A method for constructing a fingerprint of a medicine for treating gout and uric acid nephropathy

Abstract

The present disclosure provides a traditional Chinese medicine composition for treating gout and uric acid nephropathy, and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition comprises the following raw medicinal materials in parts by weight: 20-30 parts of smilax glabra, 20-30 parts of phragmites communis, 10-15 parts of atractylodes lancea, 10-15 parts of phellodendron amurense, 10-15 parts of cyathula officinalis, 20-30 parts of coicis semen, 10-20 parts of lonicera japonica, 10-15 parts of acanthopanax, 5-10 parts of eupolyphaga, 5-10 parts of rhizoma corydalis, 10-15 parts of salvia miltiorrhiza, and 15-30 parts of plantago asiatica. The traditional Chinese medicine composition can effectively treat gout and uric acid nephropathy.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of traditional Chinese medicine, in particular to a traditional Chinese medicine composition and a preparation method and application thereof. BACKGROUND

[0002] Uric acid nephropathy is also called gout nephropathy. Epidemiological results show that in recent years, with the change of the diet structure of the public, the intake of high purine diet is increasing, the prevalence of hyperuricemia is increasing year by year, and the prevalence of gout and uric acid nephropathy is also increasing, which has become a common and frequently-occurring disease in clinic. Gout and uric acid nephropathy are closely related. Literature reports that more than 40% of long-term gout patients can develop into chronic kidney disease, that is, gout patients are often accompanied by uric acid nephropathy, and one of the typical clinical symptoms of uric acid nephropathy patients is gout. Studies have shown that in the autopsy results of gout patients, 79% to 99% of gout patients have uric acid nephropathy. In the process of uric acid-induced kidney disease, the two often cause each other and form a vicious cycle.

[0003] From the perspective of traditional Chinese medicine, according to its clinical characteristics, uric acid nephropathy can be classified into the category of "gout" in traditional Chinese medicine. Traditional Chinese medicine believes that uric acid nephropathy is mainly caused by deficiency of the spleen and kidney, combined with overeating of rich food, emotional disorder, overwork, and the invasion of external pathogens, and the disease is caused by internal and external factors; the movement of qi, blood and water is obstructed, phlegm and dampness are generated internally, and stasis is caused after a long time, which causes waist pain, joint pain and arthralgia; the spleen and kidney are weak, and the water and liquid metabolism is disordered, which causes edema; phlegm turbidity, dampness and blood stasis are caused by heat, which damages the kidney collaterals and causes blood to flow, which is blood stone; the pathogenic factor penetrates deep into the body, and the urine impurities are combined into sand and stone, which is stone; if it enters the viscera, it will "exhaust the kidney", and the storage function will be lost. Deficiency of the spleen and kidney is the root, and phlegm, dampness and stasis are the branches. The root and branches are both deficiency and excess, and the disease is characterized by deficiency and excess. It can be seen that "phlegm-dampness-stasis" plays an important role in the pathogenesis of uric acid nephropathy and is the main syndrome type of uric acid nephropathy. Strengthening the prevention and treatment of uric acid nephropathy can effectively inhibit uric acid-induced kidney damage, which embodies the characteristics of traditional Chinese medicine in treating the whole body and preventing the disease.

[0004] From the perspective of Western medicine, uric acid nephropathy is caused by excessive uric acid production or uric acid excretion disorder, which leads to purine metabolism disorder, resulting in elevated blood uric acid, and then causing uric acid and its crystals to deposit in the kidney, leading to kidney damage. Therefore, the current clinical treatment method is mainly to reduce uric acid, and modern medicine prefers allopurinol and febuxostat to inhibit uric acid production, or chooses benzobromarone to promote uric acid excretion, in order to reduce the level of blood uric acid. The above three drugs are currently the first-line drugs for reducing uric acid in clinical treatment of gout and uric acid nephropathy, but in recent years, numerous studies have confirmed that these three drugs have different side effects.

[0005] Allopurinol is mainly metabolized by the kidney, and part of the patients with gout and uric acid nephropathy have impaired renal function, long-term use will increase the burden of the kidney, in addition, allopurinol can also cause hypersensitivity, drug eruption and other adverse reactions; long-term use of febuxostat will increase the risk of cardiovascular adverse events; benzbromarone reduces serum uric acid by inhibiting the reabsorption of uric acid in the proximal tubule and increasing the excretion of urate, but this kind of drug has the risk of increasing the incidence of urinary stones, and with the decrease of glomerular filtration rate, the excretion of uric acid also decreases, in addition, benzbromarone also has hepatotoxicity and cannot be used for a long time. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings of the prior art, provide a traditional Chinese medicine composition and its preparation method and application, which can effectively treat gout and uric acid nephropathy.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: in the first aspect, a traditional Chinese medicine composition is provided, which comprises the following raw medicinal materials by weight: 20-30 parts of smilax glabra, 20-30 parts of cyperus rotundus, 10-15 parts of atractylodes, 10-15 parts of phellodendron, 10-15 parts of cyathula officinalis, 20-30 parts of coicis semen, 10-20 parts of lindera aggregata, 10-15 parts of acanthopanax, 5-10 parts of eupolyphaga, 5-10 parts of rhizoma corydalis, 10-15 parts of salvia miltiorrhiza, and 15-30 parts of plantago.

[0008] The traditional Chinese medicine composition in this paper can effectively reduce the levels of test indexes such as blood uric acid, creatinine and urea nitrogen in the body by intervening mechanisms such as oxidative stress and inflammation, play the role of inhibiting renal fibrosis and relieving uric acid-induced renal damage, and thus achieve the effect of treating gout and uric acid nephropathy. The traditional Chinese medicine composition follows the treatment strategy of eliminating phlegm and turbidity, removing dampness and evil, and removing blood stasis. Among them, smilax glabra removes phlegm and turbidity, detoxifies, invigorates the stomach and dries dampness, and relieves joints; cyperus rotundus removes turbidity and dries dampness; atractylodes dries dampness, invigorates the spleen, removes phlegm, removes obstruction and unblocks collaterals; phellodendron removes heat and dries dampness, removes fire and detoxifies; lindera aggregata relaxes tendons and unblocks collaterals, removes dampness and harmonizes the stomach; acanthopanax removes dampness and benefits the liver and kidney; eupolyphaga removes phlegm and phlegm; salvia miltiorrhiza promotes blood circulation, removes blood stasis and unblocks collaterals; corydalis promotes blood circulation, removes qi stagnation and relieves pain; coicis semen and plantago remove turbidity and induce diuresis; cyathula officinalis promotes blood circulation, removes blood stasis, benefits the kidney, induces diuresis and unblocks collaterals, and also leads the blood downward; the combination of various drugs can remove phlegm and turbidity, remove dampness and evil, and remove blood stasis.

[0009] In one embodiment, the herbal composition comprises the following raw materials in parts by weight: 25-30 parts of Smilax glabra, 25-30 parts of Dioscorea hypoglauca, 12-15 parts of Atractylodes lancea, 10-15 parts of Phellodendron chinense, 10-15 parts of Achyranthes bidentata, 20-30 parts of raw Coix lacryma-jobi, 10-20 parts of Chaenomeles speciosa, 10-15 parts of Acanthopanax senticosus, 6-10 parts of Eupolyphaga sinensis, 8-10 parts of Corydalis yanhusuo, 10-15 parts of Salvia miltiorrhiza, and 15-30 parts of Plantago asiatica. The inventors have found that when the weight parts of each raw material in the herbal composition are within the above-mentioned range, the herbal composition exhibits better therapeutic effects.

[0010] In one embodiment, the raw materials include the following parts by weight: 30 parts Smilax glabra, 30 parts Dioscorea hypoglauca, 15 parts Atractylodes lancea, 12 parts Phellodendron chinense, 12 parts Achyranthes bidentata, 30 parts Coix lacryma-jobi, 20 parts Chaenomeles speciosa, 15 parts Acanthopanax senticosus, 6 parts Eupolyphaga sinensis, 9 parts Corydalis yanhusuo, 15 parts Salvia miltiorrhiza, and 30 parts Plantago asiatica. The inventors have found that when the weight parts of each raw material in the traditional Chinese medicine composition are within the above-mentioned range, the therapeutic effect of the traditional Chinese medicine composition is better.

[0011] In one embodiment, the raw materials include the following parts by weight: 30 parts Smilax glabra, 30 parts Dioscorea hypoglauca, 15 parts Atractylodes lancea, 12 parts Phellodendron chinense, 12 parts Achyranthes bidentata, 20 parts Coix lacryma-jobi, 10 parts Chaenomeles speciosa, 10 parts Acanthopanax senticosus, 10 parts Eupolyphaga sinensis, 10 parts Corydalis yanhusuo, 10 parts Salvia miltiorrhiza, and 15 parts Plantago asiatica. The inventors have found that when the weight parts of each raw material in the traditional Chinese medicine composition are within the above-mentioned range, the therapeutic effect of the traditional Chinese medicine composition is better.

[0012] Secondly, a method for preparing the aforementioned traditional Chinese medicine composition is provided, comprising the following steps: adding each raw material to water in a certain proportion and performing water extraction or alcohol extraction to obtain the traditional Chinese medicine composition.

[0013] Thirdly, the application of the aforementioned traditional Chinese medicine composition in the preparation of a medicament for treating uric acid nephropathy is provided.

[0014] Fourthly, a medicine for treating uric acid nephropathy is provided, the medicine comprising the above-mentioned traditional Chinese medicine composition.

[0015] In one embodiment, the drug further comprises a pharmaceutically acceptable carrier.

[0016] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0017] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of therapeutic agents including various excipients and diluents and the like. This term refers to carriers that are nontoxic to the subject to be treated and that do not interact deleteriously with the active ingredient. Suitable carriers are well known to persons of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Pharmaceutically acceptable carriers in the compositions can contain liquids such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances can be present in the carriers, including, but not limited to, mannitol, sorbitol, sodium pyrosulfite, sodium bisulfite, sodium thiosulfite, cysteine hydrochloride, mercaptoacetic acid, methionine, vitamin C, disodium EDTA, calcium disodium EDTA, carbonates of monovalent alkali metals, acetates of monovalent alkali metals, phosphates of monovalent alkali metals or aqueous solutions thereof, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginic acid, gelatin, polyvinylpyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate. However, one of ordinary skill in the art will also recognize that pharmaceutical carriers useful in the present application are not limited to the types described above.

[0018] In one embodiment, the dosage form of the medicament is oral liquid, capsule, pill, decoction, tablet, granule, powder.

[0019] The dosage form of the medicament of the present application is not particularly limited and can be any dosage form suitable for oral administration to mammals; preferably, the dosage form can be selected from the group consisting of oral liquid, capsule, pill, decoction, tablet, granule, powder. From the standpoints of ease of preparation and administration, liquid preparation, especially decoction, is preferred.

[0020] Specifically, the preparation method of the decoction is as follows: weigh the ingredients of Smilax glabra, Alisma orientalis, Atractylodes lancea, Phellodendron amurense, Chaenomeles speciosa, Eleutherococcus senticosus, Eupolyphaga, Salvia miltiorrhiza, Corydalis, Cyperus rotundus, Radix Cyathulae, and add 8 times the weight of water to soak for 0.5 hours, first boil with strong fire, then boil with weak fire, and combine the two water decoctions to obtain the decoction.

[0021] The method of taking and the dosage of the decoction for treating gout and uric acid nephropathy are as follows: oral administration, 200 mL, twice a day.

[0022] Specifically, the preparation method of the oral liquid is as follows: the proportions of Smilax glabra, Alisma orientalis, Atractylodes lancea, Phellodendri chinensis, Chaenomelis fruit, Eleutherococcus senticosus, Eupolyphaga, Salvia miltiorrhiza, Corydalis, Coix seed, Herba Plantaginis, Radix Cyathulae are weighed, and the total weight of the raw materials is soaked in 8 times the amount of water for 0.5 hours, decocted twice, the first time for 2 hours, and the second time for 1.5 hours, filtered, the filtrate is combined and concentrated, 50-60% ethanol is added and soaked for 10 hours, filtered, the ethanol is recovered, water and preservatives are added, and the oral liquid is obtained.

[0023] The oral liquid is used for treating gout and uric acid nephropathy, and the administration method and dosage are as follows: oral administration, 15 mL each time, 2 times a day, 7 days for an acute attack period, and 30 days for a chronic remission period.

[0024] Specifically, the preparation method of the capsule is as follows: the proportions of Smilax glabra, Alisma orientalis, Atractylodes lancea, Phellodendri chinensis, Chaenomelis fruit, Eleutherococcus senticosus, Eupolyphaga, Salvia miltiorrhiza, Corydalis, Coix seed, Herba Plantaginis, Radix Cyathulae are weighed, and the total weight of the raw materials is soaked in 6 times the amount of water for 0.5 hours, decocted twice, the first time for 2.5 hours, and the second time for 2 hours, filtered, the filtrate is combined and concentrated, 50-60% ethanol is added and soaked for 10 hours, filtered, the ethanol is recovered, the filtrate is concentrated, spray-dried into fine powder, starch and microcrystalline fiber are added, mixed uniformly, and then divided into capsule shells to obtain the capsule.

[0025] In a fourth aspect, a method for constructing a fingerprint of the medicine for treating gout and uric acid nephropathy is provided, and the method comprises the following steps:

[0026] (1) precisely weighing the medicine to prepare a medicine test sample solution;

[0027] (2) analyzing the medicine test sample solution by using a liquid chromatograph to obtain a medicine fingerprint;

[0028] The chromatographic conditions of the liquid chromatograph analysis are as follows: gradient elution is performed with water as mobile phase A and acetonitrile as mobile phase B, the flow rate is 0.75-1.5 mL / min, the column temperature is 38-42℃, the injection volume is 18-22 μL, and the detection wavelength is 300-360 nm.

[0029] In an embodiment, the gradient elution conditions are as follows: 0-5 min, 95%-78% A; 5-30 min, 78%-62% A; 30-45 min, 62%-60% A; 45-55 min, 60%-40% A; and 55-65 min, 40% A.

[0030] Specifically, the chromatographic conditions of the liquid chromatograph analysis are as follows: the chromatographic column is an Ultimate (AQC18 4.6*150 cm, 5 μm) column; the mobile phase is water (A)-acetonitrile (B); gradient elution (0-5 min, 95%-78% A; 5-30 min, 78%-62% A; 30-45 min, 62%-60% A; 45-55 min, 60%-40% A; 55-65 min, 40% A); the flow rate is 1.0 mL / min; the column temperature is 40 DEG C; the injection volume is 20 μL; and the detection wavelength is 330 nm.

[0031] In one embodiment, the test sample solution is prepared by the method comprising the following steps: taking a drug granule, a drug tablet or a drug powder, grinding, taking 1-2 g, accurately weighing, placing in a container, accurately adding 40%-50% methanol 20-30 ml, weighing, ultrasonic treatment for 20-40 minutes, cooling, re-weighing, supplementing the lost weight with 40%-50% methanol, shaking, filtering, and taking the filtrate to obtain the test sample solution.

[0032] In one embodiment, the test sample solution is prepared by the method comprising the following steps: taking a drug granule, a drug tablet or a drug powder, grinding, taking 1-2 g, accurately weighing, placing in a container, accurately adding 40%-50% methanol 20-30 ml, weighing, ultrasonic treatment for 20-40 minutes, cooling, re-weighing, supplementing the lost weight with 40%-50% methanol, shaking, filtering, and taking the filtrate to obtain the test sample solution.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] (1) The traditional Chinese medicine composition can treat gout and uric acid nephropathy by the ways of eliminating phlegm and turbidity, transforming dampness evil, and removing blood stasis, wherein, the earth-wood lotus eliminates phlegm, turbidity and detoxification, strengthens the stomach and dries dampness, and relieves joints; the rhizoma alismatis separates clear and turbid, and dries dampness; the rhizoma atractylodis dry dampness, strengthen the spleen, and transform phlegm; the cortex phellodendri clears heat and dries dampness, and purges fire and detoxifies; the chaenomeles fruit relaxes the sinews, activates the collaterals, and transforms dampness and stomach; the acanthopanax bark removes dampness and benefits the liver and kidney; the eupolyphaga transforms phlegm and eliminates phlegm; the salvia miltiorrhiza activates blood, removes blood stasis, and unblocks the collaterals; the rhizoma corydalis activates blood, moves qi, and relieves pain; the coix seed and the plantain herb eliminate turbidity and induce diuresis; the cyathula root activates blood, removes blood stasis, benefits the kidney, induces diuresis, and relieves stranguria, and leads blood downward; the combination of the above-mentioned drugs can eliminate phlegm and turbidity, transform dampness evil, and remove blood stasis. The traditional Chinese medicine composition can inhibit uric acid synthesis and promote uric acid excretion, has a wide target, has obvious treatment effect, and has small side effects.

[0035] (2) The liquid chromatography is used to construct the fingerprint of the medicine for treating gout and uric acid nephropathy, to realize the qualitative analysis of the effective components in the medicine for treating gout and uric acid nephropathy, to fully show the chemical component characteristics of the medicine for treating gout and uric acid nephropathy, to comprehensively reflect the characteristic peak information of the sample, and the method is stable, high in precision and good in reproducibility. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Figure for the influence of different conditions in Example 4 on HK-2 cells (human renal tubular epithelial cells), wherein A is the influence of different concentrations of uric acid on HK-2 cell viability for 12, 24 and 48H, B is the influence of different concentrations of DCS on HK-2 cells, C is the influence of 1 / 4 concentration of DCS on HK-2 cells for different action times;

[0037] Figure 2 Figure for the detection results of Q-PCR and Wb in Example 4, wherein A is the detection of mRNA expression in each group by Q-PCR, B is the detection of protein expression bands by Wb, C is the semi-quantitative results of the detection results by Wb;

[0038] Figure 3 Figure for the influence of DCS on fibrotic HK-2 cells in Example 4, wherein A is the treatment of fibrotic HK-2 cells by DCS for 12, 24 and 48H, B is the influence of DCS on the relative level of mitochondrial membrane potential of fibrotic HK-2 cells, C is the influence of DCS on the mitochondrial membrane potential of fibrotic HK-2 cells, D is the influence of DCS on the ultrastructure of fibrotic HK-2 cells;

[0039] Figure 4 Figure for the influence of DCS on ROS of fibrotic HK-2 cells in Example 4;

[0040] Figure 5 Figure for the relative level of mRNA and protein in each group of HK-2 cells in Example 4, wherein A is the relative level of Keap1 mRNA in each group of HK-2 cells, B is the relative level of Nrf2 mRNA in each group of HK-2 cells, C is the relative level of HO-1 mRNA in each group of HK-2 cells, D is the protein expression bands of Keap1, Nrf2, HO-1 and GAPDH in each group of HK-2 cells, E is the relative expression level of Keap1 protein in each group of HK-2 cells, F is the relative expression level of Nrf2 protein in each group of HK-2 cells, G is the relative expression level of HO-1 protein in each group of HK-2 cells, and H is the action legend;

[0041] Figure 6 Fingerprint of 10 batches of Huishi Lezhu Quyu Decoction Granules (S1-S10) and control spectrum (R) in Example 5;

[0042] Figure 7 HPLC chromatogram of the mixed control solution in Example 5, wherein 36 is cryptotanshinone, 47 is tanshinone IIA, 48 is salvianolic acid B, 61 is atractylenolide, 62 is astilbin, and 63 is plantain glycoside.

[0043] Figure 8 A cluster analysis diagram of 10 batches of Huashi Xuezhuo Quyu Decoction Granules in Example 5 is shown.

[0044] Figure 9 A principal component analysis diagram of 10 batches of Huashi Xuezhuo Quyu Decoction Granules in Example 5 is shown. DETAILED DESCRIPTION

[0045] In order to better illustrate the purposes, technical solutions and advantages of the present application, the present application will be further described below in combination with specific examples and comparative examples, which aims to understand the content of the present application in detail, rather than limiting the present application. All other examples obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present application. The experimental reagents and instruments involved in the implementation of the present application are all common ordinary reagents and instruments unless otherwise specified.

[0046] Example 1

[0047] The present embodiment provides a traditional Chinese medicine composition, which comprises the following raw medicinal materials by weight: 30 parts of Smilax glabra, 30 parts of Rehmannia chathamica, 15 parts of Atractylodes lancea, 12 parts of Phellodendron amurense, 12 parts of Cyathula officinalis, 30 parts of Coix lachryma-jobi, 20 parts of Argyoxiphium fortunei, 15 parts of Eleutherococcus senticosus, 6 parts of Eupolyphaga, 9 parts of Corydalis tuber, 15 parts of Salvia miltiorrhiza, and 30 parts of Plantago asiatica.

[0048] The present embodiment also provides a decoction medicine for treating gout and uric acid nephropathy, and the preparation method thereof is as follows: 30 parts of Smilax glabra, 30 parts of Rehmannia chathamica, 15 parts of Atractylodes lancea, 12 parts of Phellodendron amurense, 12 parts of Cyathula officinalis, 30 parts of Coix lachryma-jobi, 20 parts of Argyoxiphium fortunei, 15 parts of Eleutherococcus senticosus, 6 parts of Eupolyphaga, 9 parts of Corydalis tuber, 15 parts of Salvia miltiorrhiza, and 30 parts of Plantago asiatica are weighed; then 8 times the amount of water as the total weight of the raw medicinal materials is added to soak for 0.5 hours, and water decoction is performed twice, and the two times of water decoction liquid are combined to obtain the decoction medicine.

[0049] The embodiment also provides a granule medicine for treating gout and uric acid nephropathy, and a preparation method thereof is as follows: 30 parts of smilax glabra, 30 parts of pharbitidis, 15 parts of atractylodes, 12 parts of phellodendri, 12 parts of radix cyathulae, 30 parts of coicis, 20 parts of radix campylotropidis, 15 parts of cortex acanthopanacis, 6 parts of eupolyphaga, 9 parts of rhizoma corydalis, 15 parts of salvia miltiorrhiza, and 30 parts of plantaginis are weighed; then 8 times of water of the total weight of the raw medicinal materials is added to soak for 0.5 hours, decocted twice, 2.5 hours for the first time and 2 hours for the second time, filtered, the filtrates are combined and concentrated, 60% ethanol is added to soak for 10 hours, filtered, the ethanol is recovered, the filtrate is concentrated, spray-dried into fine powder, starch and microcrystalline fiber are added, uniformly mixed, and the granule medicine is obtained.

[0050] Embodiment 2

[0051] The embodiment provides a traditional Chinese medicine composition, which comprises the following raw medicinal materials in parts by weight: 30 parts of smilax glabra, 30 parts of pharbitidis, 15 parts of atractylodes, 12 parts of phellodendri, 12 parts of radix cyathulae, 20 parts of coicis, 10 parts of radix campylotropidis, 10 parts of cortex acanthopanacis, 10 parts of eupolyphaga, 10 parts of rhizoma corydalis, 10 parts of salvia miltiorrhiza and 15 parts of plantaginis.

[0052] The embodiment also provides a medicine for treating gout and uric acid nephropathy, and a preparation method thereof is as follows: 30 parts of smilax glabra, 30 parts of pharbitidis, 15 parts of atractylodes, 12 parts of phellodendri, 12 parts of radix cyathulae, 20 parts of coicis, 10 parts of radix campylotropidis, 10 parts of cortex acanthopanacis, 10 parts of eupolyphaga, 10 parts of rhizoma corydalis, 10 parts of salvia miltiorrhiza and 15 parts of plantaginis are weighed; then 8 times of water of the total weight of the raw medicinal materials is added to soak for 0.5 hours, decocted twice, the two times of decocted liquid are combined, and the decoction is obtained.

[0053] Embodiment 3

[0054] The embodiment provides a traditional Chinese medicine composition, which comprises the following raw medicinal materials in parts by weight: 20 parts of smilax glabra, 30 parts of pharbitidis, 20 parts of atractylodes, 10 parts of phellodendri, 18 parts of radix cyathulae, 40 parts of coicis, 10 parts of radix campylotropidis, 10 parts of cortex acanthopanacis, 20 parts of eupolyphaga, 10 parts of rhizoma corydalis, 15 parts of salvia miltiorrhiza and 10 parts of plantaginis.

[0055] The embodiment also provides a medicine for treating gout and uric acid nephropathy, and a preparation method thereof is as follows: 20 parts of smilax glabra, 30 parts of pharbitidis, 20 parts of atractylodes, 10 parts of phellodendri, 18 parts of radix cyathulae, 40 parts of coicis, 10 parts of radix campylotropidis, 10 parts of cortex acanthopanacis, 20 parts of eupolyphaga, 10 parts of rhizoma corydalis, 15 parts of salvia miltiorrhiza and 10 parts of plantaginis are weighed; then 8 times of water of the total weight of the raw medicinal materials is added to soak for 0.5 hours, decocted twice, 2.5 hours for the first time and 2 hours for the second time, filtered, the filtrates are combined and concentrated, 60% ethanol is added to soak for 10 hours, filtered, the ethanol is recovered, the filtrate is concentrated, spray-dried into fine powder, starch and microcrystalline fiber are added, uniformly mixed, and the granule medicine is obtained.

[0056] Example 4

[0057] This embodiment illustrates the mechanism of the Chinese medicine composition in treating gout and uric acid nephropathy by cell experiments. The specific method is as follows:

[0058] S1: Establishment of HK-2 cell fibrosis model

[0059] The human renal tubular epithelial cells HK-2 used in this experiment were purchased from China Type Culture Collection Center. 30 mg of uric acid (UA) was added to the serum-free RPMI-1640 solution and heated to dissolve. The UA solution had a concentration of 0.5 mg / ml and a pH value of 7.2-7.4, and was used after ultrafiltration with a small ultrafiltration machine. During the experiment, the mixed solution was diluted with RPMI-1640 to prepare UA solutions with concentrations of 0.1 mg / ml, 0.3 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, and 3 mg / ml. According to the cell viability, the optimal concentration and the best treatment time for UA solution modeling were determined, and the HK-2 cell fibrosis model was prepared by stimulating the cells with the UA solution at the optimal concentration.

[0060] S2: Construction of HK-2 cell line model overexpressing Keap1

[0061] a. Plasmid extraction: After 24 hours of competent bacteria preparation, plasmid transformation was performed according to the standard procedure. When the bacterial solution was appropriately turbid, plasmid extraction was prepared. According to the instructions of the plasmid extraction kit (Tiangen, China), 4 ml of Buffer P1 was added to the bacterial microspheres to completely resuspend them. Then, 4 ml of Buffer P2 was added, mixed gently, and incubated at room temperature for 5 min. 4 ml of pre-cooled Buffer P3 was added immediately, mixed, and placed on ice for 20 min. Then, centrifugation was performed at 4°C and 12000 rpm for 30 min. The supernatant was transferred to another centrifuge tube and centrifuged again. The QIAGEN-tip100 was balanced with 4 ml of buffer QBT, and the supernatant after the second centrifugation was transferred to the QIAGEN-tip100 column, allowing the liquid to flow out naturally. The chromatographic column was washed twice with 10 ml of Buffer QC, and then 5 ml of Buffer QF was added to elute the plasmid. 3.5 ml of isopropanol was added and mixed evenly, and then centrifugation was performed at 4°C and 11000 rpm for 30 min. The supernatant was discarded, and the resulting precipitate was washed once with 5 ml of 70% ethanol. After air-drying for 5-10 min, 100 μl of TE was added for dissolution. The plasmid concentration was determined using a UV spectrophotometer, and the plasmid was stored at -20°C for future use.

[0062] b. Lentivirus production: 24h before transfection, HK-2 cells in good growth condition were digested and inoculated in T25 culture flask, and cultured conventionally until the cell growth coverage reached 80%. The culture medium was replaced with serum-free medium for half an hour, and then packaged by liposome-mediated method according to the reagent instruction (Thermo Fisher, USA). The lentivirus produced by packaging had two types, one was lentivirus containing Keap1, and the other was blank control lentivirus.

[0063] c. Construction of HK-2 cell line overexpressing Keap1: the lentivirus containing Keap1 fragment and the control lentivirus packaged above were used to infect HK-2 cell line in good growth condition respectively. 8h later, the virus-containing culture medium was discarded, and complete culture medium was added. The infection efficiency of HK-2 cells and blank control cells was observed by inverted microscope at 24h and 48h respectively, which was used for subsequent experiments.

[0064] S3: Construction of HK-2 cell line silenced for Nrf2 and HO-1 (siRNA)

[0065] The specific small RNA interference fragments of Nrf2 and HO-1 genes were designed and synthesized using the free software provided by the Invitrogen website (https: / / maiciesigner.invitrogen.com / sima / ). The Nrf2 and HO-1 silenced HK-2 cell line was constructed by plasmid extraction and lentivirus production (method step S2), and the Nrf2 and HO-1 silenced HK-2 cells were verified by Western Blot (Wb) and qRT-PCR (qRT-PCR), method steps S6 and S7.

[0066] S4: Test grouping

[0067] A. Under the culture conditions of step S1, HK-2 cells were divided into 5 groups: control group; model group; DCS-L (low-dose traditional Chinese medicine composition); DCS-M (medium-dose traditional Chinese medicine composition); DCS-H (high-dose traditional Chinese medicine composition). This will help us determine the effect of DCS on the expression of Keap1 / Nrf2 pathway.

[0068] B. In order to further explore the potential relationship between traditional Chinese medicine composition and Keap1 / Nrf2 / HO-1, we designed the following 10 groups of experiments: control group; model group; DCS group; allopurinol intervention group (Allo); Keap1 plasmid + DCS transfection Keap1 blank plasmid + DCS transfection Nrf2 siRNA + DCS; Nrf2 siRNA negative control (NC) + DCS; HO-1 siRNA + DCS; HO-1 siRNA NC + DCS.

[0069] S5: MTT assay

[0070] When the cells in the flask were about 80% full, the medium in the flask was discarded and washed twice with PBS. 1 ml of 0.25% trypsin was used to digest the cells for 1 min to make a cell suspension. After cell counting, 1 x 10 4 Cells were cultured for 24 h. Then the medium was replaced with serum-free medium and cultured for another 12 h. According to the above grouping, each group was further divided into 5 parallel control groups. After 12 h, 24 h and 48 h of intervention, the original culture solution was aspirated, 20 ul of MTT solution (5 mg / ml) was added, and it was cultured in a 37°C incubator for 4 h. Then 150 ul of DMSO solution was added and shaken on a horizontal shaker for 10 min. The OD value at 490 nm was measured by a microplate reader, and the cell survival rate was counted.

[0071] S6: qRT-PCR

[0072] The mRNA sequences of Keap1, Nrf2 and HO-1 were obtained from the gene bank, and the primer 3.0 software (PREMIER Biosoft International, USA) was used for primer design. The primer sequences are shown in Table 1. Total RNA was used as a template to reverse transcribe into cDNA, and SYBR chimeric fluorescence detection was performed according to the requirements of the instructions. In the qRT-PCR detection process, β-Actin was used as an internal reference gene. Each sample was detected in triplicate, and amplification was performed on an ABI 7500 fluorescence quantitative PCR amplifier. The relative abundance of mRNA was calculated by the △△Ct method, and the gene specificity efficiency was explained. Then statistical analysis was performed according to the above results.

[0073] Table 1

[0074]

[0075] S7: Protein extraction and WB

[0076] Each tube of cells was added with 100 ul of lysis solution containing pmsf, and after ice bath for 30 min, homogenate was centrifuged at 4°C, 12000 rpm for 5 min, and the total protein concentration was detected by BCA (1 ug / mL) protein concentration determination kit. The protein was added to the EP tube containing 125 μL 5xSDS, and placed in 100°C water bath for 5-10 min, and after cooling to room temperature, it was stored in a-80°C refrigerator. The total protein extract was subjected to polyacrylamide gel electrophoresis with 10%-15% gel. Then the sample was transferred to PVDF membrane. After blocking in 5% skim milk for 2 h, the PVDF membrane was incubated with the following diluted primary antibody (Shanghai Kangcheng Biological, Table 2) at 4°C overnight. The specific primary antibody information is shown in Table 2. After washing the membrane with 1 times TBST, horseradish peroxidase (HRP) conjugated anti-rabbit IgG secondary antibody (1:2000, southern Biotech) was used to block at 37°C for 60 min. After washing with TBST for 3 times, the bound antibody was shown by chemiluminescence with Immobilon Western Chemilum Hrp Substrate (MILLIPORE). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The luminescence signal was detected by fluorescence imaging. The band intensity was quantified by Image J software.

[0077] Table 2

[0078] Antibody Dilution rate Company Protein size (kDa) Keap1 1:1000 Cell Signaling Technology 109 Nrf2 1:1000 Cell Signaling Technology 94 HO-1 1:1000 Cell Signaling Technology 19.6 GAPDH 1:10000 aksomics 36

[0079] S8: Ultrastructure observation

[0080] Logarithmic growth phase HK-2 cells were taken, low-speed centrifugation, and the cells were precipitated at the bottom of the centrifuge tube. The supernatant was absorbed, and 2.5% glutaraldehyde and PBS were immediately added for fixation for 2 hours. Then, 0.1M phosphate washing solution was used for washing, 1% osmium acid fixing solution was used for fixation for 1h, and then alcohol and acetone were used for dehydration step by step. Pure acetone and embedding liquid were used for embedding, toluidine blue staining was performed, and then ultrathin sectioning was performed. Finally, uranyl acetate and lead citrate were used for staining, and transmission electron microscopy observation and photography of HK-2 cell mitochondrial ultrastructure were performed.

[0081] S9: Mitochondrial membrane potential detection

[0082] Cells in logarithmic growth phase were taken, and the original complete medium was discarded. The cells were washed twice with phosphate buffer. The adherent cells were digested with 0.25% trypsin for 1 min, and the cell suspension was blown into the 6-well plate. After counting, 1 x 104 cells were inoculated in each well. After 1-2 days of cell culture, the medium was replaced with serum-free medium, and the cells were cultured for another 12 h. The cells were detected using the mitochondrial membrane potential detection kit (JC-1, Abeam) according to the grouping in 2.4. The change of mitochondrial membrane potential of the cells was determined by the ratio of green and red fluorescence intensity. The average fluorescence value of green and red fluorescence was analyzed using image analysis software. The larger the ratio, the more obvious the decrease in mitochondrial membrane potential.

[0083] S10: Detection of ROS change by fluorescent probe

[0084] DCFH-DA was used for detection. DCFH-DA has no fluorescence, but after entering the cell, it can be oxidized by intracellular oxidants to generate fluorescent DCF. The level of ROS in the cell can be determined by detecting the fluorescence of DCF. DCHF-DA was diluted into serum-free DMEM to a final concentration of 10 umol / L. After the end of the cell intervention experiment, the original culture medium was removed, and the cells were washed twice with PBS buffer. The diluted DCHF-DA medium was added in advance, and the cells were incubated in a 37°C incubator for 30 min. After incubation, the cells were washed twice with PBS, and then collected by conventional digestion and resuspended in PBS. The fluorescence value was determined by flow cytometry, and the median of the DCHF-DA fluorescence intensity represented the Ros content. The average fluorescence intensity value was analyzed by image J image analysis system software. The higher the fluorescence intensity value, the higher the level of reactive oxygen species.

[0085] S11: Statistics and analysis

[0086] Statistical analysis and graph generation were performed using GraphPad Prism 9 (San Diego, CA, USA). The data were collected from at least three independent experiments, determined by student's t-test, and all results were expressed as mean standard deviation. P<0.05 was considered statistically significant.

[0087] S12: Results

[0088] (1) As shown in Figure 1 A, a stable HK-2 cell fibrosis model can be established by culturing HK-2 cells at a uric acid concentration of 0.5 mg / mL for 24 hours. At this concentration, there is no significant change in cell survival rate. As shown in Figure 1 B, compared with the model group, the cell survival rate is the highest when the concentration of the traditional Chinese medicine composition is 1 / 4 (p-0.0001). After determining the optimal treatment concentration, as shown in Figure 1C, we detected the survival rate of cells in the model group and drug treatment group at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, respectively. From Figure 1 C, it can be seen that the best treatment time of traditional Chinese medicine composition for RF is 48 h.

[0089] (2) The effect of different concentrations of drug-containing serum on the expression level of Keap1 / Nrf2. As shown in Figure 2 A, compared with the control group, the transcription level of Keap1 gene in the model group and DCS-l group increased, but with the increase of DCS dosage, the transcription level of Keap1 gene increased, and the transcription level of Keap1 decreased (p<0.001). The transcription level of Nrf2 gene showed an opposite trend to Keap1 (p<0.05). Subsequently, we detected the protein expression level of Keap1 / Nrf2, and the results are shown in Figure 2 B and Figure 2 C, which is similar to the trend of gene transcription level. It shows that traditional Chinese medicine composition has a dose-dependent regulatory effect on Keap1 / Nrf2 in the treatment of RF.

[0090] (3) We used MTT method to detect the proliferation of HK-2 cells under different treatment times ( Figure 3 A). We found that with the passage of time, the cell proliferation rate of DCS group increased significantly, but overexpression of Keap1 or knockdown of Nrf2 significantly inhibited this trend. Healthy cells usually have a small amount of green fluorescence and a large amount of red fluorescence, representing the aggregation of JC-1, and mitochondria are not depolarized. As shown in Figure 3 B and 3C, there is almost no red fluorescence in the RF group, and the red fluorescence of the traditional Chinese medicine composition treatment group and the allow group is obviously restored. After overexpression of Keap1, there is obvious green fluorescence in HK-2 cells, and the down-regulation of Nrf2 and HO-1 also shows a similar trend. The results of ultrastructure observation show that Figure 3 D), compared with the control group, the mitochondrial membrane of the model group is damaged, and there are more ridge fractures. The traditional Chinese medicine composition can inhibit the occurrence of such damage, but it has a similar trend with the microscopic observation results. Overexpression of Keap1 and knockdown of Nrf2 and HO-1 can affect the treatment effect of traditional Chinese medicine composition.

[0091] (4) Figure 4 The results show that traditional Chinese medicine composition can reduce the ROS content of UA-treated HK-2 cells. Overexpression of Keap1 or knockdown of Nrf2 and HO-1 can inhibit the antioxidant capacity of traditional Chinese medicine composition (p<0.05 or p<0.01), which shows that traditional Chinese medicine composition can inhibit the ROS production of UA-treated HK-2 cells through Keap1 / Nrf2 / HO-1 signaling pathway.

[0092] (5) As shown inFigure 5 A, B, C, we detected the gene expression level of Keap1 / Nrf2 / HO-1 signaling pathway, the results showed that the traditional Chinese medicine composition can inhibit the expression of Keap1, promote the expression of Nrf2 / HO-1 to increase (p<0.05 or p<0.01). The experimental results also prove that we successfully established the Keapl overexpression model and Nrf2 / HO-1 knockdown model. At the same time, HO-1 is the downstream gene of Keap1 / Nrf2, and its knockdown has no significant effect on the upstream two genes. Figure 5 D, E, f, G shows the protein expression results of Keap1 / Nrf2 / HO-1 signaling pathway, which is highly consistent with the gene expression results, further proving the accuracy of our results.

[0093] Example 5

[0094] This embodiment uses high performance liquid chromatography (HPLC) to establish the fingerprint of the granules of Example 1, which illustrates the target point of the traditional Chinese medicine composition in treating gout and uric acid nephropathy.

[0095] The specific test steps are as follows:

[0096] 1. Instruments and reagents

[0097] 1.1 Instruments LC-20AT high performance liquid chromatograph (Japan Shimadzu Company); KQ-500V ultrasonic cleaner (Kunshan ultrasonic instrument Co., Ltd.); ME204 millionth electronic balance (Mettler Company of Switzerland).

[0098] 1.2 Reagents Chromatography pure acetonitrile purchased from Merck Company of Germany; anhydrous sodium acetate, glacial acetic acid are analytical pure; water is ultrapure water (18.2MΩ, self-made in the laboratory); cryptotanshinone (content ≥98%, batch number: M16GB148633), tanshinone IIA (content ≥98%, batch number: Y16M10C88487), atractyloside (content ≥98%, batch number: MFCD01075143), plantain glycoside (content ≥98%, batch number: P01N9F74010), astilbin (content ≥98%, batch number: J29GB153016), salvianolic acid B (content ≥98%, batch number: P13N11F130912) purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.; 10 batches of Huashi Xiezhuo Quyu Decoction Granules (number S1-S10; batch number: 2212001, 2212002, 2211001, 2108004, 2202007, 2210002, 2208023, 2208001, 2103003, 2211001; specification: 18g / bag, China Resources Sanjiu Pharmaceutical Co., Ltd.) purchased from the Traditional Chinese Medicine Department of the First Affiliated Hospital of Sun Yat-sen University.

[0099] 2 Method and result

[0100] 2.1 Chromatographic conditions The chromatographic column was Ultimate (AQC18 4.6 x 150 cm, 5 μm) column; the mobile phase was water (A) - acetonitrile (B); gradient elution (0-5 min, 95%-78% A; 5-30 min, 78%-62% A; 30-45 min, 62%-60% A; 45-55 min, 60%-40% A; 55-65 min, 40% A); the flow rate was 1.0 mL / min; the column temperature was 40 °C; the injection volume was 20 μL, and the detection wavelength was 330 nm.

[0101] 2.2 Preparation of reference solution A certain amount of tanshinone, tanshinone IIA, salvianolic acid B, atractylenolide, astilbin and plantain glycoside was precisely weighed, respectively, and dissolved in methanol to prepare reference solutions with concentrations of 0.22, 0.36, 1.13, 0.75, 0.66 and 0.41 mg / mL, respectively, which were stored in a 4 °C refrigerator in the dark.

[0102] 2.3 Preparation of test sample solution The formula granules were ground into powder and mixed uniformly, 1 g of the mixed powder was weighed into a conical flask with a plug, 30 mL of 50% methanol water (V / V) was accurately added, and after weighing, ultrasonic oscillation (400 W, 50 kHz) was performed for 30 min. After cooling, the weight was weighed again, the weight difference was calculated, and the solution was supplemented with 50% methanol water to the original weight, shaken uniformly, and filtered through a 0.22 μm filter membrane to obtain the test sample solution.

[0103] 2.4 Methodology investigation

[0104] 2.4.1 Precision test: The test sample solution was taken, and the chromatographic conditions in item 2.1 were continuously injected for 6 times. With No. 47 salvianolic acid B chromatographic peak as the reference peak (S), the relative retention time RSD of each common peak was calculated to be less than 0.22%, and the relative peak area RSD was less than 5.00%, indicating that the precision of the experimental instrument was good.

[0105] 2.4.2 Reproducibility test: The same batch of sample granules was taken, and 5 test sample solutions were prepared in parallel according to the method in item 2.3. The sample was injected and determined, with No. 47 salvianolic acid B chromatographic peak as the reference peak (S). The relative retention time RSD of each common peak was calculated to be less than 0.91%, and the relative peak area RSD was less than 4.99%, indicating that the method had good reproducibility.

[0106] 2.4.3 Stability test: The test sample solution was prepared, and the sample was injected and determined at 0, 3, 6, 9, 12 and 24 h, respectively, with No. 47 salvianolic acid B chromatographic peak as the reference peak (S). The relative retention time RSD of each common peak was calculated to be less than 0.22%, and the relative peak area RSD was less than 5.00%, indicating that the test sample solution had good stability within 24 h.

[0107] 2.5 Fingerprint and similarity evaluation Ten batches of sample solutions were injected and analyzed under the chromatographic conditions of item 2.1, and the chromatograms were recorded. The AIA format chromatographic data at 330 nm wavelength were introduced into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and the fingerprint of sample S1 was used as the reference chromatogram. The median method was used, the time window width was 0.1 min, and the Marker peak matching was performed after multi-point correction to establish the overlay chromatogram and the control chromatogram (R), as shown in Figure 6 63 common chromatographic peaks accounted for 80.6% to 90.8% of the total peak area. The similarity evaluation results of the 10 batches of samples are shown in Table 3. The results showed that the similarity of the 10 batches of samples to the control fingerprint was in the range of 0.842 to 0.998. Among them, 1 batch (S10) had a similarity lower than 0.900, indicating that there was a certain difference in the quality of the samples of different batches, which might be related to the production area, picking time, storage method and other conditions of the raw medicinal materials.

[0108] By comparing with the control solution, 6 components were identified, which were cryptotanshinone, tanshinone IIA, salvianolic acid B, atractylodin, astilbin and plantain glycoside. The characteristic chromatogram of the control solution is shown in Figure 7 .

[0109] Table 3 Similarity evaluation results of the fingerprint of Huishi Xiezhuo Quyu Decoction Granules

[0110]

[0111]

[0112] 2.6 Chemical pattern recognition analysis

[0113] 2.6.1 Cluster analysis: The common peak area of the 10 batches of samples was introduced into the SIMCA-P 14.1 software for cluster analysis, and the results are shown in Figure 8 . The 10 batches of samples can be divided into 2 categories, S1-S9 samples are clustered into one category, and S10 sample is clustered into one category. It is indicated that there is a certain difference in the quality of different samples, which might be related to the production area or quality of the raw medicinal materials used in the preparation.

[0114] 2.6.2 Principal component analysis: The common peak area of the 10 batches of samples was introduced into the SIMCA-P 14.1 software for principal component analysis, and the 10 batches of samples can be divided into 2 categories, which is consistent with the cluster analysis results, and the results are shown in Figure 9 .

[0115] Example 6

[0116] This example illustrates the therapeutic effect of the traditional Chinese medicine composition of the present application on treating gout and uric acid nephropathy through clinical trials.

[0117] The specific method is as follows:

[0118] The 33 patients in the treatment group were all from the outpatient clinic of the First Affiliated Hospital of Sun Yat-sen University, male 30 cases, female 3 cases, male: female was 10: 1, age 27-68 years old, average (42.3±3.16) years old, disease duration 1-26 years, average (6.5±2.66) years, among which 7 cases were 1-5 years, 13 cases were 5-10 years, and 13 cases were more than 10 years; 33 cases in the normal control group were all from the hospital, male 30 cases, female 3 cases, male: female was 10: 1; age 25-65 years old, average (38.2±5.22) years old.

[0119] The diagnostic criteria are as follows: (1) Primary hyperuricemia: all cases before treatment have hyperuricemia (male >417 μmol / L, female >357 μmol / L), and exclude secondary hyperuricemia caused by other kidney diseases, blood diseases and tumor radiotherapy, chemotherapy or thiazide diuretics. (2) At least one of the following kidney damage: ① proteinuria; ② hematuria; ③ one or several kidney function decline; ④ urinary tract stones, which can exclude other causes. Syndrome type belongs to wet turbidity, blood stasis and obstruction of collaterals: chest and abdominal distension, poor appetite, vomiting, loose stool and frequent urination, heavy limbs, heavy head, hazy face, yellow or blackish face, dark purple lips and nails, subcutaneous ecchymosis, purple tongue, or petechiae or tooth marks, white and greasy tongue fur, and stringy and rough pulse.

[0120] Treatment group: give the decoction obtained in Example 1, 1 dose / day, taken twice in the morning and afternoon; the normal control group does not use any drug intervention. Both groups take low-salt and low-fat diet, strictly prohibit alcohol and smoking, and keep the protein intake at 0.8-1.0 g / kg body weight per day. If there is significant damage to kidney function, the diet should be adjusted according to the requirements of kidney failure. One month of treatment is one course of treatment, and three courses of treatment are one treatment stage. The results are shown in Table 4.

[0121] Table 4 Effect of Example 1 Decoction on Oxidative Stress Related Indicators of UAN Patients (x±s)

[0122]

[0123] Note: compared with the normal control group, *P<0.05; compared with before treatment, #P<0.05.

[0124] From Table 4, it can be seen that the serum MDA of the patients before treatment was significantly higher than that of the normal control group, and the activities of GSH-Px, SOD and CAT were significantly lower than those of the normal control group (P<0.05), indicating that the oxidative stress level of the UAN patients was increased. Compared with before treatment, the serum MDA after treatment with the decoction of Example 1 was significantly decreased (P<0.05), and the activities of GSH-Px, SOD and CAT were significantly increased (P<0.05); compared with the normal control group, the serum MDA after treatment with the decoction of Example 1 was significantly increased (P<0.05), and the activity of GSH-Px was significantly decreased (P<0.05), and the activities of SOD and CAT had no statistically significant difference (P>0.05), indicating that the Chinese medicinal composition of the application can effectively improve the anti-oxidative stress ability of the gout and uric acid nephropathy patients to a certain extent.

[0125] Finally, it should be noted that the above examples are used to illustrate the technical solutions of the application and not to limit the protection scope of the application. Although the application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the application.

Claims

1. A method for constructing a fingerprint of a medicine for treating gout and uric acid nephropathy, characterized in that, It comprises the following steps: (1) precisely take the medicine, and prepare a medicine test sample solution; take cryptotanshinone, tanshinone IIA, salvianolic acid B, atractylodin, astilbin and plantain asistoside as control samples, and prepare a control sample solution; The test sample solution is prepared by the following method: take medicine granules, medicine tablets or medicine powders, grind them, take 1-2 g, precisely weigh, put them in a container, precisely add 40%-50% methanol 20-30 ml, weigh, ultrasonic treat for 20-40 minutes, cool, weigh again, make up the weight loss with 40%-50% methanol, shake, filter, take the filtrate, and obtain it; (2) analyze the medicine test sample solution and the control sample solution by a liquid chromatograph to obtain a medicine fingerprint; The chromatographic conditions of the liquid chromatograph analysis are as follows: the chromatographic column is an Ultimate (AQC18 4.6*150 cm, 5 μm) column; gradient elution is performed with water as mobile phase A and acetonitrile as mobile phase B, the flow rate is 0.75-1.25 mL / min; the column temperature is 38-42 ℃; the injection amount is 18-22 μL, and the detection wavelength is 300-360 nm; The gradient elution conditions are as follows: 0-5 min, 95%-78% A; 5-30 min, 78%-62% A; 30-45 min, 62%-60% A; 45-55 min, 60%-40% A; 55-65 min, 40% A; The medicine is prepared from the following raw materials by weight: Smilax glabra 20-30 parts, Phalaris maculate 20-30 parts, Atractylodes lancea 10-20 parts, Phellodendron amurense 10-15 parts, Cyathula officinalis 10-20 parts, Coix lachryma-jobi 20-40 parts, Argyoxylon reptans 10-20 parts, Eleutherococcus senticosus 10-15 parts, Eupolyphaga 5-20 parts, Corydalis tuber 5-10 parts, Salvia miltiorrhiza 10-15 parts, Plantago asiatica 10-30 parts.

2. The method for constructing the fingerprint of the medicine for treating gout and uric acid nephropathy according to claim 1, characterized in that, The medicine is prepared from the following raw materials by weight: Smilax glabra 25-30 parts, Phalaris maculate 25-30 parts, Atractylodes lancea 12-15 parts, Phellodendron amurense 10-15 parts, Cyathula officinalis 10-15 parts, Coix lachryma-jobi 20-30 parts, Argyoxylon reptans 10-20 parts, Eleutherococcus senticosus 10-15 parts, Eupolyphaga 6-10 parts, Corydalis tuber 8-10 parts, Salvia miltiorrhiza 10-15 parts, Plantago asiatica 10-30 parts.

3. The method for constructing the fingerprint of the medicine for treating gout and uric acid nephropathy according to claim 1, characterized in that, The preparation method of the medicine comprises the following steps: add each raw material into water according to the proportion, and perform water extraction, and obtain the medicine.

4. The method for constructing the fingerprint of the medicine for treating gout and uric acid nephropathy according to claim 1, characterized in that, The medicine further comprises a pharmaceutically acceptable carrier.

5. The method for constructing the fingerprint of the medicine for treating gout and uric acid nephropathy according to claim 1, characterized in that, The dosage form of the medicine is oral liquid, capsule, pill, decoction, tablet, granule and powder.

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