Application of demethyleneberberine in preparation of medicine for preventing or treating gout

Abstract

The invention relates to the field of biological medicine, in particular to application of demethyleneberberine in preparation of medicine for preventing or treating gout. In-vitro experiments show that the demethyleneberberine hydrochloride can inhibit RAW264.7 cell inflammation caused by monosodium urate crystals; in-vitro experiments show that the traditional Chinese medicine composition can relieve rat gout-like symptoms induced by MSU and relieve hyperuricemia induced by oteracil potassium combined with hypoxanthine. The result shows that the demethyleneberberine hydrochloride (DMB) can inhibit gout induced by monosodium urate crystals in the aspects of resisting inflammation and regulating uric acid metabolism, and the application range of the demethyleneberberine hydrochloride is widened.

Description

Technical field:

[0001] The present invention relates to the field of biomedicine, and in particular to the application of desmethoxyberberine in the preparation of a drug for preventing or treating gout. Technical background:

[0002] Gout is a type of sterile arthritis caused by the deposition of monosodium urate crystals, affecting more than 55 million patients worldwide. Gout attacks can cause severe pain, which can occur in joints or periarticular tissues, mostly in the joints of the lower limbs, especially the foot joints, of which the first metatarsophalangeal joint is the most common site of onset. Gout attacks are accompanied by varying degrees of swelling and fever, which severely restrict the function of the affected area and make walking difficult. Some people with persistent hyperuricemia may also develop chronic gouty arthritis and structural joint damage.

[0003] The deposition of sodium urate crystals at the site of onset is a typical feature of gout, but its pathogenesis is complex and not yet fully understood. It is the result of the interaction between multiple immune cells and synovial cells, chondrocytes, and other periarticular tissue cells, mainly manifested as inflammatory cell infiltration, synovial cell proliferation, cartilage tissue damage, etc. In the tissues of the gout-affected area, there are usually high levels of inflammatory factors, such as IL-1β, which can induce cell recruitment and activation of dendritic cells, macrophages, and neutrophils. The increase in IL-1β may be related to the activation of the NLRP3 inflammasome pathway. Macrophages are stimulated by sodium urate crystals, and the expression of NLRP3 inflammasomes and adaptor protein ASC increases, which in turn promotes the secretion of IL-1β. The secretion of IL-1β is related to the recruitment of inflammatory cells such as neutrophils, and causes further inflammatory reactions. At the same time, this process is accompanied by oxidative damage.

[0004] At present, the treatment drugs for acute gout attacks mainly reduce the pain response of patients during the onset of the disease by inhibiting the inflammatory response. Commonly used drugs include prednisone, indomethacin, colchicine, etc. These drugs have adverse reactions to varying degrees. For example, indomethacin has more serious gastrointestinal adverse reactions, glucocorticoid drugs such as prednisone have the risk of inducing diabetes, and colchicine has a certain renal burden. Biological agents for the treatment of gout mainly target IL-1β, such as anakinra and rilonacept. However, most biological agents are expensive, and the treatment effects vary greatly among different patients. Therefore, it is very necessary to develop a new type of drug for the treatment of gout.

[0005] Demethinyl berberine is 9,10-dimethoxy-5,6-dihydroisoquinolin[2,1-b]isoquinolin-7-ium-2,3-dihydroxy. Its molecular formula is C19H18NO4+, and its molecular weight is 324.35. Its Chemical Abstracts Number (CAS) is 25459-91-0. Demethinyl berberine can form a variety of salts with inorganic or organic acids, such as chloride, sulfate, phosphate, bromide, iodide, citrate, fumarate, maleate, malate and succinate.

[0006] So far, there is no record or report on the therapeutic effect of desmethicone berberine on gout in the prior art. Summary of the invention:

[0007] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a compound for treating gout, and in particular to provide the use of desmethylenylberberine hydrochloride (DMB) as a drug for preventing and treating gout.

[0008] Application of desmethoxyberberine and its pharmaceutically acceptable salts or a pharmaceutical composition in the preparation of a drug for preventing or treating gout; the pharmaceutical composition contains desmethoxyberberine hydrochloride and its pharmaceutically acceptable salts and a pharmaceutically acceptable excipient

[0009]

[0010] Desmethinylberberine.

[0011] The application is characterized in that the demethylene berberine salt is hydrochloride, phosphate or sulfate.

[0012] Further, the present invention preferably uses desmethenylberberine hydrochloride;

[0013] Desmethoxyberberine hydrochloride, as shown in formula (I):

[0014]

[0015] The present invention establishes a gout animal model to observe the therapeutic effect of desmethoxyberberine hydrochloride on gout in rats. The research results show that desmethoxyberberine hydrochloride has a good therapeutic effect on gout.

[0016] The present invention induces WISTAR rats to establish a gout model by monosodium urate crystals (25mg / 200uL / ). One week before modeling, desmethoxy berberine hydrochloride begins to be administered, and rats are given 50mg / kg, 100mg / kg, and 200mg / kg by gavage, respectively. After one week of administration, monosodium urate crystals are injected into the ankle joint, and gout modeling begins. The results of the study show that different doses of desmethoxy berberine hydrochloride have different degrees of improvement on gout. After monosodium urate injection, the ankle joint of rats swells, the walking pattern changes, and the synovium is infiltrated by inflammatory cells. After treatment with desmethoxy berberine hydrochloride, the swelling of the ankle joint of rats is relieved, the walking pattern returns to normal, and the infiltration of synovial inflammatory cells is reduced. At the same time, desmethoxy berberine hydrochloride can significantly reduce the oxidative stress of the ankle joint tissue of rats, and the above results fully show that desmethoxy berberine hydrochloride can effectively treat gout.

[0017] The present invention induces RAW264.7 cells with monosodium urate crystals, adds different doses of berberine hydrochloride, extracts cell proteins, and uses Western BOT to detect the expression of NLRP3, IL-1β, and Caspase1 proteins.

[0018] The desmethoxyberberine hydrochloride product used in the present invention is prepared by conventional chemistry and separation and purification. This laboratory uses high performance liquid chromatography (HPLC) analysis and detection, and its purity is more than 99%, and after chemical method, mass spectrometry and nuclear magnetic resonance analysis and identification, it is shown that the chemical structure of the desmethoxyberberine hydrochloride product used in this laboratory is correct. This study shows that the purity and chemical structure of desmethoxyberberine hydrochloride meet the requirements for conducting in vivo and in vitro biological activity and pharmacological action research.

[0019] The present invention also relates to a pharmaceutical composition containing desmethoxyberberine hydrochloride as an active ingredient and conventional pharmaceutical excipients or adjuvants. Usually, the pharmaceutical composition of the present invention contains 0.1 to 95% by weight of desmethoxyberberine hydrochloride. The compound of the present invention generally contains 0.1 to 100 mg in a unit dosage form.

[0020] The pharmaceutical composition of the compounds of this invention can be prepared according to methods well known in the art. When used for this purpose, if necessary, the compounds of this invention can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to prepare suitable application forms or dosage forms that can be used as human medicine or veterinary medicine.

[0021] The compound of the present invention or the pharmaceutical composition containing the same can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum.

[0022] The administration route of the compound of the present invention or the pharmaceutical composition containing the compound can be injection, which includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and acupoint injection.

[0023] The dosage form for administration can be a liquid dosage form or a solid dosage form. For example, the liquid dosage form can be a true solution, a colloid, a microparticle dosage form, an emulsion dosage form, or a suspension dosage form. Other dosage forms include tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, freeze-dried powder injections, and the like.

[0024] The compound of the present invention can be prepared into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

[0025] For example, in order to formulate a unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0026] For example, in order to prepare the dosing unit into a capsule, the active ingredient of the present compound, desmethenylberberine hydrochloride, is mixed with the above-mentioned various carriers, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule. The active ingredient of the present compound can also be prepared into a microcapsule, suspended in an aqueous medium to form a suspension, or loaded into a hard capsule or prepared as an injection for use.

[0027] For example, the compound desmethyberberine hydrochloride of the present invention is made into an injection preparation, such as a solution, a suspension solution, an emulsion, or a freeze-dried powder injection. This preparation may be aqueous or non-aqueous, and may contain one or more pharmacologically acceptable carriers, diluents, adhesives, lubricants, preservatives, surfactants, dispersants, osmotic pressure regulators, solubilizers, and pH regulators. For example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitol lipids, fatty acid esters, etc. may be used for dilution. The osmotic pressure regulator may be sodium chloride, mannitol, glycerol, glucose, phosphate, acetate, etc.; the solubilizer or cosolvent may be poloxamer, lecithin, hydroxypropyl β-cyclodextrin, etc.; the pH regulator may be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc. When preparing a freeze-dried powder injection, mannitol, glucose, etc. may also be added as a support.

[0028] In addition, if necessary, colorants, preservatives, spices, flavoring agents, sweeteners or spices, etc. can also be added to the pharmaceutical preparations. These auxiliary materials are commonly used in the art.

[0029] The sterile media used in the present invention can be prepared by standard techniques well known to those skilled in the art. They can be sterilized, for example, by filtering through a bacterial filter, by adding a sterilizing agent to the composition, by irradiating the composition, or by heating the composition. They can also be made into sterile injectable media before use.

[0030] In order to achieve the purpose of medication and increase the therapeutic effect, the medicine or pharmaceutical composition of the present invention can be administered by any known method of administration. Of course, the route of administration used to implement the compounds of the present invention depends on the disease and the part to be treated. Because the pharmacokinetic and pharmacodynamic characteristics of the compounds of the present invention will be different to some extent, the most preferred method to obtain therapeutic concentrations in tissues is to gradually increase the dosage and monitor the clinical effect. For such gradually increasing therapeutic doses, the initial dose will depend on the route of administration.

[0031] For any particular patient, the dosage of the pharmaceutical composition of the compound of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, personality and individual response of the patient or animal, the route of administration, the number of administrations, the purpose of treatment, and therefore the therapeutic dose of the present invention may vary over a wide range. Depending on the condition of the patient being treated, some changes may have to be made to the dosage, and in any case, the appropriate dosage for the individual patient is determined by the physician.

[0032] The dosage refers to the weight of the compound excluding the weight of the carrier (when a carrier is used). Generally speaking, the dosage of the pharmaceutical ingredients used in the present invention is well known to those skilled in the art. The actual drug dosage contained in the final preparation of the compound composition of the present invention can be appropriately adjusted to achieve the requirement of its therapeutically effective amount and complete the prevention or treatment purpose of the present invention. It can be administered in a single dosage form or divided into several, such as two, three or four dosage forms; this is limited by the clinical experience of the administering physician and the administration regimen including the use of other treatment methods. The compound or composition of the present invention can be taken alone, or used in combination with other therapeutic drugs or symptomatic drugs and the dosage can be adjusted.

[0033] Beneficial effects:

[0034] Gout requires long-term medication, but commonly used therapeutic drugs such as colchicine, benzbromarone, and allopurinol have significant efficacy, but due to large side effects, long-term medication causes serious consequences. Therefore, there is an urgent need for drugs with low side effects that can effectively relieve gout symptoms. The present invention first discovered that desmethyberberine hydrochloride has a therapeutic effect on gout, which not only adds new indications for the compound, but also provides new ideas for the development and research of drugs for the treatment of gout.

[0035] Specifically:

[0036] The present invention found that DMB can act on the NLRP3 inflammasome pathway to exert anti-inflammatory activity and thus treat MSU-induced gout. Among them, DMB can reduce plasma protein extravasation, reduce inflammatory cell infiltration, and improve the effect of inflammation; it can increase GSH levels, reduce MDA, and increase SOD levels, and has an antioxidant effect, that is, it can improve the oxidative damage of ankle joint tissue cells. DMB can reduce the activity of XOD in serum and liver, thereby reducing uric acid production and alleviating hyperuricemia; DMB can regulate the transcription level, and at the protein level, DMB can regulate the expression of URAT1 in the kidney.

[0037] Compared with the commonly used anti-inflammatory drugs, DMB has a better ability to relieve ankle swelling caused by MSU modeling than BBR, and there is no significant difference between the high-dose DMB group and colchicine in the treatment of ankle swelling. At 24h, there is no significant difference between the high-dose DMB group and colchicine in the treatment of gout-induced pain, and the treatment effect is better than BBR. At 48h, there is no significant difference between the medium- and high-dose DMB groups and colchicine in the treatment of gout-induced pain, and the treatment effect is better than BBR. The high-dose DMB group has a better ability to relieve gait abnormalities caused by MSU modeling than BBR, and there is no significant difference between the high-dose DMB group and colchicine in the treatment of gait abnormalities. The DMB group has a better ability to relieve hyperuricemia caused by oxonate potassium combined with hypoxanthine modeling than BBR, and the high-dose DMB group has a better ability to relieve hyperuricemia caused by oxonate potassium combined with hypoxanthine modeling than benzbromarone. DMB can improve renal dysfunction caused by hyperuricemia and restore kidney morphology. The effects of medium and high doses of DMB are equivalent to those of benzbromarone, and the side effects are less than allopurinol.

[0038] In summary, the DMB of the present invention has an anti-gout effect, among which there is no significant difference in analgesia compared with colchicine, the ability to relieve hyperuricemia is better than benzbromarone, and the improvement of renal dysfunction and restoration of kidney morphology are equivalent to benzbromarone; and DMB has low side effects and is equivalent to the efficacy of commonly used drugs, and has obvious advantages for long-term medication substitution. Description of the drawings:

[0039] Figure 1 , the effect of DMB on the ankle swelling of MSU-induced gout rats;

[0040] Figure 2 , the effect of DMB preventive treatment on ankle pain perception in MSU-induced gout rats;

[0041] Figure 3 , the effect of DMB preventive treatment on gait of MSU-induced gout rats;

[0042] Figure 4 , DMB preventive treatment of GSH in ankle joints of MSU-induced gout rats;

[0043] Figure 5 , DMB preventive treatment of MSU-induced gout rat ankle joint MDA results;

[0044] Figure 6 , DMB preventive treatment of SOD in ankle joints of MSU-induced gout rats;

[0045] Figure 7, DMB preventive treatment of MSU-induced gout rats ankle joint plasma protein permeability results;

[0046] Figure 8 , HE staining of ankle synovium in rats with MSU-induced gout treated with DMB for prevention (×100);

[0047] Fig. 9 , the effect of DMB administration on the expression of inflammasome proteins in MSU-induced RAW264.7 cells; wherein, A is the Western blot result, B is the grayscale analysis of NLRP3, C is the grayscale analysis of Caspase1, and D is the grayscale analysis of IL-1β.

[0048] Fig.10 , the effect of DMB on the blood uric acid concentration in rats with hyperuricemia induced by potassium oxonate combined with hypoxanthine;

[0049] Fig.11 , the effect of DMB on renal function and morphology in rats with hyperuricemia induced by potassium oxonate combined with hypoxanthine; A is serum creatinine concentration, B is serum urea nitrogen concentration, and C is a picture of kidney morphology;

[0050] Fig.12 Effects of DMB on serum and liver xanthine oxidase activity in rats with hyperuricemia induced by potassium oxonate combined with hypoxanthine; A is serum xanthine oxidase activity, B is liver xanthine oxidase activity

[0051] Fig.13 Effects of DMB treatment on mRNA transcription and protein expression levels of uric acid transport-related proteins in rats with hyperuricemia induced by potassium oxonate combined with hypoxanthine A is the RT-qPCR result of URAT1, OAT1, Glut9, and ABCG2, and B is the Western blot result of URAT1 / β-actin Specific implementation method:

[0052] The following examples may help those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0053] DMB: Desmethylberberine hydrochloride

[0054] IL-1β: interleukin 1-beta

[0055] NLRP3: pyrin domain-containing NOD-like receptor family 3

[0056] Caspase1: Cysteine-containing aspartate protease

[0057] MSU: Monosodium urate crystals

[0058] BBR: Berberine

[0059] COL: Colchicine

[0060] IG: intragastric administration

[0061] CMC-Na: Sodium carboxymethyl cellulose

[0062] MDA: Malondialdehyde

[0063] GSH: Glutathione

[0064] SOD: Superoxide dismutase

[0065] RT-qPCR: Real-time quantitative PCR

[0066] Western-Blot: Immuno-protein blot

[0067] BEN: Benzbromarone

[0068] ALLO: Allopurinol

[0069] SCR: serum creatinine

[0070] XOD: Xanthine oxidase

[0071] URAT1: urate transporter 1

[0072] OAT1: Organic anion transporter 1

[0073] GLUT9: glucose transporter 9

[0074] ABCG2: ATP-binding cassette transporter G2

[0075] PBS: Phosphate buffered saline

[0076] HE: Hematoxylin-eosin staining

[0077] Example 1. DMB preventive treatment can alleviate ankle swelling in MSU-induced gout rats

[0078] Methods: 25mg / 200uL MSU crystals were injected into the ankle joints of rats to prepare a rat gout model for evaluating the efficacy of DMB. Male WISTAR rats, weighing 180-200g, were randomly divided into 7 groups, 5 rats in each group, including normal control group, MSU model group, DMB low-dose group (50mg / kg, IG), DMB medium-dose group (100mg / kg, IG), DMB high-dose group (200mg / kg, IG), BBR group (100mg / kg, IG), and colchicine group (2mg / kg, IG). DMB, BBR, and colchicine were dissolved in 0.5% CMC-Na solution. Rats in the drug-treated group were given the drug by gavage once a day, and rats in the blank group and MSU model group were given 0.5% CMC-Na solution by gavage. After 7 days of preventive administration, the rats were anesthetized with isoflurane and injected with 25 mg / 200 uL MSU crystals into the ankle joints, except for the blank group, which was injected with 200 uL PBS solution. The ankle circumference of the rats was measured at 4h, 24h, and 48h after modeling.

[0079] Results: Figure 1 As shown in the results, the DMB group was better than BBR in alleviating ankle swelling caused by MSU modeling, and the therapeutic effect of the high-dose DMB group on ankle swelling was not significantly different from that of colchicine.

[0080] Example 2. DMB preventive treatment can relieve ankle pain in MSU-induced gout rats

[0081] Methods: 4h, 24h, and 48h after MSU modeling, an electronic dynamometer equipped with a blunt probe was used to stimulate the fixed position of the rat modeling site. The rat contracted its hind limbs due to pain reflex. At this time, the dynamometer automatically recorded the maximum thrust value, which was recorded as the maximum tolerance of the rat's pain perception. The average value of three measurements was taken.

[0082] Results: Figure 2 As shown, at 24 hours, there was no significant difference in the therapeutic effect of the high-dose DMB group on gout-induced pain compared with colchicine, and the therapeutic effect was better than BBR. At 48 hours, there was no significant difference in the therapeutic effect of the medium- and high-dose DMB groups on gout-induced pain compared with colchicine, and the therapeutic effect was better than BBR.

[0083] Example 3. DMB preventive treatment can improve gait abnormalities in MSU-induced gout rats

[0084] Methods: 48 hours after MSU modeling, the VisuGait animal gait analysis system was used to analyze the gait changes of rats. The rats were placed on a glass track, and the light refracted by the fluorescent lamp was recorded by a camera to obtain the footprint area of ​​the rats.

[0085] Results: Figure 3 As shown in the results, the ability of the high-dose DMB group to alleviate gait abnormalities caused by MSU modeling was better than that of BBR, and there was no significant difference between the high-dose DMB group and colchicine in the therapeutic effect on gait abnormalities.

[0086] Example 4. DMB can improve the changes in GSH levels in ankle joint tissues of gout rats induced by MSU

[0087] Methods: 48 hours after MSU modeling, rats were killed by cervical dislocation under isoflurane anesthesia, and ankle joint tissues were removed and ground with physiological saline on an ice bath to prepare 10% ankle joint tissue homogenate. The supernatant was taken after centrifugation, and the GSH level in ankle joint tissues was determined according to the instructions of Nanjing Jiancheng kit.

[0088] Results: GSH has an antioxidant effect and is mainly used to measure oxidative damage. Gout is often accompanied by oxidative damage while the inflammatory response occurs. Compared with the control group, the GSH level in the ankle joint tissue of the rats in the model group was reduced, indicating that the oxidative damage of the ankle joint tissue of the gout rats was more serious, and the high-dose DMB administration group (200 mg / kg, IG) could significantly increase the GSH content in the lung tissue. Figure 4 As shown in the figure, the research results show that DMB can increase the GSH level of gout rats and has the effect of anti-oxidative damage.

[0089] Example 5. DMB can reduce the level of MDA in ankle joint tissue of rats with gout induced by MSU

[0090] Methods: 48 hours after MSU modeling, rats were killed by cervical dislocation under isoflurane anesthesia, and ankle joint tissues were removed and ground with physiological saline on an ice bath to prepare 10% ankle joint tissue homogenate. The supernatant was taken after centrifugation, and the MDA level in ankle joint tissues was determined according to the instructions of Nanjing Jiancheng kit.

[0091] Results: MDA is a lipid peroxide formed by oxygen free radicals in the body attacking polyunsaturated fatty acids in the biomembrane. The measurement of MDA can reflect the degree of lipid peroxidation in the body and indirectly reflect the degree of cell damage. Compared with the control group, the MDA level in the ankle joints of mice in the model group increased, while the DMB administration group significantly reduced MDA compared with the model group. Figure 5 As shown, DMB has the effect of reducing MDA and can improve the oxidative damage of ankle joint tissue cells.

[0092] Example 6. DMB can improve the changes in SOD levels in ankle joint tissues of gout rats induced by MSU

[0093] Methods: 48 hours after MSU modeling, rats were killed by cervical dislocation under isoflurane anesthesia, and ankle joint tissues were removed and ground with physiological saline on an ice bath to prepare 10% ankle joint tissue homogenate. After centrifugation, the supernatant was taken and the SOD level in ankle joint tissues was determined according to the instructions of Nanjing Jiancheng kit.

[0094] Results: SOD plays a vital role in the body's oxidation and antioxidant balance. Gout is often accompanied by oxidative damage while the inflammatory response occurs. Compared with the control group, the SOD level in the ankle joint tissue of the model group rats was reduced, indicating that the oxidative damage of the ankle joint tissue of the gout rats was more serious, and the DMB administration group could significantly increase the SOD content in the ankle joint tissue. Figure 6 As shown in the figure, the research results showed that DMB can increase the SOD level of gout rats and has the effect of anti-oxidative damage.

[0095] Example 7. DMB can improve the plasma protein permeability of ankle joints in MSU-induced gout rats

[0096] Methods: 48 hours after MSU modeling, rats were killed by cervical dislocation under isoflurane anesthesia. The Achilles tendon and ankle synovium of the rats were cut to expose the joint cavity. 10uL PBS buffer was injected into the joint cavity with a pipette. After flushing the ankle joint cavity, the washing fluid was recovered and the protein concentration was measured.

[0097] Results: When inflammation occurs, the permeability of the surrounding vascular walls changes, leading to extravasation of plasma proteins. Compared with the control group, the protein concentration of the ankle joint cavity washing fluid of the model group rats increased significantly, indicating that inflammation has a more serious impact on the surrounding tissues, while the DMB administration group can significantly reduce the protein concentration of the ankle joint cavity washing fluid. Figure 7 As shown, the results of the study showed that DMB can reduce plasma protein extravasation and has the effect of improving inflammation.

[0098] Example 8. DMB can improve ankle synovial injury in gout rats induced by MSU

[0099] Methods: 48 hours after MSU modeling, rats were killed by cervical dislocation under isoflurane anesthesia, and ankle joint tissues were removed and fixed in 10% neutral formalin for 72 hours, then decalcified and cut longitudinally along the ankle joint, embedded in paraffin, sliced, and stained with HE to observe pathological changes.

[0100] Results: HE staining results showed that compared with the normal control group, the inflammatory cell infiltration in the synovium of the model group rats increased significantly, and the synovial morphology changed. However, the inflammatory cell infiltration in the DMB group decreased, such as Figure 8 shown.

[0101] Example 9. DMB acts on the NLRP3 inflammasome pathway to exert anti-inflammatory activity

[0102] Methods: RAW264.7 cells were used as the research object. DMB 10, 20, 40uM was given to the cell culture medium for pretreatment for 2h, and then MSU crystals with a final concentration of 600ug / mL were added for 24h and the cells were collected. The model group only added 600ug / mL MSU crystals, and the blank group was not treated. Cell proteins were extracted by RIPA method, and the effect of DMB on NLRP3 inflammasome pathway was detected by Western Blot method.

[0103] Results: Western Blot results showed Fig. 9 ,DMB can inhibit the protein expression of NLRP3, CASPASE1, and IL-1β. It is speculated that DMB can act on the NLRP3 inflammasome pathway to exert anti-inflammatory activity and thus treat MSU-induced gout.

[0104] Example 10. DMB can alleviate hyperuricemia in rats induced by potassium oxonate and hypoxanthine

[0105] Methods: Rats were intraperitoneally injected with 100 mg / kg potassium oxonate and intragastrically administered with 500 mg / kg hypoxanthine to prepare a rat hyperuricemia model for evaluating the efficacy of DMB. Male WISTAR rats, weighing 180-200 grams, were randomly divided into 8 groups, 5 rats in each group, including normal control group, model group, DMB low-dose group (50 mg / kg, IG), DMB medium-dose group (100 mg / kg, IG), DMB high-dose group (200 mg / kg, IG), BBR group (100 mg / kg, IG), benzbromarone group (10 mg / kg, IG), and allopurinol group (40 mg / kg, IG). DMB, BBR, benzbromarone, and allopurinol were dissolved in 0.5% CMC-Na solution. Every morning, the model group and each drug-treated group were intraperitoneally injected with potassium oxonate and gavaged with hypoxanthine, while the control group was gavaged and intraperitoneally injected with 0.5% CMC-Na. Every afternoon, the drug-treated group rats were gavaged with 0.5% CMC-Na solution, and the blank group rats were gavaged with 0.5% CMC-Na solution. After 7 days of modeling and drug administration, the rats were anesthetized with isoflurane, blood was collected from the eye sockets, and serum was obtained after centrifugation. The uric acid level in the serum was determined according to the instructions of the Nanjing Jiancheng kit.

[0106] Results: Fig.10 As shown, the ability of relieving hyperuricemia caused by potassium oxonate combined with hypoxanthine modeling in the DMB administration group was better than that of BBR, and the ability of relieving hyperuricemia caused by potassium oxonate combined with hypoxanthine modeling in the high-dose DMB group was better than that of benzbromarone.

[0107] Example 11. DMB can alleviate renal dysfunction in hyperuricemia rats

[0108] Methods: Seven days after the hyperuricemia model was established, blood was collected from the eye sockets under isoflurane anesthesia, serum was obtained after centrifugation, and kidney tissue was removed after cervical dislocation. Serum creatinine and urea nitrogen levels were determined according to the instructions of Nanjing Jiancheng kit.

[0109] Results: Hyperuricemia is accompanied by uric acid deposition in the kidneys, causing kidney diseases such as kidney stones, obstruction, interstitial nephritis, acute or chronic renal failure, etc. Creatinine is metabolized from creatine phosphate in the muscles, and its content is usually stable. The content of free creatinine mainly depends on its excretion rate. Urea is the main end product of protein metabolism in the human body, and urea nitrogen in the blood is mainly excreted from the body through the kidneys. Therefore, the determination of serum creatinine and urea nitrogen concentrations can be used to evaluate renal function. Fig.11 As shown, DMB can improve renal dysfunction caused by hyperuricemia and restore kidney morphology. The effects of medium and high doses of DMB are equivalent to those of benzbromarone, and the side effects are less than allopurinol.

[0110] Example 12. DMB can regulate xanthine oxidase activity to relieve hyperuricemia

[0111] Methods: Seven days after the hyperuricemia model was established, blood was collected from the eye sockets under isoflurane anesthesia, and serum was obtained after centrifugation. After the animals were killed by cervical dislocation, the liver tissue was removed, ground with physiological saline on an ice bath, and a 10% liver homogenate was prepared, and the supernatant was obtained after centrifugation. According to the instructions of the Nanjing Jiancheng kit, the activity of XOD in serum and liver was determined.

[0112] Results: Xanthine oxidase is a key enzyme in the synthesis of uric acid. Inhibiting the activity of this enzyme can achieve the purpose of treating hyperuricemia. Fig.12 As shown in the results, DMB can reduce the activity of XOD in serum and liver, thereby reducing uric acid production and alleviating hyperuricemia.

[0113] Example 13. DMB can regulate the expression of uric acid transporter

[0114] Methods: Seven days after the hyperuricemia model was established, the rats were killed by cervical dislocation under isoflurane anesthesia, and the renal tissues were removed. The mRNA transcription level and protein expression level of urate transporter in renal tissues were detected by RT-PCR and Western Blot.

[0115] Results: Fig.13 As shown, RT-qPCR method showed that DMB can regulate the transcription level, while at the protein level, DMB can regulate the expression of URAT1 in the kidney.

Claims

1. Use of desmethoxyberberine and its pharmaceutically acceptable salt or a pharmaceutical composition in the preparation of a drug for preventing or treating gout; the pharmaceutical composition contains desmethoxyberberine hydrochloride and its pharmaceutically acceptable salt and a pharmaceutically acceptable excipient; the structural formula of desmethoxyberberine is as follows 2. The use according to claim 1, characterized in that: The salt of desmethyberberine is chloride, sulfate, phosphate, bromide, iodide, citrate, fumarate, maleate, malate or succinate.

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