White peony root extract for treating hyperuricemia, preparation method and application thereof

Abstract

The present invention belongs to the field of medical technology related to the extraction and application of natural drug components, discloses a white peony extract for treating hyperuricemia, a preparation method thereof and an application thereof, and provides a preparation method of a white peony extract for treating hyperuricemia, wherein the obtained white peony extract has a good therapeutic effect on a hyperuricemia animal model; mainly inhibiting uric acid production by inhibiting the activity of xanthine oxidase in serum and liver, while downregulating the levels of renal tissue proteins URAT1 and GLUT9, and upregulating the levels of renal tissue proteins OAT1 and OAT3 to reduce uric acid reabsorption and promote uric acid excretion, thereby achieving the purpose of reducing uric acid levels. Furthermore, the white peony extract is synergistically used with the currently commonly used uric acid-lowering drug allopurinol to develop a more effective and safe uric acid-lowering treatment plan.

Description

Technical Field

[0001] The invention belongs to the field of medical technology related to extraction and application of natural drug components, and in particular relates to a white peony root extract for treating hyperuricemia, a preparation method and application thereof. Background Art

[0002] Hyperuricemia is a chronic metabolic disease characterized by abnormal purine metabolism, which can lead to elevated uric acid levels in the blood. Hyperuricemia is considered to be an important risk factor for a variety of diseases such as gout, hypertension and diabetes. Due to changes in people's lifestyles and dietary structures, the global prevalence of hyperuricemia has steadily increased year by year, reaching as high as 17.4% in China. The prevalence of hyperuricemia in the United States is about 20%, and it is even higher in South Korea, reaching 26.6%. Therefore, more and more studies have begun to focus on hyperuricemia.

[0003] The main strategy for preventing and treating hyperuricemia is to reduce serum uric acid levels by inhibiting uric acid formation and / or promoting uric acid excretion. The production of uric acid is the final result of purine metabolism. Purine is converted into uric acid in the liver through the action of xanthine oxidase, which is a key enzyme in the synthesis of uric acid. Therefore, xanthine oxidase is a key target for reducing uric acid production. The kidney is the main organ for uric acid excretion. If the patient's uric acid level is elevated, urate crystals will be deposited in the kidneys, leading to impaired renal function. In addition, when renal function is damaged and corresponding kidney diseases occur, it is also easy to cause uric acid excretion to be blocked, resulting in increased uric acid levels in the body, which can increase the risk of gout. The kidney plays a key role in urate metabolism, mainly through four processes: glomerular filtration, tubular reabsorption, secretion, and reabsorption after secretion. Increasing evidence shows that urate reabsorption and secretion are regulated by proteins related to urate transporters. The promotion of uric acid reabsorption is mainly mediated by uric acid transporter 1 (URAT1) and glucose transporter 9 (GLUT9). The transport and metabolism of uric acid are mainly mediated by the key roles played by organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3).

[0004] In the prior art, drugs that inhibit uric acid synthesis include allopurinol and febuxostat, and drugs that increase uric acid excretion include benzbromarone and probenecid. However, most uric acid-lowering drugs generally have large toxic side effects, causing secondary damage to the body. The white peony extract for treating hyperuricemia in the present invention can increase uric acid excretion while inhibiting uric acid synthesis, and has a very good uric acid-lowering effect; and it is a Chinese medicinal material that can be used as both medicine and food, which is safer and more reliable.

[0005] Dai Yun et al. (2016) preliminarily reported that total glycosides of white peony had a certain protective effect on the hyperuricemia mouse model, which was mainly achieved by reducing serum uric acid levels and inhibiting the activities of liver xanthine oxidase and adenosine deaminase. However, it was found through research that the uric acid-lowering active substances in white peony are not limited to total glycosides of white peony. The present invention provides a preparation method of white peony extract for treating hyperuricemia, and this preparation method can extract the active ingredients with uric acid-lowering effect in white peony to a greater extent. Further discussion was made on the mechanism of action of this white peony extract in treating hyperuricemia, confirming that the white peony extract inhibits uric acid production by inhibiting xanthine oxidase activity, regulating the expression of uric acid re-collection and secretion-related proteins, and also promotes uric acid excretion, and has a good uric acid-lowering effect, has a good therapeutic effect on hyperuricemia, and expands the application of white peony in the preparation of uric acid-lowering drugs. Currently, allopurinol is a common drug for the treatment of hyperuricemia in clinical practice. It inhibits uric acid production mainly by inhibiting the activity of xanthine oxidase and has a significant uric acid-lowering effect. However, while exerting therapeutic effects, it is often accompanied by adverse reactions such as diarrhea, gastrointestinal reactions, and renal insufficiency. In order to further enrich the application of white peony root in uric acid-lowering drugs, white peony root extract that can treat hyperuricemia is synergized with allopurinol, a common drug for the treatment of hyperuricemia in clinical practice, in order to better exert the therapeutic effect while effectively reducing the toxic side effects caused by allopurinol, making it safer and more reliable.

[0006] Through the above analysis, the problems and defects of the existing technology are as follows: it only preliminarily introduces that total glucosides of white paeony achieves therapeutic effects on hyperuricemia mice by lowering serum uric acid levels and inhibiting the activities of liver xanthine oxidase and adenosine deaminase, and does not comprehensively discuss the specific mechanism of white paeony in treating hyperuricemia. Most of the uric acid-lowering drugs introduced in the prior art have adverse reactions and will cause secondary harm to patients. A common drug for the treatment of hyperuricemia is allopurinol, which mainly inhibits uric acid production by inhibiting the activity of xanthine oxidase and has a significant uric acid-lowering effect. However, while exerting therapeutic effects, it is often accompanied by adverse reactions such as diarrhea, gastrointestinal reactions, and renal insufficiency. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a white peony root extract for treating hyperuricemia, and a preparation method and application thereof.

[0008] The present invention is achieved by: a method for preparing a white peony root extract for treating hyperuricemia comprising:

[0009] Step 1, crushing medicinal materials;

[0010] Step 2, removing impurities from ether;

[0011] Step 3, ethanol extraction;

[0012] Step 4, extraction.

[0013] Further, the medicinal material is crushed:

[0014] White peony root medicinal materials or decoction pieces are crushed into white peony root ultrafine powder with broken cell walls using a wall breaking machine.

[0015] Further, the ether is impurity-removed:

[0016] The ultrafine white peony powder was soaked in 12-15 times dry weight (W / W) ether solution at room temperature for 24 hours, and ultrasonic extraction was performed. The powder was filtered with a 300-400 mesh filter. The residue was extracted twice under the same conditions and the residue was retained.

[0017] Further, the ethanol extraction:

[0018] The filter residue is extracted once with 8-10 times the amount of 95% ethanol, 85% ethanol and 75% ethanol respectively, the extraction process is assisted by ultrasonic extraction, the extraction temperature is 60°C, the extraction time is 3 hours each time, the three filtrates are filtered with 300-400 mesh filter screens respectively, the filtrates are combined, and concentrated under reduced pressure at 60°C until there is no alcohol taste to obtain an ethanol extract.

[0019] Further, the extraction:

[0020] The ethanol extract was diluted with pure water, extracted with petroleum ether, ethyl acetate and n-butanol respectively, and each solvent was extracted three times. The extracts were combined and concentrated under reduced pressure at 60°C to obtain white peony root extract, which was freeze-dried to obtain white peony root extract dry powder.

[0021] The invention provides a system for preparing white peony root extract for treating hyperuricemia, the system comprising: a medicinal material crushing module, used for crushing white peony root medicinal materials or decoction pieces into white peony root ultrafine powder with broken cell walls; an ether impurity removal module, used for removing impurities in the white peony root ultrafine powder by soaking in an ether solution and ultrasonic extraction; an ethanol extraction module, used for extracting effective components in the white peony root by using ethanol solutions of different concentrations under the condition of auxiliary ultrasonic extraction; and an extraction module, used for extracting the ethanol extract by using different solvents to obtain the white peony root extract.

[0022] Furthermore, the medicinal material crushing module adopts a wall breaking machine for crushing to ensure that the cell walls of the white peony medicinal material or the decoction pieces are destroyed, thereby improving the subsequent extraction efficiency.

[0023] Furthermore, the ether impurity removal module effectively removes impurities in the white peony ultrafine powder by setting the soaking time, the amount of ether solution and the conditions of ultrasonic extraction to ensure the purity of the extract.

[0024] Furthermore, the system also includes a control module for automatically controlling the entire preparation process, including the degree of medicinal material crushing, the setting of conditions for ether impurity removal and ethanol extraction, the selection of extraction solvents, and the control of the number of extractions, so as to improve the preparation efficiency and product quality. At the same time, the control module also has data recording and analysis functions to optimize and improve the preparation process.

[0025] The invention provides a white peony extract, which comprises the following chemical components: paeoniflorin, debenzoyl paeoniflorin, peony lactone A, peony lactone D, white peony lactone C, 4-O-gallate albizzine, catechin, kaempferol, apigenin, quercetin, benzoic acid, protocatechuic aldehyde, protocatechuic acid, gallic acid, methyl gallate, ursolic acid and oleanolic acid; the white peony extract is obtained by a specific preparation process, which comprises the steps of crushing medicinal materials, removing impurities with ether, extracting with ethanol and extracting; the obtained white peony extract has a significant effect of treating hyperuricemia, is high in purity and has few impurities, and is suitable for preparing a medicine or health product for treating hyperuricemia

[0026] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0027] First, the purpose of the present invention is to provide a method for preparing a white peony extract for treating hyperuricemia, wherein the obtained white peony extract has a good therapeutic effect on a hyperuricemia animal model; it mainly inhibits uric acid production by inhibiting the activity of xanthine oxidase in serum and liver, and simultaneously downregulates the levels of renal tissue proteins URAT1 and GLUT9, and upregulates the levels of renal tissue proteins OAT1 and OAT3 to reduce uric acid reabsorption and promote uric acid excretion, thereby achieving the purpose of reducing uric acid levels. Furthermore, the white peony extract is synergistically used with the currently commonly used uric acid-lowering drug allopurinol to develop a more effective and safe uric acid-lowering treatment regimen.

[0028] 1. The present invention provides a method for preparing a white peony root extract for treating hyperuricemia. Compared with the existing preparation technology of white peony total glucosides with uric acid lowering effect, this preparation method can extract the active ingredients with uric acid lowering effect in white peony root to a greater extent.

[0029] 2. The obtained white peony extract inhibits uric acid production by inhibiting the activity of xanthine oxidase in serum and liver; at the same time, it reduces uric acid reabsorption by down-regulating the levels of renal tissue proteins URAT1 and GLUT9, and promotes uric acid excretion by up-regulating the levels of renal tissue proteins OAT1 and OAT3; while existing uric acid-lowering drugs usually only exert their uric acid-lowering effects through a single pathway of inhibiting uric acid production or promoting uric acid excretion, the white peony extract can achieve the purpose of lowering uric acid by inhibiting uric acid production while inhibiting uric acid reabsorption and promoting uric acid excretion, thereby having a better therapeutic effect on patients with hyperuricemia.

[0030] 3. Currently, drugs used in the clinic to treat hyperuricemia generally have large toxic side effects. In the present invention, we introduce a white peony extract that can be used as both medicine and food and treat hyperuricemia into allopurinol, a commonly used drug for treating hyperuricemia in the clinic. It is found that the combined action of white peony extract and allopurinol in the treatment of hyperuricemia can more effectively reduce the level of uric acid in the body, and at the same time can improve the gastrointestinal reactions caused by allopurinol alone, and effectively alleviate kidney damage. The present invention is the first to use white peony extract and allopurinol in combination to treat hyperuricemia, proving that the synergistic effect of the two is safer and more reliable while enhancing the therapeutic efficacy.

[0031] Second, the present invention solves the problems of low extraction efficiency, low purity and large loss of active ingredients in the prior art by optimizing the preparation method of white peony extract. First, through the medicinal material crushing step, the white peony medicinal material or decoction piece is crushed into ultrafine powder with broken cell walls using a wall breaking machine, which effectively increases the surface area of ​​white peony and improves the dissolution rate of the effective ingredients in the subsequent extraction process. This crushing method significantly improves the extraction efficiency compared with the traditional method, so that the extract has a higher content of active ingredients.

[0032] Secondly, in the ether impurity removal step, 12-15 times the dry weight of ether solution is used for room temperature soaking, and ultrasonic extraction is used to effectively remove impurities in the white peony ultrafine powder and retain the effective ingredients to the greatest extent. After multiple extractions and filtrations, it is ensured that there are basically no impurities remaining in the filter residue, providing pure raw materials for subsequent ethanol extraction. This process can significantly improve the purity of the extract compared to traditional methods.

[0033] In the ethanol extraction step, different concentrations of ethanol were innovatively used for multiple extractions, supplemented by ultrasound-assisted extraction, at a temperature of 60°C, and each extraction time was 3 hours. This multi-step extraction method effectively improved the extraction rate of the effective ingredients of white peony root, and removed ethanol by reduced pressure concentration, ultimately obtaining a high-concentration ethanol extract. Compared with traditional single-concentration ethanol extraction, the method of the present invention significantly improved the extraction efficiency and the retention rate of the effective ingredients.

[0034] Finally, in the extraction step, after the ethanol extract extract is diluted, petroleum ether, ethyl acetate and n-butanol are used for multiple extractions, and combined with reduced pressure concentration and freeze drying, a high-purity white peony extract powder is obtained. This method not only simplifies the operating process, but also effectively retains the active ingredients of white peony. Compared with traditional methods, the multi-solvent extraction method of the present invention significantly improves the purity and yield of the extract, and ensures the high quality and stability of the extract. Through these innovative steps, the present invention has achieved significant technological progress while solving the problems of the prior art, and provides a more effective and high-quality white peony extract for treating hyperuricemia.

[0035] Second, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0036] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0037] After the technical solution of the present invention is transformed, it will be transformed into products or technologies to create value, achieve market sales and obtain profits. It is expected to generate an additional sales revenue of 100 million yuan, an additional profit of 15 million yuan, and a tax of 4 million yuan. It will radiate and drive related enterprises and rural professional cooperatives to carry out standardized planting of high-quality white peony root, guide Chinese medicine enterprises to develop standardized and large-scale planting of authentic Anhui medicinal materials, promote the construction of the "Top Ten Anhui Medicines" industrial demonstration base, drive the transformation of local advantageous resources, develop characteristic industries, achieve the purpose of protecting the ecological environment and sustainable utilization of resources, and produce significant economic, social and ecological benefits.

[0038] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0039] In the current domestic reports on the uric acid-lowering effect of white peony, it is only preliminarily judged that total glycosides of white peony play a therapeutic role in hyperuremia by reducing serum uric acid levels and inhibiting the activity of liver xanthine oxidase. However, the components with uric acid-lowering effect in white peony are not limited to total glycosides of white peony. The present invention provides a preparation method of white peony extract for treating hyperuricemia. Further research has found that white peony extract inhibits uric acid production by inhibiting the activity of xanthine oxidase in serum and liver; at the same time, uric acid reabsorption is reduced by down-regulating the levels of renal tissue proteins URAT1 and GLUT9, and uric acid excretion is promoted by up-regulating the levels of renal tissue proteins OAT1 and OAT3. With this, white peony extract shows good therapeutic effect on hyperuricemia.

[0040] At present, the drugs commonly used to treat hyperuricemia abroad are mainly Western medicines such as allopurinol, but they all have a series of adverse reactions. In the present invention, the safe and non-toxic traditional Chinese medicine white peony root is introduced into the treatment of allopurinol, which increases the efficacy while reducing the adverse reactions of gastrointestinal reactions and renal insufficiency caused by the use of allopurinol alone, thereby better improving safety.

[0041] (3) The technical solution of the present invention solves the technical problems that people have been eager to solve but have never been able to solve successfully:

[0042] Due to changes in people's lifestyles and dietary structure, the global prevalence of hyperuricemia has steadily increased year by year, and is becoming younger and younger. Hyperuricemia is considered to be an important risk factor for various diseases such as gout, hypertension and diabetes. Therefore, the prevention and treatment of hyperuricemia has been a hot topic in recent years. However, the therapeutic drugs for hyperuricemia in the prior art often cause a series of adverse reactions. The white peony extract for treating hyperuricemia provided in the present invention is safer and more reliable while ensuring the efficacy, and has no adverse reactions.

[0043] (4) The technical solution of the present invention overcomes technical prejudice:

[0044] Clinically, existing uric acid-lowering drugs are usually drugs that inhibit uric acid synthesis, such as allopurinol and febuxostat, and drugs that increase uric acid excretion, such as benzbromarone and probenecid. Most of these Western medicines only play a uric acid-lowering role in inhibiting uric acid production or promoting uric acid excretion. White peony extract can achieve the purpose of lowering uric acid by inhibiting uric acid production, inhibiting uric acid reabsorption, and promoting uric acid excretion. It has a better therapeutic effect on patients with hyperuricemia. In addition, the dual-purpose white peony root material for treating hyperuricemia is safer and more reliable while enhancing the efficacy of the drug, and can significantly reduce adverse reactions of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a flow chart of a method for preparing a white peony root extract for treating hyperuricemia provided by an embodiment of the present invention;

[0046] Figure 2 : is a graph showing the effect of the white peony root extract provided in the embodiment of the present invention on the blood uric acid level in hyperuricemia rats; compared with the normal control group, ### P<0.01; compared with the hyperuricemia model group, * P < 0.5, P < 0.1, *P < 0.01;

[0047] Figure 3 : is a graph showing the effect of the white peony root extract provided in the embodiment of the present invention on the xanthine oxidase activity in the serum (A) and liver (B) of hyperuricemia rats; compared with the normal group, ## P<0.01; compared with the model group, *P<0.5, P<0.1;

[0048] Figure 4 This is a graph showing the effect of the white peony root extract provided by the embodiment of the present invention on the levels of URAT1, GLUT9, OAT1, and OAT3 proteins in the kidneys of hyperuricemia rats; compared with the normal control group, ### P<0.001; compared with the hyperuricemia model group, *P<0.05, P<0.01, *P<0.001;

[0049] Figure 5: This is a graph showing the effect of white peony root extract combined with allopurinol on the blood uric acid level in hyperuricemia rats provided by the embodiment of the present invention; compared with the normal control group, ### P<0.01; compared with the hyperuricemia model group, *P<0.01;

[0050] Figure 6 This is a graph showing the effect of the white peony root extract combined with allopurinol provided in an embodiment of the present invention on the body weight of hyperuricemia rats;

[0051] Figure 7 This is a picture of the effect of white peony extract combined with allopurinol on the renal tissue of hyperuricemia rats (400X); A: normal control group; B: hyperuricemia model group; C: allopurinol group; D: white peony extract combined with allopurinol group. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0053] Example 1: High-purity white peony root extract and its preparation process

[0054] Component ratio:

[0055] - Paeoniflorin: 30%

[0056] - Debenzoylpaeoniflorin: 10%

[0057] -Peonidin A: 5%

[0058] -Peonidin D: 3%

[0059] - Paeonia lactone C: 2%

[0060] -4-O-gallate albendazole: 2%

[0061] - Other ingredients (catechins, kaempferol, apigenin, etc.): total not exceeding 48%

[0062] Preparation process:

[0063] 1. Crush the medicinal materials: Crush the dried white peony root into fine powder and sieve to ensure uniform particle size.

[0064] 2. Ether removal: Use ether to soak the fine powder to remove some fat-soluble impurities, then filter and dry.

[0065] 3. Ethanol extraction: Add the treated fine powder into 70% ethanol and perform heating reflux extraction twice, 2 hours each time. Combine the extracts and recover the ethanol under reduced pressure until there is no alcohol taste.

[0066] 4. Extraction and purification: The concentrated extract is loaded onto a treated macroporous adsorption resin column, and impurities are first eluted with water, and then gradient eluted with ethanol solutions of different concentrations (such as 20%, 30%, and 50%), and the eluate containing the target component is collected. The eluate is concentrated under reduced pressure and vacuum dried at low temperature to obtain a high-purity white peony extract.

[0067] Effect evaluation: The content of each component in the white peony extract was verified by analytical methods such as HPLC to ensure that the purity of the main components such as paeoniflorin reached the predetermined standard and the impurity content was low. Experiments have shown that the extract has a significant therapeutic effect on hyperuricemia model animals.

[0068] Example 2: Optimization of white peony root extract and its preparation

[0069] Component ratio:

[0070] - Paeoniflorin: 40%

[0071] - Debenzoylpaeoniflorin: 8%

[0072] -Peonidin A: 6%

[0073] -Peonidin D: 4%

[0074] - Paeonia lactone C: 3%

[0075] -4-O-gallate albendazole: 2.5%

[0076] - Other ingredients: total not exceeding 36.5%

[0077] Preparation process:

[0078] 1. Pretreatment of medicinal materials: Wash, peel, slice, dry and crush into powder of appropriate particle size.

[0079] 2. Impurity removal and extraction: Mix the powder with ether to remove some impurities, filter and dry. Then use supercritical carbon dioxide extraction technology combined with ethanol extraction to improve extraction efficiency and purity.

[0080] 3. Purification step: The extract was further purified by silica gel column chromatography, using a mixed solvent of ethyl acetate and methanol in different proportions for gradient elution, and the eluate of the target component was collected.

[0081] 4. Drying and finished product: The eluate is concentrated under reduced pressure and dried in a vacuum at low temperature to obtain a high-purity white peony root extract.

[0082] Effect evaluation: This embodiment further improves the purity of main components such as paeoniflorin in white peony extract by optimizing the extraction and purification process, and effectively reduces the impurity content. The experimental results show that the extract shows better efficacy in treating hyperuricemia. At the same time, the process is also suitable for industrial production and can meet the market demand for high-quality white peony extract.

[0083] like Figure 1 As shown, a method for preparing a white peony root extract for treating hyperuricemia provided by an embodiment of the present invention comprises the following steps:

[0084] S101, medicinal material crushing;

[0085] S102, ether impurity removal;

[0086] S103, ethanol extraction;

[0087] S104, extraction.

[0088] The medicinal material crushing provided by the embodiment of the present invention:

[0089] White peony root medicinal materials or decoction pieces are crushed into white peony root ultrafine powder with broken cell walls using a wall breaking machine.

[0090] Ether impurity removal provided by the embodiment of the present invention:

[0091] The ultrafine white peony powder was soaked in 12-15 times dry weight (W / W) ether solution at room temperature for 24 hours, and ultrasonic extraction was performed. The powder was filtered with a 300-400 mesh filter. The residue was extracted twice under the same conditions and the residue was retained.

[0092] The ethanol extraction provided by the embodiment of the present invention:

[0093] The filter residue is extracted once with 8-10 times the amount of 95% ethanol, 85% ethanol and 75% ethanol respectively, the extraction process is assisted by ultrasonic extraction, the extraction temperature is 60°C, the extraction time is 3 hours each time, the three filtrates are filtered with 300-400 mesh filter screens respectively, the filtrates are combined, and concentrated under reduced pressure at 60°C until there is no alcohol taste to obtain an ethanol extract.

[0094] The extraction provided by the embodiment of the present invention:

[0095] The ethanol extract was diluted with pure water, extracted with petroleum ether, ethyl acetate and n-butanol respectively, and each solvent was extracted three times. The extracts were combined and concentrated under reduced pressure at 60°C to obtain white peony root extract, which was freeze-dried to obtain white peony root extract dry powder.

[0096] The present invention is specifically implemented:

[0097] The present invention clarifies the mechanism of action of a white peony root extract for treating hyperuricemia, comprising the following steps:

[0098] (1) Therapeutic effect of white peony root extract on hyperuricemia rats;

[0099] 40 8-12 week old SPF grade SD male rats with a body weight of (200±20) g were randomly divided into a normal control group, a hyperuricemia model group, and a low (200 mg / kg), medium (400 mg / kg), and high (800 mg / kg) dose group of white peony extract, with a total of 5 groups and 8 rats in each group.

[0100] The blank control group was given 0.5% sodium carboxymethyl cellulose solution intraperitoneally every day; the rats in the other groups were all modeled (intraperitoneal injection of a mixed solution of 300 mg / kg potassium oxonate and 100 mg / kg hypoxanthine), and appropriate amounts of potassium oxonate and hypoxanthine were weighed and dissolved in 0.5% sodium carboxymethyl cellulose solution to prepare the modeling drug. One hour after the intraperitoneal injection, the three white peony extract groups were gavaged, and the model group and the blank group were gavaged with equal doses of normal saline for 14 days.

[0101] Two hours after the last intragastric administration, the rats were anesthetized, and the abdominal aorta was opened to collect blood, and the serum was separated. The uric acid level of the rats after medication was detected using a uric acid kit. The remaining serum, liver and kidney tissues were kept for later use.

[0102] Figure 2 Effect of white peony root extract on serum uric acid level in hyperuricemia rats; compared with the normal control group, ### P<0.01; compared with the hyperuricemia model group, *P<0.5, P<0.1, *P<0.01

[0103] By the above Figure 2 It was found that the white peony extract in the treatment of hyperuricemia rats showed a significant downward trend in the blood uric acid level (P<0.05, P<0.01, P<0.001), and the effect was concentration-dependent. This shows that the white peony extract has a significant therapeutic effect on hyperuricemia rats.

[0104] (2) White peony extract inhibits uric acid production by inhibiting the activity of xanthine oxidase in the serum and liver of hyperuricemic rats;

[0105] Weigh 50 mg of liver tissue accurately, add 9 times the volume of homogenization medium (0.9% saline) at a ratio of weight (g): volume (mL) = 1:9, and mechanically homogenize in an ice-water bath to prepare a 10% homogenate. Centrifuge at 2500-3000 rpm for 10 minutes, and take the supernatant for determination. The serum test solution and liver tissue homogenate supernatant obtained above were determined using a xanthine oxidase kit. The results are as follows Figure 3 shown.

[0106] Figure 3 Effects of white peony root extract on xanthine oxidase activity in serum (A) and liver (B) of hyperuricemia rats; compared with the normal group, ## P<0.01; compared with the model group, *P<0.5, P<0.1

[0107] By the above Figure 3 Whether it is the xanthine oxidase content in serum or the xanthine oxidase content in liver tissue, white peony extract can effectively inhibit the xanthine oxidase activity of hyperuricemia rats after treatment, thereby inhibiting uric acid production and achieving the effect of lowering uric acid.

[0108] (3) White peony extract reduces uric acid reabsorption and promotes uric acid excretion by downregulating the levels of URAT1 and GLUT9 proteins in the renal tissue of hyperuricemia rats and upregulating the levels of OAT1 and OAT3 proteins in the renal tissue.

[0109] From the fresh kidney tissues taken from the above animal experiments, one rat kidney tissue was randomly selected from each group for protein extraction, including the normal control group, the hyperuricemia model group, the white peony extract low (200 mg / kg), medium (400 mg / kg), and high (800 mg / kg) dose groups. The tissue was cut into small pieces, 50 mg was weighed, the blood was washed with phosphate buffer, and the filter paper was blotted dry (operated on ice), and then the minced tissue was placed in an EP tube. 50 mg of tissue was added with 500 μl of radioimmunoprecipitation lysis buffer, and the tissue was broken using a tissue homogenizer grinder, and then placed on ice for 30 minutes. After it was fully lysed, it was centrifuged (4°C, 12,000 speed, 10 minutes), and the supernatant was taken to obtain tissue protein. After quantification by the bipyridine carbendazim quantitative method, an appropriate amount of loading buffer was added and boiled in a 100°C metal bath for 10 minutes, and then the protein sample was placed in a -20°C refrigerator for standby use.

[0110] Prepare 10% polyacrylamide gel, add protein samples to the loading wells, and perform polyacrylamide gel electrophoresis at 80 volts (30 minutes) and 120 volts (60 minutes) in sequence. Wet transfer, set the current to 200 mA, and the transfer time is determined according to the molecular weight. Transfer the protein on the polyacrylamide gel to the polyvinylidene fluoride membrane. After blocking with a fast blocking solution for 20 minutes, wash the membrane three times with TBST (Tris-buffered saline with Tween 20), each for 15 minutes. Place the polyvinylidene fluoride membrane in the primary antibody diluted in the prescribed ratio and incubate overnight at 4°C. The next day, wash the membrane three times with TBST, incubate the secondary antibody at room temperature for 1 hour, continue to wash the membrane three times with TBST, take pictures and record with a gel imaging system, and use analysis software for data processing. Repeat the experiment three times.

[0111] Figure 4Effects of white peony extract on the levels of URAT1, GLUT9, OAT1, and OAT3 proteins in the kidneys of hyperuricemia rats. ### P<0.001; compared with the hyperuricemia model group, *P<0.05, P<0.01, *P<0.001

[0112] By the above Figure 4 , it was found that the protein molecules URAT1 and GLUT9 responsible for uric acid reabsorption in the kidney tissue of hyperuricemia rats showed a significant increase trend, and after treatment with white peony extract, this increasing trend was significantly inhibited; the expression of protein molecules OAT1 and OAT3 responsible for uric acid excretion in the kidney tissue of hyperuricemia rats was significantly reduced, and after treatment with white peony extract, the expression of OAT1 and OAT3 molecules showed an increasing trend. This shows that white peony extract has a significant therapeutic effect on hyperuricemia rats by inhibiting uric acid reabsorption and promoting uric acid excretion.

[0113] 3. The present invention provides a method for treating hyperuricemia by synergistically using white peony root extract and the existing uric acid-lowering drug allopurinol, comprising the following steps:

[0114] (1) The synergistic therapeutic effect of white peony root extract and allopurinol on hyperuricemia rats;

[0115] Thirty-two SPF SD male rats aged 8-12 weeks with a body weight of (200±20) g were randomly divided into a normal control group, a hyperuricemia model group, an allopurinol group (10 mg / kg), and a combined treatment group of allopurinol (10 mg / kg) and white peony extract (400 mg / kg), with a total of 4 groups, 8 rats in each group.

[0116] The blank control group was given intraperitoneal injection of 0.5% sodium carboxymethyl cellulose solution every day; the rats in the other groups were modeled (intraperitoneal injection of a mixed solution of 300 mg / kg potassium oxonate and 100 mg / kg hypoxanthine), and appropriate amounts of potassium oxonate and hypoxanthine were weighed and dissolved in 0.5% sodium carboxymethyl cellulose solution to prepare the modeling drug. One hour after the intraperitoneal injection, the drug-dosing groups were gavaged, and the model group and the blank group were gavaged with equal doses of normal saline for 14 days.

[0117] Two hours after the last intragastric administration, the rats were anesthetized, and the abdomen was opened to collect blood from the abdominal aorta, and the serum was separated. The uric acid level of the rats after medication was detected using a uric acid kit. The kidney tissue was retained for later use.

[0118] Figure 5 Effects of white peony root extract combined with allopurinol on the level of serum uric acid in hyperuricemia rats; compared with the normal control group, ### P<0.01; compared with the hyperuricemia model group, *P<0.01

[0119] By the above Figure 5 It was found that the allopurinol group and the allopurinol combined with white peony extract group had comparable therapeutic effects on hyperuricemia rats (P<0.001), but the allopurinol combined with white peony extract group had a better effect on reducing the blood uric acid level in hyperuricemia rats.

[0120] (2) To evaluate the safety of white peony extract and allopurinol in the synergistic treatment of hyperuricemia in rats.

[0121] It has been reported that allopurinol often causes adverse reactions such as gastrointestinal reactions and renal damage when treating patients with hyperuricemia. Therefore, in the above-mentioned animal efficacy experiment, the weight of each group of rats was tested, and the kidney tissue was stained with HE pathological sections (hematoxylin-eosin staining method) to observe whether the introduction of white peony extract while treating allopurinol can improve its side effects.

[0122] Figure 6 Effect of white peony root extract combined with allopurinol on body weight in hyperuricemia rats

[0123] By the above Figure 6 , it was found that the weight of rats in the normal group, the hyperuricemia model group, and the group treated with white peony extract combined with allopurinol increased steadily and healthily during the two-week treatment period; while it was found that rats treated with allopurinol alone had a series of adverse reactions such as diarrhea and abnormal defecation, and their weight also decreased with the number of days of treatment. Therefore, the introduction of white peony extract in the treatment of hyperuricemia with allopurinol can enhance the efficacy of the drug while improving the gastrointestinal adverse reactions caused by allopurinol.

[0124] Figure 7 Effect of white peony extract combined with allopurinol on HE pathological sections of renal tissue of hyperuricemia rats (400X); A: normal control group; B: hyperuricemia model group; C: allopurinol group; D: white peony extract combined with allopurinol group.

[0125] By the above Figure 7 Compared with the normal control group, the renal tissue of rats in the hyperuricemia model group showed tubular dilatation, inflammatory infiltration, necrosis and shedding of tubular epithelial cells, reflecting obvious damage to the renal structure. Compared with the allopurinol group, the combined action of white peony extract and allopurinol significantly reversed this damage, and the introduction of white peony extract was more effective in alleviating this renal damage. This shows that the combined action of white peony extract and allopurinol is safer and more reliable than the single administration of allopurinol in the treatment of hyperuricemia.

[0126] A method for preparing total glucosides of white peony comprises the following steps: 1) extraction: taking white peony slices, extracting with ethanol, and filtering the extract; 2) concentration: heating and concentrating the extract, recovering ethanol, and obtaining a concentrated solution; 3) extraction: a, alkalization: adding alkali solution to the concentrated solution, adjusting the pH value to 5-7; b, ethyl acetate extraction and impurity removal: adding ethyl acetate for extraction, and retaining the mother liquor; c, n-butanol ethyl acetate mixed solution extraction: extracting the above medicinal liquid with a mixed solution of n-butanol ethyl acetate to obtain a n-butanol ethyl acetate extract; 4) concentration: after the n-butanol ethyl acetate is recovered from the extract, water is added to dissolve it, and then heating and concentrating it to obtain a concentrated solution; 5) drying: spray drying the concentrated solution.

[0127] The invention relates to a preparation method of total glucosides of white peony. The method comprises the following steps: 1) extraction: taking white peony slices, extracting with ethanol, and filtering the extract; 2) concentration: heating and concentrating the extract, recovering ethanol, and obtaining a concentrated solution; 3) extraction: a, alkalization: adding alkali solution to the concentrated solution, and adjusting the pH value to 5-7; b, ethyl acetate extraction and impurity removal: adding ethyl acetate for extraction, and retaining the mother liquor; c, n-butanol ethyl acetate mixed solution extraction: extracting the above medicinal liquid with a mixed solution of n-butanol ethyl acetate to obtain a n-butanol ethyl acetate extract; 4) concentration: after the n-butanol ethyl acetate is recovered from the extract, water is added to dissolve it, and it is heated and concentrated to obtain a concentrated solution; 5) drying: spray drying the concentrated solution.

[0128] The following active ingredients in white peony root that can lower uric acid are clearly identified: total glucosides of white peony root (main ingredients: paeoniflorin, paeoniflorin lactone glucosides, benzoylpeoniflorin, oxidized paeoniflorin), catechins, quercetin, kaempferol, ursolic acid, oleanolic acid, benzoic acid, gallic acid, protocatechuic aldehyde, and apigenin.

[0129] The monoterpenes and glycosides are debenzoylated paeoniflorin;

[0130] Triterpenes and their glycosides 11α,12α-epoxy-3β-hydroxyoleic acid-28,13β-lactone;

[0131] Mixed terpenes TCA 15B type;

[0132] Phenylethanol glycosides are 2-phenylacetyl[-α-L-rhamnopyranoside (-1→6)]-β-D-glucoside;

[0133] The volatile oil is pentadecanoic acid;

[0134] Tannins are 1-O-galloyl-β-D-glucoside;

[0135] The flavonoids are kaempferol 3-O-(6-O-galloyl)-β-D-glucoside;

[0136] The steroid is caudatin-3-O-(4-O-methyl-β-D-cyclopyranosyl)(-1→4)-α-D-enosylpyranosyl(-1→4)-β-D-glucopyranosyl(1→4)-α-L-rhamnosylpyranose;

[0137] The organic acid is ethyl gallate.

[0138] The following are two specific embodiments according to the above claims:

[0139] Embodiment 1:

[0140] Step 1: Crush the herbs

[0141] Take 100 grams of white peony root medicinal material or decoction pieces, use a wall breaking machine to crush it, and obtain white peony root ultrafine powder with broken cell walls.

[0142] Step 2: Ether removal

[0143] The superfine powder of white peony root after crushing was soaked at room temperature for 24 hours with 1200 grams of ether solution (i.e. 12 times of dry weight), during which ultrasonic extraction was assisted once every 6 hours for 30 minutes each time. After 24 hours, filtered with a 300 mesh filter, and filter residue was retained. The filter residue was extracted twice with ether under the same conditions, using new ether solution each time, and assisted ultrasonic extraction, and then filtered and retained the filter residue.

[0144] Step 3: Ethanol Extraction

[0145] The residue obtained in step 2 was extracted with 800 g of 95% ethanol at 60°C for 3 hours, with ultrasonic extraction as an auxiliary. After the extraction is completed, filter with a 300-mesh filter and retain the filtrate. Then, the above extraction process was repeated with 800 g of 85% ethanol and 75% ethanol respectively. The filtrates extracted three times were combined and then concentrated under reduced pressure at 60°C until there was no alcohol taste, to obtain an ethanol extract.

[0146] Step 4: Extraction

[0147] The ethanol extract was diluted to 1000 ml with pure water, and then extracted three times with petroleum ether, ethyl acetate, and n-butanol, using 500 ml of solvent each time. After the extraction was completed, the extracts of each solvent were combined and concentrated under reduced pressure at 60°C to obtain the white peony extract. Finally, the extract was freeze-dried to obtain the white peony extract dry powder.

[0148] Embodiment 2:

[0149] Step 1: Crush the herbs

[0150] Take 200 grams of white peony root medicinal materials or decoction pieces, use a wall breaking machine to crush them, and obtain white peony root ultrafine powder with destroyed cell walls.

[0151] Step 2: Ether removal

[0152] 3000 g of ether solution (i.e. 15 times of dry weight) was used to soak the white peony root powder at room temperature for 24 hours, and ultrasonic extraction was performed once an hour during the period. After 24 hours, the powder was filtered with a 400-mesh filter and the residue was retained. The residue was extracted twice with ether under the same conditions and the residue was filtered and retained.

[0153] Step 3: Ethanol Extraction

[0154] The residue was extracted with 1600 g, 1700 g and 1800 g of 95% ethanol, 85% ethanol and 75% ethanol respectively, the extraction temperature was 60°C, each extraction was 3 hours, and ultrasonic extraction was assisted. After three extractions, they were filtered with a 400-mesh filter, the three filtrates were combined, and concentrated under reduced pressure at 60°C until there was no alcohol taste, to obtain an ethanol extract.

[0155] Step 4: Extraction

[0156] After the ethanol extract was diluted to 2 liters with pure water, it was extracted three times with petroleum ether, ethyl acetate, and n-butanol, respectively, with the amount of solvent used for each extraction being 1 liter. Each extraction solution was combined, and concentrated under reduced pressure at 60°C to obtain the white peony extract, and the white peony extract dry powder was obtained after freeze drying.

[0157] like Figure 1 The extraction method shown in the figure obtains a white peony extract, which includes the following chemical components: paeoniflorin, debenzoyl paeoniflorin, peony lactone A, peony lactone D, white peony lactone C, 4-O-gallic acid albizzia alkaloid, catechin, kaempferol, apigenin, quercetin, benzoic acid, protocatechuic aldehyde, protocatechuic acid, gallic acid, methyl gallate, ursolic acid, oleanolic acid and other chemical components.

[0158] Figure 2 The effect of the white peony root extract on the blood uric acid level in hyperuricemia rats. The uric acid level in the hyperuricemia group was higher than that in the normal control group. ### P<0.01, indicating that the hyperuricemia model was successfully constructed; compared with the hyperuricemia model group, the efficacy of white peony extract (low (200 mg / kg), medium (400 mg / kg), and high (800 mg / kg) doses) was *P<0.5, P<0.1, and *P<0.01, respectively. This shows that white peony extract has the effect of reducing blood uric acid in hyperuricemia rats, and the effect is concentration-dependent. It shows that white peony extract has a significant therapeutic effect on hyperuricemia rats.

[0159] Figure 3The effect of white peony extract on the activity of xanthine oxidase in the serum (A) and liver (B) of rats with hyperuricemia. Xanthine oxidase is a key enzyme in the synthesis of uric acid, which can catalyze the precursor substrate hypoxanthine into the substrate xanthine, and at the same time catalyze the substrate xanthine into uric acid. Therefore, uric acid production can be inhibited by inhibiting the activity of xanthine oxidase in the body, thereby achieving the effect of lowering uric acid. Figure 3 The results showed that white peony extract could effectively inhibit the xanthine oxidase activity in both serum and liver tissue of hyperuricemia rats, thereby inhibiting uric acid production and achieving the effect of lowering uric acid.

[0160] Figure 4 The effect of white peony extract on the levels of URAT1, GLUT9, OAT1, and OAT3 proteins in the kidneys of hyperuricemic rats. The reabsorption and secretion of urate are regulated by proteins related to urate transporters. The promotion of uric acid reabsorption is mainly mediated by uric acid transporter 1 (URAT1) and glucose transporter 9 (GLUT9). The transport and metabolism of uric acid are mainly mediated by the key roles played by organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3). Figure 4 It was found that the protein molecules URAT1 and GLUT9 responsible for uric acid reabsorption in the kidney tissue of hyperuricemia rats showed a significant increase trend, and after treatment with white peony extract, this increasing trend was significantly inhibited; the expression of protein molecules OAT1 and OAT3 responsible for uric acid excretion in the kidney tissue of hyperuricemia rats was significantly reduced, and after treatment with white peony extract, the expression of OAT1 and OAT3 molecules showed an increasing trend. This shows that white peony extract has a significant therapeutic effect on hyperuricemia rats by inhibiting uric acid reabsorption and promoting uric acid excretion.

[0161] Figure 5 The effect of white peony extract combined with allopurinol on the blood uric acid level in hyperuricemia rats. It was found that the allopurinol group and the allopurinol combined with white peony extract group had similar therapeutic effects on hyperuricemia rats (P<0.001), but the allopurinol combined with white peony extract group had a better effect on reducing the blood uric acid level in hyperuricemia rats.

[0162] Figure 6This is the effect of white peony extract combined with allopurinol on the body weight of hyperuricemia rats. It was found that the body weight of rats in the normal group, the hyperuricemia model group, and the white peony extract combined with allopurinol administration group showed a steady and healthy growth during the two-week administration period; while in the observation, it was found that when allopurinol was administered alone, rats had a series of adverse reactions such as diarrhea and abnormal defecation, and their body weight also decreased with the number of days of administration. From this point of view, the introduction of white peony extract in the treatment of hyperuricemia with allopurinol can not only enhance the efficacy of the drug, but also improve the gastrointestinal adverse reactions caused by allopurinol.

[0163] Figure 7 Results of HE pathological section staining of renal tissue of hyperuricemia rats with white peony extract combined with allopurinol. Compared with the normal group, the renal tissue of rats in the hyperuricemia model group showed dilatation of renal tubules, inflammatory infiltration, necrosis and shedding of renal tubular epithelial cells, reflecting obvious damage to the renal structure. Compared with the allopurinol group, the combination of white peony extract and allopurinol significantly reversed this damage, and the introduction of white peony extract can more effectively alleviate this renal damage. Therefore, it is shown that the combination of white peony extract and allopurinol is safer and more reliable than the single administration of allopurinol in the treatment of hyperuricemia.

[0164] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a white peony root extract for treating hyperuricemia, characterized in that: The following steps are involved: Step 1: Crush the herbs Take 100 grams of white peony root medicinal material or decoction pieces, and crush them using a wall-breaking machine to obtain white peony root ultrafine powder with broken cell walls; Step 2: Ether removal The crushed white peony root ultrafine powder is soaked in 1200 g of ether solution, i.e., 12 times of dry weight, at room temperature for 24 hours, during which ultrasonic extraction is performed once every 6 hours for 30 minutes each time; after 24 hours, the powder is filtered with a 300-mesh filter and the filter residue is retained; the filter residue is extracted twice with ether under the same conditions, each time with a new ether solution and ultrasonic extraction, and then filtered and the filter residue is retained; Step 3: Ethanol Extraction The residue obtained in step 2 was extracted with 800 g of 95% ethanol at 60° C. for 3 hours, with the aid of ultrasonic extraction; after the extraction was completed, it was filtered with a 300-mesh filter and the filtrate was retained; Then, the above extraction process was repeated with 800 g of 85% ethanol and 75% ethanol respectively; the filtrates from the three extractions were combined, and then concentrated under reduced pressure at 60° C. until there was no alcohol taste, to obtain an ethanol extract; Step 4: Extraction The ethanol extract was diluted to 1000 ml by adding pure water, and then extracted three times with petroleum ether, ethyl acetate and n-butanol respectively, using 500 ml of solvent each time; after the extraction was completed, the extracts of each solvent were combined and concentrated under reduced pressure at 60°C to obtain the white peony extract; finally, the extract was freeze-dried to obtain the white peony extract dry powder.

2. A method for preparing a white peony root extract for treating hyperuricemia, characterized in that: The following steps are involved: Step 1: Crush the herbs Take 200 grams of white peony root medicinal material or decoction pieces, and crush them using a wall-breaking machine to obtain white peony root ultrafine powder with destroyed cell walls; Step 2: Ether removal Use 3000 grams of ether solution, i.e. 15 times the dry weight, to soak the white peony superfine powder at room temperature for 24 hours, and assist with ultrasonic extraction once an hour during the period; after 24 hours, filter with a 400-mesh filter and retain the filter residue, extract the filter residue with ether twice under the same conditions, and filter and retain the filter residue; Step 3: Ethanol Extraction The residue was extracted with 1600 g, 1700 g and 1800 g of 95% ethanol, 85% ethanol and 75% ethanol respectively, the extraction temperature was 60°C, each extraction was 3 hours, and ultrasonic extraction was assisted; after three extractions, they were filtered with a 400-mesh filter, the three filtrates were combined, and concentrated under reduced pressure at 60°C until there was no alcohol taste, to obtain an ethanol extract; Step 4: Extraction The ethanol extract was diluted to 2 liters with pure water, and then extracted three times with petroleum ether, ethyl acetate and n-butanol, respectively, with 1 liter of solvent used for each extraction; the extracts from each extraction were combined, concentrated under reduced pressure at 60°C to obtain the white peony extract, and then freeze-dried to obtain the white peony extract dry powder.

3. A white peony root extract, characterized in that The white peony root extract is obtained by the preparation method of the white peony root extract for treating hyperuricemia as claimed in claim 1 or claim 2.

Images