression was reduced by 27.5%, 39.1%, 45.5%, 22.1%, 35.2% for each group of rats compared to the model group, respectively, with the high dose of anserine composition having the best inhibitory effect on CNT2 transporter expression. CNT2 transporter is a concentration-dependent protein that exists in the small intestine and, upon ingestion of foods containing a large amount of purine compounds, the expression level of CNT2 transporter is increased accordingly to promote absorption of purine compounds. CNT2 transporter expression was inhibited, indicating that the anserine composition has an effect of inhibiting absorption of food-borne purines.
5) Intestinal flora detection
The cecal content of the mice was thawed by taking out from the refrigerator at-80℃and total DNA of microorganisms in the intestinal content of the mice was extracted using FAST DNA SPIN KIT for Feces stool gene extraction kit. The V3-V4 region of 16S rRNA in the genome of the microorganism was amplified by Polymerase Chain Reaction (PCR). And (3) carrying out electrophoresis inspection after amplification is finished, wherein the PCR products are qualified and the positive control and the negative control of the same batch are normal. And (3) mixing samples and library QC, and finally detecting the qualified library for high-throughput sequencing, and sequencing by using a Illumina NovaSeq platform. The diversity of the flora is analyzed by adopting alpha diversity and beta diversity, and the microbial population difference is analyzed from the phylum and genus level.
The results show that the anserine composition can effectively improve the intestinal microbial flora disorder of rats and restore the flora structure. Compared with the model group, the relative abundance of the thick-walled bacteria phylum of the rats of the goose carnosine composition group, the goose carnosine control group, the chicory and the Euglena binary extract group is obviously increased, the relative abundance of the bacteroidetes phylum is obviously reduced, and the relative abundance of various microorganisms such as the Proteobacteria phylum, the actinomycota and the like is obviously reduced (figure 4). At the subordinate level, the relative abundance of lactobacillus was significantly increased (fig. 5), and the relative abundance of lactobacillus was reduced by 86.1% in the model group rats compared with the normal rats, and the relative abundance of lactobacillus in the anserine composition group, the anserine control group, the chicory and the euglena binary extract group rats was restored to 72.2%, 52.8% and 55.6% of the normal levels respectively. The lactobacillus is a main strain for decomposing purine compounds in the intestinal tract, so that the compound provided by the invention can regulate intestinal flora and help to restore the normal intestinal flora structure.
Example 3
This example provides the inhibitory effect of anserine compositions on food-borne purine compounds in gout patients.
Test article: the goose carnosine composition provided by the invention comprises, by mass, goose carnosine, chicory extract and euglena extract=75:15:10.
The subject: referring to the guidelines of clinical study of new traditional Chinese medicines, 120 gout patients which are treated by the auxiliary hospitals of Guangdong medical university are selected from 2022, 5 and 2023, 6, and the serum uric acid level is rechecked, and the fasting blood sampling is carried out for 2 times on the same day under the condition of not changing daily diet. According to blood sampling detection results, 56 men with the average serum uric acid value of more than or equal to 420 mu mol/L and women with the average serum uric acid value of more than or equal to 357 mu mol/L are selected, and the patients are aged 18-60, and have no serious diseases such as heart, liver, kidney, hematopoietic system and other metabolic diseases, no allergic constitution or allergy to the tested sample.
Test protocol: the 56 patients were divided into a treatment group and a placebo group using a random number table method, with 28 persons in each group. Placebo group 18 men, 10 women; treatment group 16 men and 12 women. The 56 subjects were dosed with 50g of fried chicken liver (purine content: about 312mg/100 g) and 100g of fried hairtail (purine content: about 385mg/100 g) on a daily basis at lunch without changing their daily diet, and were treated with the anserine composition powder, respectively, by administering to the patients in the treatment group, and with the placebo powder (main ingredients: alanine, histidine, glucan, levan) by administering to the patients in the placebo group. The treatment time period of the treatment group and the placebo group is 14d, each patient takes 1 time per day, 3g each time, and the patients are taken with warm water after breakfast.
Purine compound content and blood uric acid content detection: the subjects were taken 5mL of a blood sample at 2h after the meal at treatment 7d and 14d, centrifuged at 4000r/min for 10min, and the supernatant was collected and stored at-80℃for testing. After the sample is completely collected, the content of the serum purine compounds in the test subjects is detected according to the method in the example 2, and the average value of the detection results of 2 times of sampling is taken for comparison. Serum uric acid levels of subjects were measured as in example 2, and the measurement data at 28d was compared.
Test results: the purine and uric acid detection results are shown in tables 4 and 5.
TABLE 4 purine content Change
TABLE 5 uric acid content variation
From the results shown in tables 4 and 5, it is apparent that the anserine composition provided by the invention can significantly reduce serum purine and uric acid levels of patients and inhibit abnormal increase of uric acid levels caused by high purine diets. In addition, the number of gout symptoms in placebo patients was 1.1 and the number of gout symptoms in treatment patients was 0.3 within treatment 14d, which also suggests that the anserine composition can reduce the incidence of gout by inhibiting the absorption of food-borne purine compounds.
By combining the experimental results, the anserine composition provided by the invention shows the ability of inhibiting the absorption of purine compounds and reducing uric acid in animal experiments and clinical experiments. In addition, the composition can help to recover the intestinal microbial flora disorder, increase the abundance of beneficial bacteria such as lactobacillus and the like, and has remarkable effect.
The embodiments described above are only some, but not all, embodiments of the invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments obtained without inventive effort by a person skilled in the art, which are related deductions and substitutions made by the person skilled in the art under the condition of the inventive concept, are within the scope of protection of the present invention.
Claims (1)
1. The application of the anserine composition in preparing medicines for preventing and/or treating gout or hyperuricemia is characterized in that the anserine composition inhibits the absorption of food-borne purine compounds, or reduces the expression level of CNT2 transport proteins, or promotes the metabolism of purine compounds into allantoin, or regulates intestinal flora;
The goose carnosine composition comprises goose carnosine, chicory extract and euglena extract, wherein the mass ratio of the goose carnosine to the chicory extract to the euglena extract=75-85:10-20:2-12;
the preparation method of the chicory extract comprises the following steps: crushing the roots of the chicory, sieving the crushed roots of the chicory with a 100-mesh sieve to obtain chicory powder, extracting the chicory powder with water at 80-90 ℃ for 4-5 times, each time for 1-2 hours, merging filtrate, concentrating under reduced pressure, carrying out alcohol precipitation on the water extract of the chicory powder with 40-50% ethanol for 20-40 hours, and carrying out freeze drying on alcohol precipitation after filtering to obtain the chicory extract;
The preparation method of the euglena extract comprises the following steps: sieving Euglena dry powder with 80 mesh sieve, soaking in acetic acid aqueous solution, heating at 40deg.C under stirring for 6 hr, adding equal volume of sodium hydroxide aqueous solution, heating under stirring for 4 hr, standing, filtering to obtain filtrate, adding absolute ethanol for alcohol precipitation, standing, filtering to collect alcohol precipitate, and freeze drying to obtain Euglena extract;
The anserine is prepared by chemical synthesis or extraction, and the extraction and preparation method of the anserine comprises the following steps: mincing fish meat with water, homogenizing, performing enzymolysis according to the addition amount of papain 500U/g and flavourzyme 200U/g, inactivating enzyme, centrifuging at 4deg.C and 3500×g for 15min, deoiling, decolorizing, separating, purifying, and lyophilizing to obtain anserine dry powder.
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