WO2024087259A1 - 一种防治高血尿酸症的中药复方组合物 - Google Patents

一种防治高血尿酸症的中药复方组合物 Download PDF

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WO2024087259A1
WO2024087259A1 PCT/CN2022/131219 CN2022131219W WO2024087259A1 WO 2024087259 A1 WO2024087259 A1 WO 2024087259A1 CN 2022131219 W CN2022131219 W CN 2022131219W WO 2024087259 A1 WO2024087259 A1 WO 2024087259A1
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group
formula
blood
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罗晓斌
李明辉
侯俊玲
王文全
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北京擎天数康科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/076—Poria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/288—Taraxacum (dandelion)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/484—Glycyrrhiza (licorice)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/489—Sophora, e.g. necklacepod or mamani
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to a traditional Chinese medicine compound composition for preventing and treating hyperuricemia.
  • uric acid is the end product of dietary and endogenous purine metabolism, so uric acid levels are closely related to purine metabolism in the body. Excessive production of uric acid in the body is often caused by two reasons: (1) abnormal purine metabolism; (2) excessive intake of high-purine foods.
  • XOD xanthine oxidase
  • PRS phosphoribosyl pyrophosphate synthetase
  • HGPRT hypoxanthine-guanine phosphoribosyltransferase
  • APRT adenine phosphoribosyltransferase
  • hyperuricemia is also related to metabolic diseases, kidney disease, cardiovascular and cerebrovascular diseases, nervous and respiratory diseases, psoriasis, etc.
  • the purpose of the present invention is to provide a universal and highly effective Chinese medicine compound composition for preventing and treating hyperuricemia.
  • a Chinese herbal compound composition for the prevention and treatment of hyperuricemia comprising chicory root, Poria cocos, dandelion, corn silk, Sophora japonica flower and licorice, wherein, by weight, the chicory root comprises 3 to 6 parts; Poria cocos comprises 1.5 to 3.5 parts; Dandelion comprises 1.5 to 3.5 parts; Corn silk comprises 2.5 to 4.5 parts; Sophora japonica flower comprises 1.5 to 3.5 parts; and licorice comprises 1 part.
  • the compound prescription of the present invention has the following effects: clearing away heat and detoxifying, removing dampness and turbidity, promoting diuresis and reducing swelling, cooling blood and relieving pain.
  • the prescription is explained as follows:
  • Chicory root (main ingredient) clears away heat and detoxifies, promotes diuresis and reduces swelling; Poria cocos (minister ingredient) strengthens the spleen, eliminates dampness, promotes diuresis and reduces swelling; Dandelion (auxiliary ingredient) clears heat and detoxifies, promotes diuresis and reduces swelling; Corn silk (auxiliary ingredient) promotes diuresis and reduces swelling, promotes urination and eliminates turbidity; Sophora japonica (assistant ingredient) clears the liver and drains fire, and cools the blood; Licorice (assistant ingredient) clears heat and detoxifies, relieves urgency and stops pain, and harmonizes all the medicines.
  • the main treatment principle of the present invention is to purge and eliminate turbid blood stasis: because the occurrence of gout is turbid blood stasis, therefore, the treatment principle of purge and eliminate turbid blood stasis is used, and turbid blood stasis can be gradually purge, and blood uric acid will also decline thereupon, so that the function of clearing and secreting turbid is restored, and health recovery is tending.
  • Heat-clearing drugs Chicory, which contains verbascoside, promotes uric acid excretion and can increase the intestinal excretion of uric acid; dandelion, whose water extract has a protective effect on kidney damage caused by persistent hyperuricemia; Sophora japonica, whose flavonoid extracts such as rutin, dendrobium, kaempferol and sophora japonica amine have a good therapeutic effect on hyperuricemia.
  • Diuretic drugs Poria cocos has diuretic, analgesic and uric acid reduction effects; corn silk, its flavonoid extract has the effect of inhibiting the expression of inflammatory factors such as interleukins, regulating signal pathways, thereby reducing edema and inflammatory cell infiltration, and relieving the inflammatory symptoms caused by gouty arthritis.
  • Tonic Licorice, whose flavonoids such as liquiritigenin can inhibit the AQP4/NF-kB signaling pathway and NLRP3 inflammasome activation in HUA rats, to reduce and improve renal inflammation.
  • the Chinese medicine compound composition of the present invention has been verified to be effective in preventing and treating hyperuricemia.
  • the composition of the present invention is composed of medicinal materials that are both medicinal and edible as raw materials, so it is particularly suitable for production and sale as a functional food.
  • the present invention also provides a preparation for preventing and treating hyperuricemia, comprising the aqueous extract of the above composition.
  • the preparation of the present invention can be in any suitable form, such as oral liquid or capsule, etc.
  • the composition corresponding to the recommended daily dosage for an adult is: 10g of chicory root, 5g of Poria cocos, 10g of dandelion, 10g of corn silk, 10g of Sophora japonica flower and 3g of licorice.
  • FIG1 shows the results of blood uric acid content determination under different treatments in the first efficacy experiment
  • FIG2 shows the results of measuring urea nitrogen content under different treatments in the first efficacy experiment
  • FIG3 shows the results of measuring blood creatinine content under different treatments in the first efficacy experiment
  • FIG4 shows the results of xanthine oxidase content determination under different treatments in the first efficacy experiment
  • FIG5 shows the HE injury scores of rat liver sections under different treatments in the first drug efficacy experiment
  • FIG6 shows a graph showing the injury scores of rat kidney HE sections under different treatments in the first drug efficacy experiment
  • FIG7 shows the results of blood uric acid content determination under different treatments in the second efficacy experiment
  • FIG8 shows the results of measuring urea nitrogen content under different treatments in the second efficacy experiment
  • FIG9 shows the results of measuring blood creatinine content under different treatments in the second efficacy experiment
  • FIG10 shows a graph of injury scores of rat liver slices under different treatments in the second drug efficacy experiment
  • FIG11 shows the HE injury scores of rat kidney sections under different treatments in the second drug efficacy experiment
  • FIG12 shows the results of blood uric acid content determination under different treatments in the third drug efficacy experiment
  • FIG13 shows the results of measuring urea nitrogen content under different treatments in the third efficacy experiment
  • FIG14 shows the results of measuring blood creatinine content under different treatments in the third drug efficacy experiment
  • FIG15 shows HE sections of rat kidneys and their injury scores under different treatments in the third drug efficacy experiment
  • FIG16 shows the results of blood uric acid content determination under different treatments of preventive administration
  • FIG17 shows the results of measuring urea nitrogen content under different treatments of preventive administration
  • FIG18 shows the results of measuring blood creatinine content under different treatments of preventive administration.
  • FIG. 19 shows HE sections of rat kidneys and their injury scores under different treatments of preventive administration.
  • HWS26 electric constant temperature water bath (Shanghai Hengke Instrument Co., Ltd.);
  • GZX-GF101-3-BS electric constant temperature blast drying oven (Shanghai Yuejin Medical Instrument Co., Ltd.);
  • CA-1116A cooling water circulation device (Shanghai Ailang Instrument Co., Ltd.);
  • SHB-B95A circulating water multi-purpose vacuum pump (Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd.)
  • Modeling agent acetyl butanol hydrochloride (Hangzhou Minsheng Pharmaceutical Co., Ltd., batch number: T21C068, T21D024); potassium oxonate (Shanghai McLean Biochemical Technology Co., Ltd., batch number: C12677304, C12677411); yeast extract (Shanghai McLean Biochemical Technology Co., Ltd., batch number: C12434523); adenine (Shanghai McLean Biochemical Technology Co., Ltd., batch number: C12642922); sodium carboxymethyl cellulose (Shanghai McLean Biochemical Technology Co., Ltd., batch number: C12286568).
  • Positive drugs (1) Benzbromarone tablets, Hermann Pharmaceuticals, Germany, batch number: 1914725; (2) Allopurinol tablets, Shanghai Xinyi Wanxiang Pharmaceutical Co., Ltd., batch number 07130806.
  • Kits and reagents urea nitrogen determination kit (Sino-Biotech Co., Ltd., batch number: 100020070); uric acid and serum creatinine determination kits (Nanjing Jiancheng Bioengineering Institute, batch numbers: 20200911, 20220111; 20210818; 20201106, 20220111); anhydrous ethanol (Beijing Chemical Plant, batch number 201909412); 10% neutral formalin solution (Beijing Solebold Technology Co., Ltd., 20210817); physiological saline (Shandong Hualu Pharmaceutical Co., Ltd., batch number: H19010913); xylene (Beijing Chemical Plant, batch number: 20190113); neutral gum (Shanghai Instrument Factory, batch number: 201901208); hematoxylin Plain stain (Zhuhai Beso Biotechnology Co., Ltd., batch number: 718072); eosin stain (Zhuhai Beso Biotechnology Co., Ltd., batch number: 7180
  • SPF grade SD male rats weighing 180-220 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2017-0020. This study was approved by the Ethics Committee of the Experimental Animal Center of the Institute of Medicinal Plants, Peking Union Medical College, and the experimental ethics review number is: SLXD-20210826005. The rats were kept in the animal room of the Institute of Medicinal Plants, Peking Union Medical College, and were free to drink water and eat every day.
  • CMC-Na preparation weigh 5g CMC-Na powder and dissolve it in 1000mL distilled water, mix well to make a transparent solution, and store at 4°C. Before use, it needs to be warmed to 30-35°C before gavage.
  • Model I Modeling agent preparation Preparation of potassium oxonate saline intraperitoneal injection solution: Dissolve 3g potassium oxonate crystals in 100ml of saline solution.
  • Yeast extract solution (1g/100g) preparation Weigh 100g yeast extract and dissolve it in 100mL 0.5% CMC-Na, prepare homogenate with a high-speed homogenizer, store at 4°C, and warm it to 30-35°C before use before gavage.
  • Adenine solution preparation Weigh 6.7g adenine and dissolve it in 100ml 5% CMC-Na, prepare homogenate with a high-speed homogenizer, store at 4°C, and warm it to 30-35°C before use before gavage.
  • Preparation of model II modeling agent Preparation of potassium oxonate + ethambutol intragastric solution: Weigh 5g potassium oxonate and 1.67g ethambutol and dissolve them in 0.5% CMC-Na solution to make 100mL potassium oxonate solution, prepare the homogenate with a high-speed homogenizer, and store at 4°C. Before use, it needs to be warmed to 30-35°C before intragastric administration.
  • Model III modeling agent preparation yeast extract + adenine + potassium oxonate gavage solution preparation: weigh 66.7g yeast extract, 0.667g adenine powder and 2g potassium oxonate powder and dissolve in 100ml 0.5% CMC-Na, prepare homogenate with high-speed homogenizer, and store at 4°C. Before use, it needs to be warmed to 30-35°C before gavage.
  • Model group I 3 ml yeast extract 0.5% CMC-Na solution was gavaged in the morning, and 3 ml adenine 0.5% CMC-Na solution was gavaged in 2 hours. On the 1st, 3rd, 7th, 11th and 14th days, 2 ml potassium oxonate saline solution was intraperitoneally injected.
  • Model II group was gavaged once a day in the morning with 3 ml of a mixed solution of potassium oxonate and ethambutol 0.5% CMC-Na.
  • Model III group was gavaged once a day in the morning with 3 ml of a 0.5% CMC-Na solution mixed with potassium oxonate, adenine and yeast extract.
  • Preparation of decoction of Chinese herbal medicines According to the experimental requirements, determine the experimental formula and purchase Chinese herbal medicines. Use formula 2-1 and 2-2 to calculate the amount of decoction pieces. According to the material-liquid ratio of 1:8, decoction (reflux extraction method) is performed twice, the first decoction is 40 minutes, and the second decoction is 30 minutes. The decoction is filtered through 8 layers of gauze, the two decoctions are combined, and the rotary evaporation is concentrated to a certain concentration. The volume of the concentrated liquid T is accurately measured (rement: T ⁇ V ⁇ N ⁇ X, see formula 2-4).
  • Preparation of the test solution of Chinese medicine Calculate using formula 2-3 and 2-4, add distilled water to the decoction solution (T) to make the total solution (T) for animal administration, and the concentration of the solution is calculated as (C/V) based on the amount of the decoction piece. Seal and refrigerate in a 4°C refrigerator for future use, and administer the drug according to the daily requirement (V) of each animal during the experimental treatment.
  • C is the total amount of Chinese herbal medicine slices required for rats per day (g); R is the total amount of human herbal medicine slices required for rats per day (g); 60 is the average body weight parameter (Kg); M is the rat weight (Kg); 7 is a parameter, the dosage per unit body weight of rats is 7 times the human dosage.
  • Formula 2-2 A is the total amount of slices required for N rats in a treatment group during a planned period of X days (7 days). To prevent operational losses, the daily dosage for 5 rats (5C) needs to be increased, and the actual total weight of slices is A+5C.
  • V is the daily dosage (maximum volume) of the animal
  • 15 is the specified parameter mL/kg
  • Benzbromarone tablets Dissolve in distilled water by ultrasonication, convert the dosage into rat dosage according to the instructions, and then administer by gavage, and store at 4°C.
  • Allopurinol tablets Dissolve in distilled water by ultrasonication, convert the dosage into rat dosage according to the instructions, and then administer by gavage, and store at 4°C.
  • Experimental sample collection is divided into two categories, one is blood samples, and the other is liver and kidney tissue samples.
  • Serum sample collection Orbital blood was collected at the specified time point, with a blood volume of 0.5 mL. The collected blood was allowed to stand at 4°C for 30 min, centrifuged at 3500 rpm ⁇ min -1 for 15 min to obtain serum, which was then stored in a -80°C refrigerator.
  • Serum sample determination Serum uric acid (SUA), serum creatinine (Cre), blood urea nitrogen (BUN) and xanthine oxidase (XOD) were detected using a kit and the content was detected on an ELISA reader according to the kit instructions.
  • SAA Serum uric acid
  • Re serum creatinine
  • BUN blood urea nitrogen
  • XOD xanthine oxidase
  • the experimental setting is divided into 5 treatment groups, namely: blank group, model II group, and 3 formula groups (formula group codes are AC1, AC2, AC3).
  • the formulas and dosages are shown in the above content.
  • the experimental grouping and dosing scheme are shown in Table 3 below.
  • the experimental period is 28 days, blood samples are collected 12 times, and liver and kidney tissue test samples are collected at the end of the experiment.
  • Figure 2 shows that the effect of modeling on urea nitrogen content was late.
  • the model did not show significant differences from the blank until the 11th day of modeling, and AC2 and AC3 did not show significant differences from the model group until the 15th day.
  • the three formulas all had a certain effect on reducing urea nitrogen content. From a comprehensive analysis of the intensity, duration and stability of the effect, AC2 was the best and AC1 was the worst.
  • Figure 4 shows that the content of xanthine oxidase in rats is somewhat unstable. Only on days 11-20, the model group and the blank group showed different degrees of difference. Among the 12 test points, only 2 of the 3 formulas and the model group showed a significant decrease in content. Since the important indicator of this study is the blood uric acid index, according to a large number of research results, the xanthine oxidase index is unstable, so this index was not tested in the later experiments.
  • the modeling has a certain damaging effect on the liver and kidneys, and the three formulas have a certain protective effect on liver and kidney tissues.
  • the liver is less damaged, and the modeling only has a slight impact on the liver, while the damage to the kidneys has reached an extremely significant degree.
  • the three formulas have slightly different effects on reducing kidney damage, with AC1 and AC2 being significantly stronger and AC3 being relatively weaker.
  • the results of the preliminary formula experiment show that the three Chinese medicine formulas can reduce the levels of blood uric acid, urea nitrogen and blood creatinine to varying degrees, and can reduce the damage to the kidneys caused by high blood uric acid.
  • AC2 has the best effect, followed by AC3.
  • the two formulas will be improved in the later stage to further verify their efficacy, and AC1 will be abandoned for further research.
  • the test results show that the xanthine oxidase content is unstable. Although modeling can cause it to increase to a certain extent, it is not enough to evaluate the efficacy of Chinese medicine compound. Therefore, the three blood indicators of blood uric acid content, urea nitrogen content, and blood creatinine content and the degree of damage to kidney HE sections are used as comprehensive evaluation indicators for the prevention and treatment of hyperuricemia by Chinese medicine compound.
  • AC1 was relatively poor in efficacy and was abandoned for further experiment, while AC2 and AC3 continued to undergo improved experiments; based on the previous clinical experience, the taste and compatibility of the newly designed formula of the project were improved, and 6 new formulas were designed. Together with the improved formulas of AC2 and AC3, a total of 8 improved formulas were determined for efficacy experiments in the second batch of efficacy experiments.
  • the composition and code of each formula are shown in Table 1, and the codes are: BC2, BC3, BC4, BC5, BC6, BC7, BC8, BC9. According to the previous experimental analysis, benzbromarone was selected as the positive drug, and the preparation method and dosage of the tested drug were the same as before.
  • Model II method was used to establish the model, and a total of 11 treatment groups were set up, including a blank group, a model II group, a positive drug group, and 8 Chinese medicine formula groups. After adaptive feeding, the mice were divided into groups and given drugs. The groups and experimental treatments are shown in Table 4.
  • Figure 7 shows that at the five sampling time points, there was a significant difference in the blood uric acid content between the model group and the blank group, indicating that the modeling was successful and can be used to evaluate the efficacy of the formula. Overall, the eight formulas all showed varying degrees of efficacy in lowering blood uric acid. Comprehensive analysis showed that BC7 and BC9 performed poorly, BC2 and BC3 performed best, followed by BC4, BC5, and BC8. At the four time points of 14-21 days, blood uric acid levels all dropped to the blank level; the positive drug (benzbromarone) also showed the effect of lowering blood uric acid, but after stopping the modeling, its uric acid-lowering effect was not as good as BC2 and BC3.
  • the results of urea nitrogen content detection are shown in Figure 8.
  • the model group significantly increased the urea nitrogen content.
  • Each Chinese medicine formula group had different degrees of effect in reducing the urea nitrogen content.
  • the BC7 group and the BC9 group were still at the same level as the model group at some time points.
  • the other formulas were significantly reduced compared with the model group at most time points, and the degree of decline varied with the time nodes.
  • the efficacy of the positive drug benzbromarone was generally not significantly different from that of most Chinese medicine formulas.
  • Some Chinese medicine formulas had better content than benzbromarone at different measurement points.
  • the 8 improved formulas all have certain therapeutic effects on hyperuricemia, can significantly reduce the blood uric acid content, and can significantly reduce the damage to kidney tissue and function caused by hyperuricemia.
  • Overall analysis shows that the efficacy of BC9 in all aspects is relatively low, while BC2, BC3, BC4, BC5, and BC8 are relatively good.
  • the blood uric acid content of BC2 and BC3 formulas at multiple detection time points has been reduced to the blank level, which is better than the positive drug benzbromarone.
  • the traditional Chinese medicine formulas of BC2, BC4, BC5 and BC8 were selected for optimization, and then pharmacodynamic experiments were carried out to further evaluate and verify their efficacy.
  • the experimental period was 28 days.
  • the treatment mode was: the modeling agent and the test drug were given at the same time from 7 to 21 days, and only the test drug was given from 22 to 28 days;
  • the prevention mode was: the test drug was given from 1 to 6 days, the modeling agent and the test drug were given at the same time from 7 to 21 days, and the modeling agent was no longer given from 22 to 28 days, but the test drug was continued.
  • the experiment set up a blank group and a positive drug group.
  • the model group and the ZC2, ZC4, ZC5, and ZC8 formula groups were set up in the treatment group and the prevention group, respectively, for a total of 12 treatment groups.
  • the model group adopted the model II modeling method. After adaptive feeding, the test rats were randomly divided into groups and administered. The groups and experimental treatments are shown in Table 5.
  • the experimental design cycle was 21 days. After adaptive feeding, the animals were divided into model group, positive drug group and treatment group.
  • the model group was given modeling agent on days 1-14, and the formula group and positive drug group were given modeling agent and formula extract or positive drug at the same time on days 1-14.
  • the modeling agent was stopped on days 15-21, but the formula extract or positive drug was continued.
  • the sampling time points were: dynamic blood sampling and testing on days 7, 14, 17 and 21 after the modeling agent was started, and kidney samples were collected on day 21 to make HE sections.
  • Formulas ZC2, ZC4, ZC5 and ZC8 were reduced by 32.37%, 27.85%, 31.78% and 26.19% respectively compared with the model group; 3 Compared with the blank group, the blood uric acid content of the rats that took the modeling agent and the formula extract at the same time could be restored to the level of the blank group. Sampling and detection on the 7th and 14th days showed that the blood uric acid content of formulas such as ZC2 was consistent with that of the control group. After the modeling agent was stopped (on the 17th and 21st days), the blood uric acid content of each optimized formula group returned to the control level. 4 Compared with the positive drug group, the functions of each optimized formula group in controlling rat blood uric acid were basically the same as those of the positive drug.
  • the urea nitrogen level of the model group increased significantly from 7 to 17 days (P ⁇ 0.01), indicating that the model was successfully established.
  • the urea nitrogen value of the rats was significantly higher than that of the blank group, reaching the highest on the 14th day, an increase of 58.36% over the blank group.
  • the urea nitrogen level of the rats showed a downward trend, and there was no significant difference between the urea nitrogen level of the model group and the blank group on the 21st day;
  • each optimized formula group could significantly reduce the urea nitrogen level of rats after 4 consecutive samplings (P ⁇ 0.01), with the highest level on the 14th day.
  • the ZC2, ZC4, ZC5 and ZC8 formulas still reduced the urea nitrogen level by 37.69%, 41.47%, 37.52% and 37.69% compared with the model group;
  • the urea nitrogen content of rats taking the formula extracts at the same time as the modeling agent in each optimized formula group can be restored to the level of the blank group; 4
  • the function of each optimized formula group in controlling the urea nitrogen of rats is basically the same as that of the positive drug.
  • ZC2 and ZC4 have the strongest effect on reducing urea nitrogen.
  • the urea nitrogen content is at the same level as the blank, and on the 21st day, its level is significantly lower than that of the blank and the model.
  • the blood creatinine level of the model group increased significantly from 7 to 17 days (P ⁇ 0.01), indicating that the model was successful.
  • the blood creatinine value of the model rats was significantly higher than that of the blank group, reaching the highest on the 14th day, an increase of 29.92% over the blank group.
  • the blood creatinine content showed a downward trend. It was still able to maintain a high level on the third day after stopping the modeling agent, and returned to the level of the blank group on the 21st day.
  • each optimized formula group could significantly reduce the blood creatinine level of rats after 4 consecutive sampling measurements (P ⁇ 0.01).
  • the formulas ZC2, ZC4, ZC5 and ZC8 reduced the blood creatinine level by 15.74%, 12.94%, 13.35% and 12.87% respectively compared with the model group.
  • the model group had obvious renal damage, mainly in the renal tubules, with severe tubular dilatation, large areas of tubular casts, thickened tubules, thinning epithelium, several times increase in cavity area, and inflammatory cell infiltration and renal tissue fibrosis around the tubules.
  • each optimized formula group significantly improved kidney damage. Specifically, only a small amount of inflammatory cell infiltration and renal tubular casts were seen in the sections.
  • Formulas ZC2, ZC4, ZC5 and ZC8 reduced kidney damage by 33.93%, 32.14%, 35.71% and 25.00% respectively compared with the model group.
  • the optimized formula groups were basically the same as the positive drug group in terms of the protective effect of the modeling agent-induced kidney damage. All of them significantly reduced the degree of kidney damage caused by hyperuricemia, but none of the groups recovered to the level of the blank group. Relatively speaking, the protective effect of ZC8 formula was the weakest.
  • FIG. 16 The results of measuring blood uric acid content based on 4 dynamic blood sampling are shown in Figure 16, which shows that: 1 Compared with the blank group, the UA content in the model group was significantly increased from 7 to 21 days after modeling (P ⁇ 0.01). The blood uric acid value of rats on the 7th day was significantly higher than that of the blank group, and reached the highest on the 14th day, which was 45.33% higher than that of the blank group. After stopping the modeling agent on the 14th day, the blood uric acid level of rats showed a downward trend, and there was still a significant difference between the blank group and the model group on the 21st day; 2 Compared with the model group, the values of the four consecutive sampling measurements of each optimized formula group were significantly reduced (P ⁇ 0.01).
  • the urea nitrogen level of the model group increased significantly from 7 to 17 days (P ⁇ 0.01), indicating that the model was successful.
  • the urea nitrogen value of the model rats was significantly higher than that of the blank group, reaching the highest value on the 14th day, an increase of 37.98% over the blank group, and returned to the blank level on the 21st day.
  • the results of dynamic blood sampling and determination of blood creatinine content of rats in each experimental treatment are shown in Figure 18.
  • the results show that: 1 Compared with the blank group, the model group had a significantly higher blood creatinine level than the blank group on days 7-17 (P ⁇ 0.01), and was significantly higher than the blank group on the 7th day, reaching the highest value on the 14th day. After the modeling agent was stopped on the 14th day, the content began to decrease, and there was still a significant difference on the 17th day. On the 21st day, there was no significant difference between the blank group and the model group; 2 Compared with the model group, the three consecutive sampling measurements of each formula group were significantly lower than the model group level (P ⁇ 0.01).
  • formulas ZC2, ZC4, ZC5 and ZC8 were reduced by 16.38%, 9.85%, 9.91% and 12.07% respectively compared with the model group; 3 Compared with the blank group, the blood creatinine content of rats that were given the modeling agent and formula extracts at the same time, except ZC4, could be restored to the blank group level on the 21st day.
  • each formula group has different degrees of improvement on the kidney damage caused by the modeling agent, which is specifically manifested in that only a small amount of inflammatory cell infiltration and tubular casts can be seen in the sections. From the results of quantitative statistical analysis, except for ZC8, other formulas can reduce the degree of damage by 40%-59%.
  • the therapeutic administration method has a significant therapeutic effect on hyperuricemia caused by model II.
  • the therapeutic effect can reach a significant level on the 7th day of taking the modeling agent and the formula drug at the same time.
  • Most formulas can maintain the blood uric acid content at the blank level.
  • the effects of each formula are at the same level.
  • Each formula has a good protective function in reducing the kidney damage caused by the modeling agent.
  • the two indicators of urea nitrogen and blood creatinine were sampled three times on the 7th, 14th and 17th days.
  • the content levels of most formula groups were significantly different from those of the model group, but not significantly different from the blank group, which reduced the degree of kidney tissue damage by nearly half, but did not reach the blank level.
  • the optimized formulas whether for preventive or therapeutic administration, have extremely significant effects on reducing the levels of blood uric acid, urea nitrogen and blood creatinine, and can also significantly reduce the renal damage caused by modeling.
  • the efficacy of each formula in the two administration methods of prevention and treatment is slightly different at different time nodes, there is no significant difference overall.

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Abstract

一种防治高血尿酸症的中药复方组合物，包括菊苣根、茯苓、蒲公英、玉米须、槐花和甘草，其中以重量份计，菊苣根：3～6份；茯苓：1.5～3.5份；蒲公英：1.5～3.5份；玉米须：2.5～4.5份；槐花：1.5～3.5份；以及甘草：1份。

Description

一种防治高血尿酸症的中药复方组合物  技术领域
本发明涉及一种高血尿酸症防治中药复方组合物。
背景技术
对于人和其他灵长类动物来说，尿酸是饮食及内源性嘌呤的代谢终产物，因此尿酸水平与体内嘌呤的代谢密切相关。体内尿酸生成过多，往往是两种原因造成:(1)嘌呤代谢异常；(2)体内摄入大量高嘌呤食物。
约10％原发性高尿酸血症是尿酸生成增多所致。在嘌呤代谢中，多种催化酶参与了人体的嘌呤代谢过程。目前的研究结果表明，酶活性异常是导致尿酸生成增多的主要原因,包括黄嘌呤氧化酶(XOD)、磷酸核糖焦磷酸合成酶(PRS)活性增高,次黄嘌呤-鸟嘌呤磷酸核糖转移酶(HGPRT)和腺嘌呤磷酸核糖转移酶(APRT)活性降低等，均可引起高尿酸血症和痛风的发生。
研究表明，高尿酸血症还与代谢性疾病、肾病、心脑血管疾病、神经和呼吸系统疾病、银屑病等均有关。
传统中药复方制剂在防治高血尿酸症方面具有独到优势，但寻求更为精准高效的中药配方一直是该领域的努力方向。
发明内容
本发明的目的是提供一种普适高效的高血尿酸症防治中药复方组合物。
根据本发明的第一方面，提供了一种高血尿酸症防治中药复方组合物，包括菊苣根、茯苓、蒲公英、玉米须、槐花和甘草，其中以重量份计，菊苣根：3～6份；茯苓：1.5～3.5份；蒲公英：1.5～3.5份；玉米须：2.5～4.5份；槐花：1.5～3.5份；以及甘草：1份。
本发明的复方功效为：清热解毒，利湿泄浊，利水消肿，凉血止痛。方解如下：
菊苣根(君)清热解毒，利水消肿；茯苓(臣)健脾渗湿、利水消肿；蒲公英(佐)清热解毒，利湿消肿；玉米须(佐)利水消肿，利尿泄浊；槐花(使)清肝泻火，凉血；甘草(使)清热解毒，缓急止痛，调和诸药。
由于痛风的主要病机是湿浊瘀滞内阻，且此湿浊之邪，不受之于外，而生之于内。患者多为形体丰腴之痰湿之体，并有嗜酒、喜啖之好，导致脏腑功能失调，升清降浊无权，因之痰湿滞阻于血脉之中，难以泄化，与血相结而为浊瘀，滞留于经脉，则骨节肿痛，结节畸形，甚则溃破。本发明的主要治则是泄化浊瘀：由于痛风之发生，是浊瘀为患，因此运用泄化浊瘀这一治则，浊瘀即可逐渐泄化，而血尿酸亦将随之下降，从而使分清泌浊之功能恢复，而趋健复。
本发明的复方各药味现代药理学研究分析如下：
清热药：菊苣，内含毛蕊花糖苷，促进尿酸排泄，可增加肠道对尿酸的排泄作用；蒲公英，其水提物对持续性高尿酸血症引起的肾脏损伤具有保护作用；槐花，其黄酮提取物如芦丁、斛皮素、山奈酚与槐米甲素等，对高尿酸血症具有教好的治疗作用。
利湿药：茯苓，具有利尿、镇痛，减少尿酸合成的作用；玉米须，其黄酮提取物所具有的作用是：通过抑制白细胞介素等炎性因子的表达,调控信号通路,以减轻水肿及炎性细胞浸润,缓解痛风性关节炎导致的炎症症状。
补虚药：甘草，其黄酮类化合物如甘草素，可以抑制HUA大鼠AQP4/NF-kB信号转导通路和NLRP3炎性体激活，以消减和改善肾脏炎症。
本发明的中药复方组合物已验证可以有效防治高血尿酸症。另外，本发明的组合物以药食同源的药材为原料组成，因此尤其适合作为功能性食品进行生产和销售。
本发明还提供了一种防治高血尿酸制剂，包含上述组合物的水提物。本发明的制剂可以采用任何合适形式，例如口服液或胶囊等，成人(以60kg计)一天推荐用量所对应的组合物为：菊苣根10g、茯苓5g、蒲公英10g、玉米须10g、槐花10g以及甘草3g。
附图说明：
图1示出了第一次药效实验的不同处理下血尿酸含量测定结果；
图2示出了第一次药效实验的不同处理下尿素氮含量测定结果；
图3示出了第一次药效实验的不同处理下血肌酐含量测定结果；
图4示出了第一次药效实验的不同处理下黄嘌呤氧化酶含量测定结果；
图5示出了第一次药效实验的不同处理下的大鼠肝脏HE切片损伤分值图；
图6示出了示出了第一次药效实验的不同处理下的大鼠肾脏HE切片损伤分值图；
图7示出了第二次药效实验的不同处理下血尿酸含量测定结果；
图8示出了第二次药效实验的不同处理下尿素氮含量测定结果；
图9示出了第二次药效实验的不同处理下血肌酐含量测定结果；
图10示出了第二次药效实验的不同处理下的大鼠肝脏切片损伤分值图；
图11示出了第二次药效实验的不同处理下的大鼠肾脏HE切片损伤分值图；
图12示出了第三次药效实验的不同处理下血尿酸含量测定结果；
图13示出了第三次药效实验的不同处理下尿素氮含量测定结果；
图14示出了第三次药效实验的不同处理下血肌酐含量测定结果；
图15示出了第三次药效实验的不同处理下的大鼠肾脏HE切片及其损伤分值图；
图16示出了预防给药方式的不同处理下血尿酸含量测定结果；
图17示出了预防给药方式的不同处理下尿素氮含量测定结果；
图18示出了预防给药方式的不同处理下血肌酐含量测定结果；以及
图19示出了预防给药方式的不同处理下大鼠肾脏HE切片及其损伤分值图。
上述图中：与空白组比较，*P＜0.05；**P＜0.01；***P＜0.001；****P＜0.0001(与模型组比较的 #亦同)
具体实施方式
下面结合具体实验实例和附图对本发明做进一步说明，本领域技术人员 应该理解，这些实例和附图只是为了更好地理解本发明，并不用来做出任何限制。
实验仪器
HWS26型电热恒温水浴锅(上海恒科仪器有限公司)；
AL204型电子分析天平(梅特勒-托利多仪器(上海)有限公司)；
GZX-GF101-3-BS型电热恒温鼓风干燥箱(上海跃进医疗器械有限公司)；
MPR-414F-PC型药品冷库冷冻保存箱(Panasonic)；
Multiskan MK3酶标仪(Thermo Scientific)；
AU480型全自动生化分析仪(美国贝克曼库尔特有限公司)；
Epoch涡旋混合器(BioTek)；
RE-3000型旋转蒸发器(上海亚荣生化仪器厂)；
DZF-6020型真空干燥箱(上海精宏实验设备有限公司)；
KH-500DE型数控超声波清洗器(昆山禾创超声仪器有限公司)；
CA-1116A型冷却水循环装置(上海爱朗仪器有限公司)；
SHB-B95A型循环水式多用真空泵(郑州长城科工贸有限公司)
实验材料
造模剂：盐酸乙酰丁醇(杭州民生药业有限公司，批号：T21C068、T21D024)；氧嗪酸钾(上海麦克林生化科技有限公司，批号：C12677304、C12677411)；酵母膏(上海麦克林生化科技有限公司，批号：C12434523)；腺嘌呤(上海麦克林生化科技有限公司，批号：C12642922)；羧甲基纤维素钠(上海麦克林生化科技有限公司，批号：C12286568)。
中药饮片：采购自北京本草方源药业集团有限公司，GMP许可证BJ20190426，生产许可证：京20150136。按配方需要采购。
阳性药：(1)苯溴马隆片，德国赫曼大药厂，批号：1914725；(2)别嘌醇片，上海信谊万象药业股份有限公司，批号07130806。
试剂盒及试剂：尿素氮测定试剂盒(中生北控生物科技股份有限公司， 批号：100020070)；尿酸、血肌酐测定试剂盒(南京建成生物工程研究所，批号：20200911、20220111；20210818；20201106、20220111)；无水乙醇(北京化工厂，批号201909412)；10％中性福尔马林溶液(北京索莱宝科技有限公司，20210817)；生理盐水(山东华鲁制药有限公司，批号：H19010913)；二甲苯(北京化工厂，批号：20190113)；中性树胶(上海仪器厂，批号：201901208)；苏木素染液(珠海贝索生物技术公司，批号：718072)；伊红染液(珠海贝索生物技术公司，批号：718091)；盐酸乙胺丁醇片(杭州民生药业有限公司，批号：T21C068、T21D024)；氧嗪酸钾(上海麦克林生化科技有限公司，批号：C12677304)；酵母膏(上海麦克林生化科技有限公司，批号：C12434523)；腺嘌呤(上海麦克林生化科技有限公司，批号：C12642922)；羧甲基纤维素钠(上海麦克林生化科技有限公司，批号：C12286568)。
实验动物
SPF级SD雄性大鼠，体质量180～220g，购自北京维通利华实验动物技术有限公司，许可证编号：SCXK(京)2017-0020。本研究由北京协和医学院药用植物研究所实验动物中心伦理委员会批准通过，实验伦理审查编号为：SLXD-20210826005。在北京协和医学院药用植物研究所动物房饲养，每天自由饮水、进食。
表1 实验配方设计与药物组成优化迭代过程表
Figure PCTCN2022131219-appb-000001
Figure PCTCN2022131219-appb-000002
高尿酸血症致病模型造模方法
造模剂溶解液的配制：0.5％CMC-Na配置：称量5g CMC-Na粉末溶于1000mL蒸馏水中混匀制成透明溶液，4℃存放。使用前需先经温水浴至30～35℃再行灌胃。
模型Ⅰ造模剂配制：氧嗪酸钾生理盐水腹腔注射溶液配制：将3g氧嗪酸钾晶体溶于100ml的生理盐水溶液中。酵母膏溶液(1g/100g)配制：称量100g酵母膏溶于100mL 0.5％CMC-Na中，高速匀浆机制备匀浆，于4℃存放，使用前需先经温水浴至30～35℃再进行灌胃。腺嘌呤溶液配制：称量6.7g腺嘌呤溶于100ml 5％CMC-Na中，高速匀浆机制备匀浆，于4℃存放，使用 前需先经温水浴至30～35℃再行灌胃。
模型Ⅱ造模剂配制：氧嗪酸钾+乙胺丁醇灌胃溶液配制：称取5g氧嗪酸钾和1.67g乙胺丁醇溶于0.5％CMC-Na溶液中配成100mL氧嗪酸钾溶液，高速匀浆机制备匀浆，4℃存放。使用前需先经温水浴至30～35℃再行灌胃。
模型Ⅲ造模剂配制：酵母膏+腺嘌呤+氧嗪酸钾灌胃溶液配制：称量66.7g酵母膏，0.667g腺嘌呤粉末和2g氧嗪酸钾粉末溶于100ml 0.5％CMC-Na中，高速匀浆机制备匀浆，4℃存放。使用前需先经温水浴至30～35℃再行灌胃。
动物造模方法：动物适应性饲养7天后，在无菌原则下，连续14天给予造模剂，具体操作如下：
模型Ⅰ组：上午灌胃3ml酵母膏0.5％CMC-Na溶液，2h后灌胃3ml腺嘌呤0.5％CMC-Na溶液，在第1，3，7，11，14天腹腔注射2ml氧嗪酸钾生理盐水溶液。
模型Ⅱ组，每天上午灌胃1次3ml氧嗪酸钾+乙胺丁醇0.5％CMC-Na混合溶液。
模型Ⅲ组，每天上午灌胃1次3ml氧嗪酸钾+腺嘌呤+酵母膏的混合0.5％CMC-Na溶液。
各组正常饮水，连续饲喂两周。
中药受试药物制备及实验处理方法
中药饮片煎煮制备：根据实验要求，确定实验配方，采购中药饮片。采用式2-1和2-2计算称取饮片量。按料液比为1∶8，水煎煮(回流提取法)两次，一煎40min，二煎30min，水煎液用8层纱布过滤，合并两次水煎液，旋转蒸发浓缩至一定浓度，精准测量浓缩药液体积T 缩(要求：T 缩≤V×N×X,见式2-4)。
中药受试药液制备：采用式2-3和2-4计算，将煎煮药液(T 缩)加蒸馏水定容配成动物给药的总药液(T)，药液浓度按饮片量计算为(C/V)。密封冷藏于4℃冰箱保存备用，实验处理时按每只动物日需量(V)吸取给药。
Figure PCTCN2022131219-appb-000003
A＝C×N×X      式2-2
V＝15×M      式2-3
T＝V×N×X     式2-4
式2-1：C为大鼠每日需给中药饮片总量(g)；R为配方每日人饮片总量(g)；60为人均体重参数(Kg)；M为大鼠体重(Kg)；7为参数，单位体重大鼠给药量为人用量的7倍。
式2-2：A为计划某时段X天(7天)，某处理组N只大鼠共需饮片总量。为防操作损失，需增加5只大鼠的日用量(5C)，实际饮片总重量为A+5C。
式2-3：V为动物日给药量(最大容积)，15为规定参数mL/kg，M为动物体重(Kg)。按大鼠体重200g计算，每天灌胃量为15mL/kg×0.2kg＝3mL。
式2-4：T为N只动物X天需给药总药液量。注意饮片称取时增加了5只动物的量，实际计算时，T＝V×N×X+5V＝V(N×X+5)大鼠体重按平均200g，每天灌胃3ml，每只大鼠每天3mL灌胃药液，需要提取的中药饮片量根据式2-1计算。本研究3次药效实验，各配方需要的生药量计算结果见表2。
表2 中药配方3次药效实验大鼠日给药饮片量
Figure PCTCN2022131219-appb-000004
阳性药受试药物制备及实验处理方法
选择两种阳性药物进行实验，具体制备和实验处理方法如下。
苯溴马隆片：超声溶解于蒸馏水，按说明书剂量换算成大鼠剂量后灌胃，4℃保存。
别嘌醇片：超声溶解于蒸馏水，按说明书剂量换算成大鼠剂量后灌胃，4℃保存。
实验样品的采集
实验样品采集分为两大类，一类为血液样品，另一类为肝脏和肾脏组织样品。
(1)血清样品采集：在规定采血时间点进行眼眶采血，采血量为0.5mL，采集血液4℃静置30min，用3500rpm·min -1离心15min取血清，置于-80℃冰箱保存。
(2)器官组织采集：在实验结束时取材，用7％水合氯醛麻醉后，将实验动物进行解剖，迅速摘取其肝脏和肾脏置于预冷的生理盐水中漂洗数次，再用滤纸吸干器官表面生理盐水后称量重量；然后再将器官用10％中性福尔马林溶液固定一天，后将样品外送具备相关资质的公司做HE染色切片。
检测指标及测定方法
(1)血清样品测定：血清中检测血尿酸(SUA)、血肌酐(Cre)、尿素氮(BUN)和黄嘌呤氧化酶(XOD)。测定方法采用试剂盒，按照试剂盒说明在酶标仪上进行含量检测。
(2)肝肾组织HE切片制作与观测：肝肾组织HE切片制作详见1.3.3.5(肝脏和肾脏HE染色切片制作)。并委托北京信诺嘉科技发展有限公司检测。肾脏组织HE切片受损伤程度评分规则：①肾小管上皮细胞空泡化；②肾小管萎缩、扩张；③肾小管内蛋白管型、细胞管型；④肾小管内结晶沉积；⑤间质炎细胞浸润(淋巴细胞浸润、巨噬细胞聚集、异物肉芽肿反应等)；⑥肾间质纤维化。评分标准为：无明显病变，记0分；病变范围不超过1/4，记1 分；病变范围1/4至1/2，记2分；病变范围1/2至3/4，记3分；病变范围超过3/4，记4分。
一.初选配方治疗高尿酸血症的功效分析(第一次药效实验)
实验设置分为5个处理组，分别是：空白组、模型Ⅱ组、还有3个配方组(配方组代号分别为AC1、AC2、AC3)，配方及给药量等见前述内容。实验分组和给药方案见下表3。实验周期28天，采集血样12次，实验结束采集肝脏肾脏组织检测样品。
表3：初选配功效评价实验分组及给药处理方案
Figure PCTCN2022131219-appb-000005
不同配方对血尿酸(UA)含量的影响
造模第4-28天的各时间节点采样测定血尿酸(UA)结果如图1所示，模型与空白组血尿酸含量差异显著，AC2和AC3第6-22天与模型组产生了极显著差异，有些测点降低到了空白组水平，说明两个配方对大鼠血尿酸含量具有良好的调降作用。AC1与AC2和AC3相比，AC1降尿酸的作用展现较早，在给造模剂期间作用较强，停止给造模剂后(第15天)作用明显减弱，始终与空白含量存在明显差异，到第22天与模型处于相同水平。而AC2和AC3表现的降尿酸效果比较稳定且显著，在第18天以后基本和空白组无差 异。
不同配方对尿素氮(BUN)含量的影响
图2显示，造模对尿素氮含量的影响表现较晚，模型在造模第11天才与空白产生明显差异，AC2和AC3第15天才与模型组产生明显差异。3个配方对尿素氮含量均具有一定调降作用，从作用强度、持续时间及稳定性等方面综合分析，AC2最好，AC1最差。
不同配方对血肌酐(Cre)含量的影响
实验处理对血肌酐含量的影响趋势，与尿素氮基本相同，造模后的第14天检测，模型组与空白组之间，以及3个配方组与模型组之间的差异才显著出现。且3个配方组之间的作用略有不同，在某些测定节点其含量可下降到空白水平，但3者之间没有产生明显区别，如图3所示。
不同配方对黄嘌呤氧化酶(XOD)含量影响
图4显示，黄嘌呤氧化酶在大鼠体内含量有些不稳定，仅在第11-20天模型组与空白组出现了不同程度的差异，3个配方与模型组在12个检测点中只有2个含量明显降低。由于本研究特别关注的重要指标是血尿酸指标，根据大量研究结果对于黄嘌呤氧化酶指标存在着不稳定性，所以后期实验对该指标就不予检测了。
造模对肝脏肾损脏伤程度分析
由图5和图6可看出，造模对肝脏和肾脏都有一定损伤作用，3个配方对肝肾组织都有一定保护作用。相较而言，肝脏受损较轻，造模只对肝脏产生轻微影响，而对肾脏的伤害作用达到了极显著程度。3个配方对肾脏的损伤消减作用稍有不同，AC1和AC2明显较强，AC3相对较弱。
相较而言，肝脏受损较轻，造模只对肝脏产生轻微影响，而对肾脏的伤害作用达到了极显著程度。3个配方对肾脏的损伤消减作用稍有不同，AC1和AC2明显较强，AC3相对较弱。
初选配方实验结果说明，3个中药配方受试物不同程度上均能低血尿酸、尿素氮和血肌酐的含量，可减轻高血尿酸对肾脏造成的损伤，其中AC2效果最佳，其次是AC3，两个配方在后期再进行改进，进一步验证其功效，AC1放弃继续研究。另外，检测结果表明，黄嘌呤氧化酶含量不稳定，造模虽然在一定程度上会导致其升高，但不足以用来评价中药复方功效。因此，后面以血尿酸含量、尿素氮含量、血肌酐含量3个血液指标和肾脏HE切片的受损程度，作为综合评价中药复方防治高尿酸血症的检测指标。
二.改进配方的高尿酸血症治疗效果分析(第二次药效实验)
参考初选配方功效实验分析结果，AC1的功效相对较差放弃继续实验，AC2和AC3继续进行改进实验；根据前期临床经验论证，对项目新设计的配方药味和配伍进行了改进，又新设计了6个配方，加上AC2和AC3的改进配方，第二批药效实验共确定8个改进配方进行功效实验，各配方组成及代号详见表1，其代号分别为：BC2、BC3、BC4、BC5、BC6、BC7、BC8、BC9。根据前面实验分析，选择苯溴马隆为阳性药，受试药物制备方法和给药剂量等同前。
动物造模与分组给药
采用模型Ⅱ方法造模，共设置空白组、模型Ⅱ组、阳性药组、8个中药配方组，共计11个处理组。经过适应性饲养后分组给药，分组及实验处理见表4。
表4 实验分组与给药处理方案
Figure PCTCN2022131219-appb-000006
Figure PCTCN2022131219-appb-000007
改进配方调降血尿酸(UA)含量功效分析
图7说明，5个采样时间点，模型组与空白组血尿酸含量都产生了明显差别，说明造模成功，可用作对配方功效进行评估。8个配方总体上均表现出不同程度的具有一定降低血尿酸功效。综合分析，BC7和BC9表现较差，BC2和BC3表现最好，其次为BC4、BC5、BC8，在14-21天4个时间点血尿酸都下降到了空白水平；阳性药(苯溴马隆)也表现出具有降低血尿酸的功效，但在停止造模后其降尿酸功效的表现不如BC2和BC3好。
改进配方对降低尿素氮(BUN)含量的作用
尿素氮含量检测结果如图8所示，模型组使尿素氮含量明显升高，各中 药配方组都有不同程度的降低尿素氮含量的作用，但相比较而言，BC7组和BC9组在部分时间点上与模型组仍处于同一水平，其他配方在大部分时间点上与模型组相比具有显著降低，各自的下降程度因时间节点不同而异。阳性药苯溴马隆的疗效总体来看与多数中药配方没有显著差异，有的中药配方在不同测量点的含量比苯溴马隆还好。
改进配方对降低血肌酐(Cre)含量的作用
各组处理对血肌酐含量的测定结果如图9所示，其结果说明各时间点模型组含量略有增加，但与空白组相比未产生显著差异；各中药配方组与模型组相比，均未展现出显著的下调作用，仅在第14、16和18天检测有些配方的含量有显著下降，在第21天检测时，模型组和空白组无差别，但所有配方与模型组和空白组相比，均有极显著的降低。阳性药组的检测结果与配方基本相同。从本项目多次实验结果来看，本次实验结果有些异常，产生原因有待研究，不宜作为评价配方功效的依据。
改进配方对减轻肝肾损伤(HE切片)的作用
各组处理对肝脏、肾脏的HE切片的分析结果如图10-11所示，对肝脏损伤的分析结果说明，造模对肝脏组织产生了一定的影响；各个中药配方组对肝损伤都有消减作用，其效果比阳性药还好，通过统计分析与阳性药组相比，未达到显著差异程度。对肾脏损伤的分析结果说明，各中药配方均展现出极显著的消减作用，体现了对肾脏的保护作用；各中药配方组与阳性药相比较，只有BC9的消减作用稍弱，其余7个中药配方组均与阳性药处于相同水平，但阳性药组和所有中药配方组在研究的时间内均未达到空白水平。
综合以上分析，改进后的8个配方对高尿酸血症都具有一定治疗功效，都可显著降低血尿酸含量，都可以显著降低高尿酸血症所致的对肾脏组织及功能造成的损害。整体分析，BC9各方面的功效均较低，BC2、BC3、BC4、BC5、BC8相对较好，BC2、BC3配方多个检测时间点的血尿酸含量降低到了空白水平，较阳性药苯溴马隆的疗效还好。为了遴选最佳疗效配方，选择 BC2、BC4、BC5和BC8中药配方再进行优化，再进行药效学实验，进一步评价和验证其功效。
三.优化配方防治高尿酸血症功效验证分析(第三次药效实验)
根据改进配方药效实验结果及项目需要，淘汰了功效较差的BC6、BC7、BC9，停止了BC3的实验。最终确定了对4个配方再进行功效验证研究，其代号分别为ZC2、ZC4、ZC5、ZC8，详细调整过程及配方见表1；阳性药为苯溴马隆；受试药物制备方法和给药剂量同前。
实验动物分组与分组给药方案
本次实验设计了治疗给药方式和预防给药方式两种，实验周期为28天。(1)治疗给药方式是：7-21天同时给造模剂和受试药物，22-28天只给受试药物；(2)预防给药方式是：1-6天给受试药物，7-21天同时给造模剂和受试药物，22-28天不再给造模剂，但继续给受试药物。实验共设置空白组、阳性药组，在治疗组和预防组中分别设置了模型组和ZC2、ZC4、ZC5、ZC8配方组，共12个处理组。模型组采用模型Ⅱ造模方法。受试大鼠经过适应性饲养后再随机分组给药，分组及实验处理见表5。
表5 实验分组与给药处理方案
Figure PCTCN2022131219-appb-000008
Figure PCTCN2022131219-appb-000009
本实验设计周期为21天，动物经适应性饲养后开始进行进行分组，分为模型组、阳性药组和治疗处理组。模型组在第1-14天给造模剂，配方组和阳性药组在第1-14天边给造模剂的同时也给配方提取物或阳性药，在第15-21天停止给造模剂，但继续给配方提取物或阳性药。取样时间点是：开始给造模剂的第7、14、17和21天动态取血采样检测，在第21天采集肾脏样品制作HE切片。
治疗给药方式的优化配方降低血尿酸(UA)含量功效分析
各实验处理组大鼠4次动态取血测定血尿酸含量结果如图12所示。由此可看出：①模型组与空白组相比，模型组在7-21天UA水平均显著高于其他各组(P＜0.01)，说明造模成功；实验观察可见，在实验处理第7天时，造模大鼠血尿酸值已显著高于空白组，在第14天达到最高，较空白组提高了64.50％。在第14天停止给造模剂以后，大鼠血尿酸水平慢慢呈下降趋势，但在第21天与空白组之间仍然存在显著差异(高15.24％)；②各优化配方组与模型组相比，根据连续4次取样测定结果说明，各优化配方组均可显著降低大鼠血尿酸水平(P＜0.01)，在第14天时相差达到最大值，配方ZC2、ZC4、ZC5和ZC8较模型组分别降低了32.37％、、27.85％、31.78％和26.19％；③各优化配方组与空白组相比，给造模剂同时服用配方提取物的大鼠血尿酸含量可恢复到空白组水平，第7和14天取样检测，ZC2等配方血尿酸含量与对照组水平一致，停止给造模剂后(第17和21天)各优化配方组的血尿酸含量均恢复到对照水平。④各优化配方组与阳性药组相比，各优化配方组控制 大鼠血尿酸的功能与阳性药作用基本相同，ZC2配方在有些点测定结果反映其功效比阳性药还好，如第7天测定阳性药组血尿酸较空白组明显偏高，而ZC2配方组则与空白组处于相同水平。综合以上分析，经过两次实验优化出的几个配方，对治疗高尿酸血症均具有良好效果，可显著控制因造模剂诱发的大鼠高尿酸血症状，各优化配方的调控能力排序为：ZC2>阳性药>ZC5>ZC4＝ZC8。
治疗给药方式的优化配方降低尿素氮(BUN)含量功效分析
实验处理各组大鼠4次动态取血测定尿素氮(BUN)含量结果如图13所示。由此可看出：
①模型组与空白组相比，模型组在7-17天尿素氮水平均显著升高(P＜0.01)，说明造模成功，造模处理第7天，大鼠尿素氮值已显著高于空白组，第14天达到最高，较空白组提高58.36％。第14天停止给造模剂后大鼠尿素氮水平呈下降趋势，在第21天与空白组之间已经没有显著差异；
②各优化配方组与模型组相比，连续4次取样测定各优化配方组均可显著降低大鼠尿素氮水平(P＜0.01)，第14天最高。在停止给造模剂3天，即第17天ZC2、ZC4、ZC5和ZC8配方，仍较模型组降低37.69％、41.47％、、37.52％和37.69％；
③各优化配方组与空白组相比，给造模剂同时服用配方提取物的大鼠尿素氮含量可恢复到空白组水平；④各优化配方组与阳性药组相比，各优化配方组控制大鼠尿素氮的功能与阳性药作用基本相同。4个配方中，ZC2和ZC4降低尿素氮作用最强，第7、14、17天，尿素氮含量与空白处于相同水平，第21天其水平明显较空白和模型还低。
治疗给药方式的优化配方降低血肌酐(Cre)含量功效分析
实验各处理4次动态取样血肌酐含量测定结果见图14所示。由此可看出：
①模型组与空白组相比，模型组在7-17天血肌酐水平均显著升高(P＜0.01)，说明造模成功。实验处理第7天，造模大鼠血肌酐值已显著高于空白组，第14天达到最高，较空白组提高29.92％，第14天停止给造模剂后血肌 酐含量呈下降趋势，停止给造模剂后第三天仍能够维持较高水平，第21天恢复到了空白组水平。
②各优化配方组与模型组相比，连续4次取样测定各优化配方组均可显著降低大鼠血肌酐水平(P＜0.01)，在停止给造模剂继续给受试药物的第三天(实验开始第17天)配方ZC2、ZC4、ZC5和ZC8较模型组分别降低了15.74％、12.94％、13.35％和12.87％。
③各优化配方组与空白组相比，给造模剂同时服用配方提取物的大鼠血肌酐含量可恢复到空白组水平，实验处理第17天，所有配方血肌酐含量与空白均无明显差异。
④各优化配方组与阳性药组相比，各优化配方组控制大鼠血肌酐的功能与阳性药作用基本相同，总体分析ZC2作用较阳性药略强，而ZC5和ZC8的作用较阳性药、ZC2和ZC4起效稍晚，但可持续时间较长。
治疗给药方式的优化配方减轻肾损伤(HE切片)功效分析
实验结束时(第21天)，大鼠肾脏HE切片检测分析结果见图15所示。从切片及量化分析结果可以看出：
①模型组与空白组相比，模型组存在明显的肾脏损伤，损伤主要在肾小管，产生了严重的肾小管扩张，肾小管大面积的管型，肾小管体积粗大，上皮变薄，空腔面积数倍增大，且在肾小管周围存在炎症细胞浸润和肾组织纤维化症状。
②各优化配方组与模型组相比，各优化配方组均明显改善了肾脏的损伤，具体表现为在切片中，仅可见少量炎症细胞浸润和肾小管管型，配方ZC2、ZC4、ZC5和ZC8较模型组分别降低了33.93％、32.14％、35.71％和25.00％。
③各优化配方组与阳性药组相比，从造模剂导致肾脏损伤的保护作用来看，各优化配方组与阳性药组基本相同，均显著降低了高尿酸血症对肾脏的损伤程度，但各组均未恢复到空白组水平，相对而言，ZC8配方的保护作用最弱。
优化配方对高尿酸血症的预防效果分析
本轮实验同时设计了治疗给药方式和预防给药方式两种，其不同点在于，预防组在给造模剂之前的前6天连续灌胃配方受试物，其他时间的给药和造模剂及取样时间方法完全相同。实验结果分析如下。
预防给药方式的优化配方降低血尿酸含量分析
根据4次动态取血测定血尿酸含量结果见图16所示，由此可见：①模型组与空白组相比，在造模后7-21天UA含量均显著升高(P＜0.01)，第7天大鼠血尿酸值已显著高于空白组，第14天达到最高，比空白组提高45.33％。在第14天停止给造模剂后大鼠血尿酸水平呈下降趋势，在第21天与空白组之间仍然存在显著差异；②各优化配方组与模型组相比，各优化配方组连续4次取样测定值均显著降低(P＜0.01)，在第17天，配方ZC2、ZC4、ZC5和ZC8较模型组分别降低了24.58％、15.40％、19.09％和13.77％；③各优化配方组与空白组相比，给造模剂同时服用配方提取物的大鼠血尿酸含量可恢复到空白组水平，第7天、第14天和第17天取样检测，各配方组血尿酸含量与空白组处于同一水平。综合以上分析，经过两次实验优化出的几个配方，对预防高尿酸血症均具有良好效果，各配方效果排序为：ZC2＝ZC5>ZC4>ZC8。
预防给药方式的优化配方降低尿素氮(BUN)含量分析
各实验处理组4次动态取血测定尿素氮含量结果如图17所示，由其可知：
①模型组与空白组相比，模型组在7-17天尿素氮水平均显著升高(P＜0.01)，说明造模成功。实验处理的第7天，造模大鼠尿素氮值已显著高于空白组，第14天达到最高值，较空白组提高37.98％，在第21天恢复到了空白水平。
②各配方组与模型组相比，各配方组连续4次取样测定尿素氮水平均显著低于模型组(P＜0.01)，在第7天配方ZC2、ZC4、ZC5和ZC8较模型组分别降低了22.96％、16.19％、19.20％和13.53％。
③各配方组与空白组相比，各配方组尿素氮含量可恢复到空白组水平。
预防给药方式的优化配方降低血肌酐含量分析
各实验处理大鼠4次动态取血测定血肌酐含量结果如图18所示。结果显示：①模型组与空白组相比，模型组在7-17天血肌酐水平较空白组显著升高(P＜0.01)，在第7天就已显著高于空白组，在第14天时达到最高值，第14天停止给造模剂后含量开始下降，在第17天仍有显著差异，到第21天时与空白组之间已经没有显著差异；②各配方组与模型组相比，各配方组连续3次取样测定均可显著低于模型组水平(P＜0.01)，在第7天配方ZC2、ZC4、ZC5和ZC8较模型组分别降低了16.38％、9.85％、9.91％和12.07％；③各配方组与空白组相比，给造模剂同时服用配方提取物的大鼠血肌酐含量在第21天除ZC4外，都可恢复到空白组水平。
预防给药方式的优化配方减轻肾损伤功效分析
各实验处理组的肾脏HE切片及其受损程度量化指标直方图如图19所示，由此可看出：①造模可导致明显的肾脏损伤，损伤主要在肾小管，严重的肾小管扩张，肾小管大面积的管型，肾小管体积粗大，上皮变薄，空腔面积数倍增大并且空腔，在肾小管周围存在炎症细胞浸润和肾组织纤维化；②各配方组与模型组相比，各配方组对造模剂引起的肾脏损伤均有不同程度的改善，具体表现为在切片中，仅可见少量炎症细胞浸润和肾小管管型，从量化统计分析结果来看，除ZC8外其他配方均可使损伤程度降低40％-59％。
对于优化出的ZC2、ZC4、ZC5、ZC8这4个配方，经过给药方式不同的实验得出：
(1)采用治疗给药方式，对于模型Ⅱ导致的高尿酸血症均有显著的治疗作用，其治疗作用在同时服用造模剂和配方药物的第7天可达到显著程度，多数配方均可使血尿酸含量维持在空白水平。与阳性药物溴苯马龙相比，各配方的作用与其在同一水平。各配方对降低造模剂所导致的肾脏损伤均具有良好的保护功能，尿素氮和血肌酐两个指标，第7、14、17天3次取样，多数配方组含量水平与模型组有极显著差异，与空白组差异不明显，使肾脏组 织的受损程度降低了近一倍，但未达到空白水平。各配方的功效综合排序为：ZC2>阳性药>ZC4＝ZC5>ZC8。
(2)采用预防给药方式，对于模型Ⅱ产生的高尿酸血症均有显著的防治作用，在给造模剂的第7、14、17和21天，血尿酸含量较模型组均有极显著的降低，到达了与空白组无显著差异的程度。各配方组对造模剂所造成的肾脏损伤均具有良好的保护功能，尿素氮和血肌酐两个指标，第7、14、17天3次取样，多数配方含量较模型有显著降低，ZC2等配方的含量可维持在空白水平上。肾脏HE切片显示，预防给药大大降低了肾脏组织的受损，但受损程度与空白还存在着显著差异。
(3)通过对预防给药方式和治疗给药方式的实验结果总体来看，两种给药方式对血尿酸、尿素氮和血肌酐含量的降低作用幅度，以及对肾损伤的减轻作用程度均差异不大，但其作用起效时间节点略有不同。预防给药在前期作用较强，治疗在后期功效更高，这可能是预防组提前6天给药产生的效果。由此推断本项目经过迭代优化的4个配方，除了具有显著的治疗作用外，也均具有一定的预防作用。即经过优化的各配方，无论是预防还是治疗给药方式，对于降低血尿酸、尿素氮和血肌酐含量均有极显著的作用，还可极显著减轻因造模而导致的肾损伤作用。预防和治疗两种给药方式，各配方功效，虽然在不同时间节点上显示略有不同，但总体上不存在显著差异。
综合分析，经过两次优化的4个配方，ZC2效果最佳，ZC8相对最差。ZC2的预防给药组，第7天血尿酸和血肌酐值均呈现明显的预防效果，其肾脏受损(HE切片)程度，较其治疗组以及其他3个配方的预防和治疗组都明显减轻。

Claims (3)

  1. 一种高血尿酸症防治中药复方组合物，包括菊苣根、茯苓、蒲公英、玉米须、槐花和甘草，其中以重量份计，菊苣根：3～6份；茯苓：1.5～3.5份；蒲公英：1.5～3.5份；玉米须：2.5～4.5份；槐花：1.5～3.5份；以及甘草：1份。
  2. 一种高血尿酸症防治制剂，包含权利要求1所述组合物的水提物。
  3. 根据权利要求2的制剂，其中成人一天用量所对应的组合物为：菊苣根10g、茯苓5g、蒲公英10g、玉米须10g、槐花10g以及甘草3g。
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