Composition for reducing blood uric acid and preparation method and application thereof
阅读说明：本技术 一种降血尿酸的组合物及其制备方法和应用 (Composition for reducing blood uric acid and preparation method and application thereof ) 是由 谭余庆 马海 杨适 赵庆贺 韩国君 刘丽 杨米一 于 2021-03-19 设计创作，主要内容包括：本发明涉及一种降血尿酸的组合物及其制备方法和应用,具体涉及药物治疗领域。本发明提供了一种降血尿酸的组合物,所述组合物的有效成分由质量百分含量为37％的酚酸组成。本发明所述组合物能够达到降低血尿酸水平的效果,同时毒副作用小。(The invention relates to a composition for reducing blood uric acid and a preparation method and application thereof, and particularly relates to the field of drug treatment. The invention provides a composition for reducing blood uric acid, wherein the effective component of the composition comprises phenolic acid with the mass percentage of 37%. The composition disclosed by the invention can achieve the effect of reducing the blood uric acid level, and is small in toxic and side effects.)
1. The composition for reducing the blood uric acid is characterized in that the effective component of the composition is phenolic acid, and the mass of the phenolic acid is 37% of the mass of the composition.
2. A method of preparing the composition of claim 1, comprising the steps of:
mixing the sunflower disc without seeds with water, soaking, decocting for 2h, and filtering with a 300-mesh filter sieve to obtain filtrate;
concentrating the filtrate, passing through a macroporous resin column, sequentially eluting with water and ethanol aqueous solutions with the volume percentage of 10%, 20%, 60% and 70%, and sectionally collecting the eluent of the ethanol aqueous solution to obtain the composition for reducing the uric acid in blood; the water and the ethanol water solution with the volume percentage of 10 percent, 20 percent, 60 percent and 70 percent are all 3 times of the volume of the macroporous resin column.
3. The method of claim 2, further comprising chopping the sunflower discs prior to soaking, wherein the size of the chopped sunflower discs is 3 cm.
4. The method according to claim 2, wherein the number of times of the decoction and filtration is 2; the water mass in each decoction is 20 times of the sunflower disc block mass.
5. The method of claim 2, wherein the concentration is carried out at a temperature of 80 ℃.
6. The method according to claim 2, wherein the concentration further comprises centrifugation; the rotation speed of the centrifugation is 4000rpm, and the time is 2 min.
7. The preparation method according to claim 6, wherein after the stepwise collecting of the ethanol solution eluate, the method further comprises sequentially concentrating and drying the collected eluate.
8. Use of the composition according to claim 1 or the composition prepared by the preparation method according to any one of claims 2 to 7 in the preparation of a medicament for reducing uric acid in blood.
9. A medicament for reducing uric acid in blood, which comprises the composition as defined in claim 1 or the composition prepared by the preparation method as defined in any one of claims 2 to 7.
Technical Field
The invention relates to the field of drug therapy, in particular to a composition for reducing blood uric acid and a preparation method and application thereof.
Background
Uric acid is the end product of purine metabolism in humans, and is produced from xanthine and hypoxanthine by the action of Xanthine Oxidase (XOD). Human beings are very prone to Hyperuricemia (HUA) because of the lack of urate oxidase and the inability to further break down the urate oxidase into soluble allantoin.
In recent years, with the occurrence of significant changes in lifestyle and dietary structure of people, the prevalence rate of the disease has increased significantly, and the onset age has continued to be low. It was found that 30% of patients with primary HUA have renal damage and are independent risk factors affecting renal function. HUA is an independent risk factor for type 2diabetes mellitus (T2 DM), hypertension, atherosclerosis, cardiovascular events, stroke and chronic kidney disease, etc., and is the most major biochemical basis and the most direct cause of gout attack.
At present, HUA has become a serious metabolic disease threatening human health. However, the drugs for treating the HUA are very limited, and no radical treatment method exists, wherein western medicine treatment mainly depends on an XOD inhibitor allopurinol and UA excretion promoting drugs such as probenecid and benzbromarone, the drugs have high toxic and side effects, are easy to cause allergy, liver and kidney injury, bone marrow suppression, gastrointestinal tract reaction, arthralgia, rash and the like, and patients are often intolerant, and the use of the drugs is limited to a certain extent. The compound research of the traditional Chinese medicine for preventing and treating the HUA is less, no scale research is found, the traditional Chinese medicine can be really and widely applied to clinical application, and at present, almost no medicine related to the traditional Chinese medicine for treating gout is available on the market. Therefore, the search for new highly effective and low toxic anti-HUA drugs is still a hot spot of current research.
Disclosure of Invention
In order to solve the problems, the invention provides a composition for reducing blood uric acid, and a preparation method and application thereof. The composition disclosed by the invention can achieve the effect of reducing the blood uric acid level, and is small in toxic and side effects.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a composition for reducing blood uric acid, wherein the effective component of the composition is phenolic acid, and the mass of the phenolic acid is 37% of that of the composition.
The invention provides a preparation method of the composition, which comprises the following steps:
mixing the sunflower disc without seeds with water, soaking, decocting for 2h, and filtering with a 300-mesh filter sieve to obtain filtrate;
concentrating the filtrate, passing through a macroporous resin column, sequentially eluting with water and ethanol aqueous solution with the volume percentage of 10%, 20%, 60% and 70%, and collecting the eluent of the ethanol aqueous solution in sections to obtain the composition for reducing the uric acid in blood; the water and the ethanol water solution with the volume percentage of 10 percent, 20 percent, 60 percent and 70 percent are all used in an amount which is 3 times of the volume of the macroporous resin column.
Preferably, before the soaking, the sunflower discs are cut into pieces, and the particle size of the cut sunflower discs is 3 cm.
Preferably, the number of times of decoction and filtration is 2; the water mass in each decoction is 20 times of the sunflower disc block mass.
Preferably, the temperature of the concentration is 80 ℃.
Preferably, after the concentration, centrifugation is further included; the rotation speed of the centrifugation is 4000rpm, and the time is 2 min.
Preferably, after the step of collecting the ethanol solution eluate, the step of sequentially concentrating and drying the collected eluate is further included.
The invention provides an application of the composition or the composition prepared by the preparation method in preparing a medicament for reducing blood uric acid.
The invention provides a medicine for reducing blood uric acid, which comprises the composition or the composition prepared by the preparation method.
Has the advantages that: the invention provides a composition consisting of 37% by mass of phenolic acid, and the composition has the effect of reducing blood uric acid and has small toxic and side effects. The experimental results show that: the composition can remarkably reduce the expression of URAT1 protein by intragastric administration, and can remarkably reduce the blood uric acid level of mice.
The sunflower disc is used as a raw material for extraction, so that the waste of resources can be avoided.
Drawings
FIG. 1 is a standard curve of caffeic acid;
FIG. 2 shows the regulation and control effects of the compositions extracted in example 1 and comparative examples 1-3 on mouse kidney URAT1 protein, wherein Blank is Blank, Model is Model, Benz is positive drug benzbromarone, relative protein expression/β -actin is relative protein expression/β -actin, compared with Blank,###p<0.001; compared with the model control group,*p<0.05，**p<0.01，***p<0.001。
Detailed Description
The materials used in the present invention are all those conventionally purchased by those skilled in the art, unless otherwise specified.
The invention provides a composition for reducing blood uric acid, wherein the effective component of the composition is phenolic acid, and the mass of the phenolic acid is 37% of that of the composition. The inhibitory action of the composition on URAT1 protein is better than that of benzbromarone on URAT1 protein, so that the effect of reducing uric acid is achieved, and the side effect is small.
The invention provides a preparation method of the composition, which comprises the following steps:
mixing the sunflower disc without seeds with water, soaking, decocting for 2h, and filtering with a 300-mesh filter sieve to obtain filtrate;
concentrating the filtrate, passing through a macroporous resin column, sequentially eluting with water and ethanol aqueous solution with the volume percentage of 10%, 20%, 60% and 70%, and collecting the eluent of the ethanol solution in sections to obtain the composition for reducing the uric acid; the water and the ethanol water solution with the volume percentage of 10 percent, 20 percent, 60 percent and 70 percent are all used in an amount which is 3 times of the volume of the macroporous resin column.
The invention mixes and soaks the sunflower disc without seeds with water, and filters the sunflower disc after decocting for 2 hours to obtain filtrate. The method for decocting the Chinese medicinal herbs is not limited in any way, and can be realized by adopting a method which is well known by a person skilled in the art.
Before the soaking, the sunflower discs are preferably cut into pieces; the particle size of the sunflower discs after cutting is preferably 3 cm.
In the present invention, the soaking time is preferably 30 min; the temperature of the soaking is preferably room temperature. In the present invention, the number of times of the decoction and filtration is preferably 2; the water mass for each of the decoctions is preferably 20 times of the sunflower disc mass. The filtration method is not limited in any way, and can be performed by a method known to those skilled in the art.
Concentrating the filtrate, passing through a macroporous resin column, eluting by sequentially adopting water and ethanol water solution with the volume percentage of 10%, 20%, 60% and 70%, and collecting the eluent of the ethanol solution in sections to obtain the composition for reducing the uric acid; the water and the ethanol water solution with the volume percentage of 10 percent, 20 percent, 60 percent and 70 percent are all used in an amount which is 3 times of the volume of the macroporous resin column. In the invention, the way of passing through the macroporous resin column is preferably to slowly pour the supernatant obtained by centrifugation into the macroporous resin column along the wall of the macroporous resin column. The supernatant is slowly poured into the macroporous resin column, so that the macroporous resin can be effectively prevented from being washed up, the adsorption of the macroporous resin to active ingredients is reduced, and the effect of the composition prepared by the method on the blood uric acid is further ensured.
According to the invention, ethanol solutions with different concentrations are adopted for elution, and the way of collecting the eluent in sections is beneficial to separating compounds with different polarities and different adsorption capacities, so that the compounds required by the invention can be obtained.
In the present invention, the temperature of the concentration is preferably 80 ℃. In the present invention, after the filtrate is concentrated, it is preferable to further include centrifuging the concentrated solution obtained by the concentration; the rotation speed of the centrifugation is preferably 4000 rpm; the time for the centrifugation is preferably 2 min.
In the present invention, the model of the macroporous resin is preferably HPD 500; the macroporous resin is preferably washed by 95% ethanol, water, 1N HCl, water, 1N NaOH, water and 95% ethanol in sequence; the volume of the macroporous resin column is preferably 120 ml. In the present invention, the 95% ethanol solution is preferably 95% ethanol solution by volume.
After the elution, the method preferably further comprises the step of concentrating and drying the eluent obtained by eluting the ethanol solutions with different concentrations to obtain the composition for reducing the uric acid, namely the solid composition. In the present invention, the temperature of the concentration and drying is preferably 80 ℃.
After the drying, the invention also comprises the step of mixing the obtained dry composition to obtain the composition for reducing the blood uric acid. The mixing method is not limited in any way, and can be a method known to those skilled in the art.
The composition can obviously reduce the expression of URAT1 protein, so that the effect of reducing blood uric acid is ensured, and the composition can be used for preparing medicines for reducing blood uric acid.
The invention provides an application of the composition or the composition prepared by the preparation method in preparing a medicament for reducing blood uric acid.
The invention provides a medicine for reducing blood uric acid, which comprises the composition or the composition prepared by the preparation method.
For further illustration of the present invention, the following detailed description of the composition for lowering uric acid in blood, the preparation method and the application thereof are provided with reference to the drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Preparation of composition for reducing blood uric acid
(1) Cutting 100g of sunflower discs into small pieces of 3cm, putting the small pieces into a 2L round-bottom flask, adding 20 times of water by mass of the sunflower discs, soaking for 30min, heating and decocting for 2h, filtering and collecting filtrate, continuously adding 20 times of water to decoct medicine residues for 2h, filtering and combining the filtrates, putting the filtrate into a water bath kettle, concentrating the filtrate at 80 ℃ to 700mL for later use, taking 50mL of concentrated solution to evaporate in an evaporation dish, and calculating the solid yield to be 51.6%.
(2) Cleaning a macroporous resin with the model of HPD500 according to the sequence of 95% ethanol-water-1N HCl-water-1 NNaOH-water-95% ethanol, then filling the treated macroporous resin into a column, wherein the volume of the filled column is 120mL, the amount of a sample liquid is 700mL, slowly pouring a concentrated solution into the column along the wall of the column, eluting by sequentially using water, 10%, 20%, 60% and 70% ethanol solutions, wherein the using amounts of the water, the 10%, the 20%, the 60% and the 70% ethanol solutions are 3 times of the volume of the column, discarding a water phase, collecting eluents in sections, concentrating and drying ethanol eluents with different concentrations in a water bath kettle at 80 ℃ respectively to obtain a solid, and mixing the solid compositions to obtain the composition for reducing uric acid.
2. Determination of phenolic acid content in composition
(1) Preparation of caffeic acid reference solution
Accurately weighing appropriate amount of caffeic acid reference substance, and adding anhydrous methanol to obtain solution containing 90 μ g of caffeic acid reference substance per lmL.
(2) Preparation of the Standard Curve
Precisely measuring reference substance solutions 0.25mL, 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL, respectively placing the solutions in 25mL measuring bottles, adding water to 6mL, adding 5% sodium nitrite solution 1mL, shaking uniformly, placing for 6min, adding 10% aluminum nitrate solution 1mL, shaking uniformly, placing for 6min, adding sodium hydroxide test solution 10mL, adding water to a scale, shaking uniformly, placing for 15min, taking a corresponding reagent as a blank, immediately adopting an ultraviolet-visible spectrophotometry, measuring absorbance at the wavelength of 500nm, taking the absorbance as an ordinate and the concentration as an abscissa, drawing a standard curve, and obtaining the result shown in Table 1 and figure 2, wherein the standard curve is: 11.169x-0.2483, R20.9902, the standard curve has good linearity and can be used to calculate phenolic acid content.
TABLE 1 absorbance results for caffeic acid at various concentrations
Content (mg)
0.0225
0.045
0.09
0.135
0.18
0.225
Absorbance (Abs)
0.358
0.834
1.325
1.794
2.273
2.696
(3) Preparation of test solution
Precisely measuring the solid composition for reducing uric acid in blood 200mg, adding a small amount of 70% ethanol for dissolving, transferring into a 50mL measuring flask, adding 70% ethanol to scale, and shaking to obtain the final product
(4) Assay method
Precisely measuring 0.5mL of the test sample, placing the test sample in a 25mL measuring flask, adding absolute ethyl alcohol to supplement 5mL, adding 2.0mL of 0.3% sodium dodecyl sulfate and 1.0mL of a mixed solution of 0.6% ferric chloride and 0.9% potassium ferricyanide (1:0.9), uniformly mixing, placing the test sample in the dark for 5min, adding 0.1mol/L hydrochloric acid solution to the scale, shaking the test sample, placing the test sample in the dark for 20min, taking the corresponding reagent as the blank, namely all reagents except the test sample solution, measuring the absorbance at the wavelength of 700nm by adopting an ultraviolet-visible spectrophotometry method, reading the weight of caffeic acid in the test sample solution from a standard curve, and calculating to obtain the phenolic acid content.
The preparation process of the composition for reducing blood uric acid is repeated for 3 times, the composition for reducing blood uric acid obtained by each extraction is measured, and the experimental results are shown in table 2.
TABLE 2 determination of phenolic acid content in the compositions
Numbering
Phenolic acid (mg)
Mass of solid (mg)
Is in percentage by weight
1
288.0
778.4
37.0
2
282.8
768.5
36.8
3
285.2
762.6
37.4
As can be seen from the experimental data shown in Table 2, the phenolic acid proportion of the method provided by the invention is 36.8-37.4, and the preparation method provided by the invention is stable.
Comparative example 1
1. Sunflower disc 60% alcohol extraction process
Preparing the sunflower disc alcohol extract: cutting 100g of sunflower discs into 3cm small pieces, putting into a 2L round-bottom flask, adding 60% ethanol solution which is 10 times of the weight of decoction pieces, soaking for 30min, heating and decocting for 1.5h, filtering and collecting filtrate, continuously adding 60% ethanol solution which is 10 times of the weight of decoction pieces, decocting the decoction dregs for 1.5h, filtering and combining the filtrates, concentrating and drying the filtrate to obtain the composition.
2. Determination of phenolic acid content in composition
The experimental procedure was the same as for the content determination procedure 2 in example 1.
The sunflower disc 60% alcohol extraction process was repeated 3 times, and the composition obtained from each extraction was determined, the results of which are shown in table 3.
TABLE 3 determination of phenolic acid content in the composition extracted by 60% alcohol extraction process
Numbering
Phenolic acid (mg)
Mass of solid (g)
Is in percentage by weight
1
213.8
40.7
0.55
2
220.9
39.8
0.56
3
215.4
39.1
0.55
As is clear from the test data shown in Table 3, the ratio of phenolic acid in the composition prepared by the method of this comparative example is between 0.55 and 0.56.
Comparative example 2
1. 80% alcohol extraction process for sunflower discs
Preparing the sunflower disc alcohol extract: cutting 100g of sunflower discs into small pieces of 3cm, putting the small pieces into a 2L round-bottom flask, adding 80% ethanol solution which is 10 times of the mass of the sunflower discs, soaking for 30min, heating and decocting for 1.5h, filtering and collecting filtrate, continuously adding 80% ethanol solution which is 10 times of the mass of the sunflower discs, decocting the dregs for 1.5h, filtering and combining the filtrate, concentrating and drying the filtrate to obtain the composition.
2. Determination of phenolic acid content in composition
The experimental procedure was the same as for the content determination procedure 2 in example 1.
The sunflower disc 80% alcohol extraction process described above was repeated 3 times, and the composition obtained from each extraction was determined, and the experimental results are shown in table 4.
TABLE 4 determination of phenolic acid content in compositions obtained by 80% alcohol extraction process
Numbering
Phenolic acid (mg)
Mass of solid (g)
Is in percentage by weight
1
372.5
32.1
1.16
2
381.1
34.5
1.10
3
396.4
34.8
1.06
As is clear from the test data shown in Table 4, the ratio of phenolic acids in the composition prepared by the method of this comparative example is 1.06-1.16.
Comparative example 3
1. Sunflower disc 80% alcohol extraction and water precipitation process
(1) Preparing 80% alcohol extract of sunflower disc: the experimental procedure was the same as that of example 1 of comparative example 2 to obtain an extract.
(2) Preparation of sunflower disk alcohol extraction precipitate
Concentrating the above extractive solution to density of 1.1g/mL, adding 40 times of water, standing for 12 hr, filtering, and oven drying the filtrate to obtain the composition.
2. Determination of the content of the composition
The experimental procedure was the same as for the content determination procedure 2 in example 1.
The sunflower disc 80% alcohol extraction and water precipitation process is repeated 3 times, the composition obtained by each extraction is measured, and the experimental results are shown in table 5.
TABLE 5 determination of phenolic acid content in the composition extracted by alcohol extraction and water precipitation
Numbering
Phenolic acid (g)
Mass of solid (g)
Is in percentage by weight
1
2.18
21.5
10.1
2
2.04
21.7
9.4
3
1.97
21.8
9.0
As is clear from the test data shown in Table 5, the ratio of phenolic acids in the composition prepared by the method of this comparative example is 9.0 to 10.1.
Application example 1
Pharmacodynamic experiment
1. Experimental Material
(1) Test article
The compositions prepared in example 1 and comparative examples 1-3 were prepared by four different processes.
(2) Positive control drug
Benzbromarone: germany hemman pharmaceutical factory (Excella GmbH & co. kg), kunshan dragon lantern ruddi pharmaceutical ltd, production lot number: 1806602, national standard J20180056, effective period is 22 months at 5 months in 2023;
(3) selection of the vehicle
In view of the differences in dissolution and dispersion of the positive drug in different solvents, the present study used 0.5% sodium carboxymethylcellulose (w/w) solution to dissolve the positive drug and the sunflower disc extract in the yeast combined potassium oxonate induced hyperuricemia in mice experiments, and also used 0.5% sodium carboxymethylcellulose as the solvent for the yeast solution preparation and the potassium oxonate solution preparation involved in the experiments.
(4) Laboratory animal
Based on the existing research and clinical manifestations, the prevalence rate of the male HUA is higher than that of the female, and for the pathogenesis, it is considered that the male HUA is mainly caused by the difference of sex hormone levels in bodies of different sexes, androgen is an XDH/XO activity inducer, estrogen is an enzyme activity inhibitor, and the androgen and the enzyme activity inhibitor play a regulating role in the transcription level, thereby influencing the activity of enzymes related to purine metabolism and further influencing the metabolism of blood UA. Therefore, in the research on the HUA and the gout at the later stage, the male animals are selected in order to obtain more clinical research effect.
The experimental animals participating in the final experiment were 70 male C57BL/6N mice, 6 weeks old, SPF grade, all purchased from beijing weitonglihua experimental animals technologies ltd, license No.: SCXK (Kyoto) 2016-.
The number of the experimental animals is determined according to ethical welfare of the experimental animals, the experimental animals are selected to be bred in a barrier environment, the license number of an experimental unit SYXK (Jing) 2019-.
The animal feed is SPF-level rat maintenance feed provided by Australian cooperative feed Co., Ltd of Beijing, and has a product batch number of: 20043213, date of manufacture: 20200410, has a shelf life of six months.
The experimental animal drinking water is purified water and is prepared by an RO-300 type reverse osmosis water making machine produced by Shenzhou (Beijing) science and technology limited company.
(5) Reagent
Potassium oxonate (Oxonic acid potassium salt), a product of Sigma Co., Lot: STBH 8632;
yeast powder, product of OXOID, Lot: 2746246-02;
sodium carboxymethylcellulose (300-: 20181211, respectively;
RIPA tissue/cell lysate, Solarbio product, Lot: r0020;
SDS-PAGE gel rapid configuration kit, pecan product, Lot: p0012 AC;
BCA protein concentration assay kit, Solarbio product, Lot: PC 0020;
PageRulerPrestated Protein Ladder, thermal scientific product, Lot: 26616;
difco TM Skim Milk, product Biotopped, Lot: 232100;
beta Actin Rabbit Polyclonal antibody, Proteitech product, Lot: 20536-1-AP;
URAT1 Rabbit Polyclonal antibody, product of Proteitech, Lot: 14937-1-AP;
uric acid determination kit (uricase method), zhongsheng bei zhi bio-technology gmbh, lot number: 190902, effective period till 2021 year 9 months.
(6) Instrument for measuring the position of a moving object
BTM21C Handheld Infrared thermometer, manufactured by BSIDE;
synergyhio microplate reader, produced by Biotek corporation;
centrifuge, product of KUBOTA corporation, japan, model number KUBOTA 5922;
the electronic balance Sartorius BP211D is used for quantitative weighing of trace substances in 210 laboratories;
the electronic balance Sartorius BSA3202S-CW (serial numbers 24790283, 24790266 and 36892266) is used for weighing the weight and the feed amount of experimental animals in a mouse experiment respectively.
Electrophoresis apparatus, manufactured by Bio-RadPowerPacTM Basic, USA;
a gel Imaging analyzer (MP Imaging System), manufactured by BD ChemiDocTM, USA;
a microplate reader, BioTek Synergy H1 microplate reader.
2. Dosage, mode and configuration of administration
Intragastric volume: 0.2mL/10g (yeast), 0.1mL/10g (benzbromarone, composition extracted in example 1, composition extracted in comparative examples 1-3).
Volume of intraperitoneal injection: 0.1mL/10 g.
(1) Dosage of test article
According to the related documents (the effects of a Hepatoprotective effect of calcium devices saline disease in mice by inhibiting oxidative stress and apoptosis of proteins), Wenxi H, Yanjiao X, Chengliang Z, et al. drug Design, Development and Therapy,2017, Volume 11: 3449-property 3460, DBZ a viral gamma acid precursor viral gene product di-induced objective, insulin resistance and Therapy Xu P, Wang F, Wang J, et al. Biochimica Acta (BBA) -serum nutrient and gut biosystems, 2017,1861, the effects of a Hepatoprotective effect of cholesterol toxin and glucose oxidase on renal cell damage, peroxidase, the administration dosage of WangRepo, Lijiao, Zhao (38932), Wangjieqiong, Caberxia, Dianthus superbus, natural product research and development, 2012, 24(02):172-175+202) is subjected to gradient experiment on the administration concentration, and the consumption cost of process raw materials is considered, so that the low dosage has the effect of reducing uric acid, but has no statistical significance, and the optimal administration dosage is determined to be 200 mg/kg.
(2) Dosage of positive control drug
Benzbromarone: the administration dose of mice was calculated as 50mg/70kg × 70kg × 0.0026/0.02kg as 6.5mg/kg, calculated as human dose 50mg (tablet)/human/day, converted to the surface area of human and animal intermediates.
(3) Drug configuration
(a) 0.5% sodium carboxymethylcellulose (CMC-Na)
Weighing 1g of sodium carboxymethylcellulose powder, dissolving in 199mL of deionized water, magnetically stirring, and hermetically storing at normal temperature after the powder is completely dissolved.
(b)1g/mL Yeast solution
50g of yeast powder is weighed and dissolved in 50ml of 0.5 percent sodium carboxymethyl cellulose solution, and the mixture is stirred uniformly and is ready to use.
(c)30mg/mL oteracil potassium solution
1.2g of oteracil potassium powder is weighed and dissolved in 40mL of sodium carboxymethyl cellulose, and the mixture is stirred evenly and used.
(d) Benzbromarone (adult oral 50 mg/day)
Mebremazone 19.5mg was weighed out and dissolved in 30mL of 0.5% CMC-Na.
(e)200mg/kg sunflower disc extract
The dosage concentration: 10mg/mL
Weighing 200mg of the composition extracted from the sunflower disc in the embodiment 1 and the comparative examples 1-3, dissolving the composition in 8mL of sodium carboxymethyl cellulose, metering the volume to 10mL, and uniformly mixing the solution for later use.
3. Experiment grouping
The yeast and oteracil potassium induced mouse hyperuricemia experiment is divided into 7 groups, 10 groups are divided into a blank group, a model group, a benzbromarone group (6.5mg/kg) and the composition (T) extracted in example 11) (200mg/kg), composition (T) extracted in comparative example 12) (200mg/kg), composition (T) extracted in comparative example 23) (200mg/kg), composition (T) extracted in comparative example 34)(200mg/kg)。
4. Statistical treatment
The experimental data all adopt the mean value plus or minus standard deviationThe comparison of the differences between the groups of the experiment was performed by Student's test, statistical analysis and data processing using the SPSS 20 software.
5. Experimental methods and results
Taking male C57BL/6N small70 mice with weight of 17-20g were divided into 10 mice per group, each group was composed of blank group, model group, positive drug benzbromarone group (6.5mg/kg), and composition extracted in example 1 (T)1) (200mg/kg), composition (T) extracted in comparative example 12) (200mg/kg), composition (T) extracted in comparative example 23) (200mg/kg), composition (T) extracted in comparative example 34)(200mg/kg)。
After the adaptive feeding is finished, starting an experiment, from the 1 st day (D1) to the 5 th day (D5) of the experiment, sequentially performing intragastric and intraperitoneal injection on 0.5% sodium carboxymethylcellulose (blank solvent) in a blank group, performing molding on other experimental animals by using yeast in combination with oteracil potassium, and immediately performing intraabdominal injection on oteracil potassium after intragastric administration of yeast, wherein the administration dose of the yeast is 15g/kg, the intragastric volume is 0.2mL/10g (body weight), the administration dose of the oteracil potassium is 300mg/kg, the administration volume is 0.1mL/10g (body weight), from the 3 rd day to the 5 th day of the experiment, different medicines are administered 1h before molding every day, whether the sunflower disc extract has the effect of reducing the blood uric acid is evaluated, namely, except that the blank group and model group experimental animals are intragastric administered with 0.5% sodium carboxymethylcellulose solution with corresponding volume, the other experimental animals are administered with corresponding therapeutic medicines, the gavage volume was 0.1mL/10g (body weight).
On the 5 th day of the experiment, 1h after molding, the mouse picks eyeballs and takes blood, blood samples are stood in a refrigerator at 4 ℃ for one night and then centrifuged at 3500r/min for 10min, upper serum is collected, the concentration of UA in the serum is detected by using a uricase kit, and the specific results are shown in Table 6.
TABLE 6 influence of the compositions extracted in example 1 and comparative examples 1 to 3 on UA content in blood of yeast combined potassium oxonate HUA mice (x. + -. SD)
Note: blank group ratioIn comparison, the method has the advantages that,###p<0.001; compared with the model control group,*p<0.05,**p<0.01,***p<0.001.
as can be seen from the results in Table 6, the serum UA level of the model group is remarkably increased, and compared with the blank group, the difference has statistical significance (p is less than 0.05), which indicates that the yeast combined with Potassium Oxonate successfully replicates the mouse HUA model; after the sunflower disc extracts in four different processes are administrated, the serum UA level of an experimental animal is obviously reduced, compared with a model control group, the differences have statistical significance (p is less than 0.001, p is less than 0.01, and p is less than 0.05), and 0.2g/kg of the sunflower disc extracts in the four different processes have obvious UA reducing effect on a mouse HUA model reproduced by yeast combined with oteracil potassium. The positive drug benzbromarone also has the effect of reducing blood UA, and the difference has statistical significance (p < 0.05). In addition, the experimental result shows that the extract obtained by extracting the extract with water through the macroporous resin process in the experiment has better UA (UA) effect reduction than a positive control medicament of benzbromarone on a mouse HUA model reproduced by combining yeast and oteracil potassium.
Application example 2
Mouse kidney tissue is taken, and protein in the tissue is extracted.
The operation method comprises the following steps: tissue was weighed 50mg, minced and placed in 300. mu.L of RIPA protein lysate. Properly grinding, centrifuging at 12000r/min at 4 deg.C for 10min, sucking supernatant into a new centrifuge tube, and storing at-20 deg.C for use.
2. Protein quantification:
(1) preparing a working solution: according to the number of the standard and the sample, 50 volumes of BCA reagent and 1 volume of Cu reagent (BCA kit) (50:1) are prepared into BCA working solution, and the BCA working solution is fully mixed (turbidity may occur during mixing, but the BCA working solution disappears after mixing). The BCA working solution is stable within 24 hours at room temperature. (9 samples +24 standards from preliminary experiments 6600. mu.L, made up of 7140. mu.L, i.e. 7000. mu. LBCA reagent: 140. mu.L Cu reagent)
(2) Dilution standard (3 replicates): 30 μ LBSA standards were diluted to 300 μ L with PBS (samples can be diluted with PBS in general) to a final concentration of 0.5 mg/mL. The standard was added to protein standard wells of a 96-well plate at 0. mu.L, 2. mu.L, 4. mu.L, 6. mu.L, 8. mu.L, 12. mu.L, 16. mu.L and 20. mu.L, and PBS was added to make up to 20. mu.L.
(3) Add 200. mu.L of BCA working solution to each well, followed by 20. mu.L to the sample wells of a 96-well plate. Because pipettes have a large error when taking small amounts of sample, the point in front of the standard line may not be very accurate, so that the sample point falls as far as possible behind the standard line 1/2.
(4) Standing at 37 ℃ for 15-30 min. And (5) measuring A562 nm by using a microplate reader, and calculating the protein concentration according to a standard curve. When using incubator to incubate, should pay attention to prevent to influence the testing result because of moisture evaporation.
3. Protein denaturation: 80 mul of the protein with the adjusted concentration) is taken into a new centrifugal tube, 20 mul of 5 Xloading Buffer is added according to the proportion of 4:1, the opening is sealed, and the centrifugal tube is placed in a metal bath with the temperature of 95 ℃ for denaturation for 5 min.
4. SDS-PAGE gel electrophoresis (gel kit)
(1) Preparing concentrated gel with the concentration of 5 percent and separation gel with the concentration of 12 percent according to the following table, filling the separation gel solution into an electrophoresis plate, and adding distilled water to level the upper layer.
The procedure for the preparation of the electrophoretic gel and the gel concentrate in this application example is shown in Table 7.
TABLE 7 electrophoretic separation gel and gel concentration procedure
12 percent of separation gel
5 percent of concentrated gum
Total volume
15mL
6mL
30% glue solution (29:1)
6mL
1mL
1MTris-HCl(PH6.8)
0
0.725mL
1.5MTris-HCl(PH8.8)
3.8mL
0
10％SDS
150μL
60μL
10% PAGE gel coagulant
150μL
60μL
PAGE gel Accelerator (TEMED)
6μL
6μL
ddH2O
4.9mL
4.1mL
Optimum separation Range
12～60kD
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(2) After observing the complete polymerization of the separation gel (about 30min), the distilled water was removed, the excess solution was removed through filter paper, the concentrated gel was added and a clean comb was quickly inserted therein.
(3) After the concentrated gel is completely polymerized (about 40min), the comb is pulled out and placed in an electrophoresis plate, and electrophoresis buffer is added for sample loading and electrophoresis. The concentrated gel is placed under a voltage of 90V for about 30min, the separation gel is kept under a constant voltage of 120V for about 60min, and then the electrophoresis is stopped.
5. Transferring and sealing (Filter paper and sponge are soaked in the transferring liquid in advance)
(1) Film transfer: shearing the gel, preparing into PVDF mold with the same size, soaking in methanol for 10s for activation treatment, and standing in Transfer Buffer for 20 min. The film treatment was carried out for 120min at 200mA in accordance with the sequential mounting of black → sponge → filter paper → gel → PVDF film → filter paper → sponge → white. Wash 3 times with TBST solution for 15min each time. After the film is completely rotated and the power supply is cut off, the film is taken out, and a small opening is cut at the upper left side.
(2) And (3) sealing: putting PVDF membrane into sealing liquid (5% skimmed milk powder, TBST diluted), and sealing on a shaking table for 1h at normal temperature.
6. URAT1 antibody (primary antibody, secondary antibody) incubation process
(1) Primary antibody incubation: adding primary antibody into the sealing liquid according to the proportion of 1: 500. After the overnight incubation was complete, membrane washing with TBST was repeated three times for 10 min.
(2) And (3) secondary antibody incubation: adding the secondary antibody into the newly-prepared confining liquid according to the proportion of 1:2000, and incubating for 1h on a shaking table at room temperature. After the incubation was completed, membrane washing with TBST was repeated three times for 10 min.
7. Protein detection
(1) And (3) uniformly mixing the ECL developing solution A, B liquid in equal amount to prepare a developing working solution, dripping the working solution on a membrane, reacting for 2min in a dark place, taking beta-actin as an internal reference, and exposing in a developing instrument.
(2) Quantitative analysis was performed by grey value of each protein band, and this experiment was repeated 3 times.
The experimental results are shown in fig. 2: through western blot, the influence of the compositions in the 4 groups of the compositions in the example 1 and the comparative examples 1-3 on the expression level of the mouse kidney uric acid transporter URAT1 is detected. The results show that compared with the control group, the kidney URAT1 protein of the model group mice is obviously increased, and the difference has statistical significance (P < 0.001); compared with the model group, the sunflower disc extracts of four different processes remarkably reduce the expression of URAT1 protein, and the difference has statistical significance (p is less than 0.001, and p is less than 0.001). The positive drug benzbromarone can also inhibit the expression of URAT1 protein, and the difference has statistical significance (p < 0.001). In addition, experimental results show that the inhibitory action of the extracts of the four processes on URAT1 protein is better than that of the positive drug benzbromarone on URAT1 protein, but the drug effect of the composition prepared by the method is the best, namely the extract obtained by the process of extracting water through macroporous resin.
In conclusion, the experiment adopts a hyperuricemia mouse model to investigate the uric acid reduction of the sunflower disc extracts of four different processes, and the result shows that the sunflower disc extracts of different processes can obviously reduce the blood uric acid level of the mouse through intragastric administration, and can obviously reduce the expression of URAT1 protein. The composition obtained by the method has the best drug effect, and the effect is superior to that of a positive control.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
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