Application of xanthohumol in preparation of drugs for reducing uric acid and resisting gout

Abstract

The invention relates to the technical field of medicines, in particular to application of an active ingredient xanthohumol (XAN) derived from a traditional Chinese medicine humulus lupulus in preparation of medicines for reducing uric acid and resisting gout, and the xanthohumol (XAN) can be prepared into various oral preparations, injection preparations and external preparations with the assistance of pharmaceutically acceptable auxiliary materials and has good development and application prospects. The inventor finds that the xanthohumol has relatively strong effects of reducing blood uric acid, resisting inflammation, resisting bone damage and the like, and the mechanism of the effect of reducing the blood uric acid is that the activity of xanthine oxidase is inhibited so as to reduce the generation of uric acid, and the renal function of an organism is improved so as to enhance uric acid excretion; according to the anti-inflammatory effect, the inflammatory reaction is relieved mainly by down-regulating the levels of inflammatory factors such as IL-1beta, IL-6, TNF alpha and PEG2; the mechanism for resisting gout bone damage is realized through the fact that RANKL / OPG / RANK signal pathway participates in bone metabolism regulation.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine, and particularly relates to the application of xanthohumol in the preparation of drugs for reducing uric acid and treating gout. Background Art

[0002] Gout is a metabolic syndrome caused by disordered purine metabolism in the body. Excessive production or reduced excretion of uric acid (UA) leads to elevated blood uric acid levels (>420 μmol / L). The deposition of monosodium urate (MSU) crystals in joints and their surrounding connective tissues triggers an inflammatory disease. Clinical findings show that long-term high blood uric acid (HUA) levels can induce gout. In the late stage of gout, obvious bone erosion can be seen, which can cause osteoporosis, osteoarthritis, and increase the risk of fractures. According to the "Chinese Guidelines for the Diagnosis and Treatment of Hyperuricemia and Gout (2019)" newly promulgated by the Endocrinology Branch of the Chinese Medical Association (hereinafter referred to as the "Guidelines"), "When blood uric acid exceeds its saturation in blood or tissue fluid, sodium urate crystals can form and deposit locally in joints, inducing local inflammatory reactions and tissue damage, that is, gout." The "Guidelines" also state that "In asymptomatic hyperuricemia patients, the diagnosis of subclinical gout can be made if urate crystals deposition and / or gouty bone erosion are found by joint ultrasound, dual-energy CT, or X-ray." Thus, the onset of gout is mainly divided into hyperuricemia and gouty arthritis. Among them, hyperuricemia is often characterized by excessive elevation of blood uric acid levels; while gouty arthritis is mainly manifested by repeated swelling, pain, heat, and dysfunction of the affected joints, with the deposition of MSU in joints as the typical pathological feature. It can also involve kidney damage or serious situations such as joint deformity, and increase the risk of other metabolic diseases, such as coronary heart disease, obesity, metabolic syndrome, and bone metabolism disorders (such as osteoporosis, bone erosion), seriously affecting physical health. According to the pathogenesis of gout, currently commonly used clinical treatment drugs include: (1) Uric acid production inhibitors: the xanthine oxidase inhibitor allopurinol (ALLO); (2) Uric acid excretors: benzbromarone; (3) Anti-inflammatory and analgesic drugs: non-steroidal anti-inflammatory drugs, colchicine, and glucocorticoids, etc. Although these drugs can partially relieve gout symptoms, they still face adverse reactions such as liver and kidney toxicity, bone marrow transplantation, and fatal hypersensitivity reactions, severely restricting their clinical application. For example, the side effects of xanthine oxidase inhibitors include: (1) Liver function disorders: fever, rash, loss of appetite, general fatigue, yellowing of the skin, yellowing of the eyes, and skin itching, etc.; (2) Aplastic anemia: sore throat, fever, red spots on hands and feet, and easy bleeding of the skin; (3) Granulocytopenia: high fever accompanied by chills and a sore throat. The use of uric acid excretors is limited in patients with hyperuricemia accompanied by renal insufficiency, and there are pain points in clinical medication such as increased safety risks in combined use. Common side effects of colchicine include gastrointestinal reactions such as nausea, vomiting, abdominal pain, and diarrhea, and it also has certain damage to the kidneys. In severe cases, oliguria, hematuria, etc. may occur. It can also cause muscle weakness, numbness, and granulocyte deficiency, and in severe cases, aplastic anemia may occur. Non-steroidal anti-inflammatory drugs can cause upper abdominal discomfort, leading to side effects such as nausea and vomiting, as well as liver, nervous system toxicity, and adverse effects on the cardiovascular and cerebrovascular systems.The side effects of glucocorticoids include central obesity, moon face, buffalo hump, hirsutism, acne, hypertension, diabetes, hyperlipidemia, hypokalemia, osteoporosis, etc. It can also induce or exacerbate infections, spread potential foci in the body, and induce or exacerbate gastric and duodenal ulcers. Therefore, developing drugs that can reduce blood uric acid while also treating gouty arthritis and its associated complications such as bone metabolism disorders or renal insufficiency has broad market prospects.

[0003] Xanthohumol (XAN) is an isoprenyl flavonoid compound isolated from the female inflorescences of Humulus lupulus, a characteristic resource plant in Xinjiang. Its chemical structure is shown in Formula I. Research has found that xanthohumol has good antioxidant, anti-tumor, antibacterial, anti-osteoporosis and other activities, but there are no reports on its effects of reducing blood uric acid and treating gout.

[0004] Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide the use of xanthohumol in the preparation of drugs for reducing blood uric acid and treating gout.

[0006] The inventors have been engaged in the research of traditional Chinese medicine pharmacology and resource development for a long time. During the screening of the anti-gout activities of natural products, it was repeatedly confirmed that the active ingredient xanthohumol derived from traditional Chinese medicine Humulus lupulus has strong effects of reducing blood uric acid, anti-inflammatory, protecting renal function, anti-bone injury, and regulating bone metabolism. On this basis, the mechanism of its action in reducing blood uric acid and treating gout was clarified.

[0007] The xanthohumol described in the present invention can be extracted from plants or obtained by purchase, and has the advantages of convenient source and high safety. Using it in the preparation of drugs for reducing blood uric acid and treating gout has broad prospects.

[0008] The xanthohumol described in the present invention, supplemented with pharmaceutically acceptable excipients, can be prepared into various oral, injection, and topical preparations. Brief Description of the Drawings

[0009] Figure 1 Showing the effect of xanthohumol on blood uric acid levels in rats (n = 8).

[0010] Figure 2 Showing the effect of xanthohumol on blood XOD activity in rats (n = 8).

[0011] Figure 3 Showing the effect of xanthohumol on the CRE level of renal function indexes in rats (n = 8).

[0012] Figure 4 Showing the effect of xanthohumol on the BUN level of renal function indexes in rats (n = 8).

[0013] Figure 5 Show the effects of xanthohumol on bone metabolism indexes in rats (n = 8).

[0014] Figure 6 Show the effects of xanthohumol on the RANKL / OPG signaling pathway in rats (n = 8).

[0015] Figure 7 Show the effects of xanthohumol on the swelling degree of the ankle joints in rats (n = 10).

[0016] Figure 8 Show the effects of xanthohumol on the levels of inflammatory factors in rats (n = 10).

[0017] Figure 9 Show the effects of xanthohumol on the morphology of bone tissue adjacent to joints and bone microstructural parameters (n = 10).

[0018] Figure 10 Show the effects of xanthohumol on the pathological histomorphological evaluation of joints (n = 10).

[0019] Figure 11 Show the effects of xanthohumol on the expression of proteins related to bone metabolism (n = 10).

[0020] Figure 12 Show the effects of xanthohumol on the expression of proteins related to bone metabolism signaling pathways (n = 10).

[0021] Figure 13 Show the effects of xanthohumol on the proliferation of osteoblasts (n = 10).

[0022] Figure 14 Show the effects of xanthohumol on BCIP / NBT staining and ALP activity of osteoblasts (n = 10).

[0023] Figure 15 Show the effects of xanthohumol on the staining of bone mineralized nodules and mineralization levels of osteoblasts (n = 10).

[0024] Figure 16 Show the effects of xanthohumol on the expression of proteins related to the bone metabolism pathway of osteoblasts (n = 3).

[0025] Figure 17 Show the effects of xanthohumol on the proliferation and differentiation of osteoclasts (n = 10).

[0026] Figure 18 Show the effects of xanthohumol on TRAP staining and its activity of osteoclasts (n = 10).

[0027] Figure 19 Show the effects of xanthohumol on the expression of proteins related to the bone metabolism pathway of osteoclasts (n = 3). Detailed implementation mode

[0028] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. These embodiments should be understood as only for illustrating the present invention and not for limiting the protection scope of the present invention. After reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0029] Example 1 - Study on the regulatory effect of xanthohumol on blood uric acid level and bone metabolism in hyperuricemia (HUA) rats.

[0030] The xanthohumol used in the embodiments of the present invention is a standard product, purchased from Dingrui Chemical Industry (Shanghai) Co., Ltd. (batch number: 8065102). The positive drug allopurinol is purchased from TMstandard (batch number: 78683). The experimental animals used are SPF-grade 6-week-old male Wistar rats, with a body weight of 150±10 g, provided by Shanghai Slack Experimental Animal Co., Ltd., certificate number: 20220004024235; the animal production license number is SCXK (Shanghai) 2022-0004. Forty-eight rats were randomly divided into 6 groups: blank control group (CON), model group (MOD), allopurinol (ALLO) group, low-dose xanthohumol group (XAN-L), medium-dose xanthohumol group (XAN-M), and high-dose xanthohumol group (XAN-H), with 8 rats in each group. The model was established by intragastric administration of potassium oxonate (200 mg / kg / d) combined with hypoxanthine (250 mg / kg / d). The positive drug group was given ALLO 20 mg / kg / d by intragastric administration, and each dose group of xanthohumol was given 5 mg / kg / d, 15 mg / kg / d, and 45 mg / kg / d by intragastric administration, respectively, for continuous intervention for 14 days. During the experiment, blood was taken from the orbital cavity on the 3rd, 7th, 10th, and 14th days, and relevant indicators were detected and analyzed. The successful establishment of the model was based on the significant increase in the blood uric acid (UA) level in the MOD group (P<0.05).

[0031] 1. Effect of xanthohumol on blood uric acid level.

[0032] The blood UA concentrations of rats in each xanthohumol treatment group decreased to varying degrees ( Figure 1 A, B), and except for the XAN-L group having no significant statistical difference from the model group on the 3rd day ( Figure 1 C), there were significant differences in each dose group at other time points ( Figure 1 C, D, E, F, P<0.01 for all). In addition, there was no significant statistical difference in the effect of reducing blood uric acid between the XAN-M, XAN-H, and ALLO groups on the 14th day ( Figure 1 F). Thus, it can be seen that XAN has a good effect of reducing blood uric acid.

[0033] 2. Effects of xanthohumol on xanthine oxidase (XOD).

[0034] The XOD activities of rats in the XAN treatment groups all decreased significantly ( Figure 2 A, B), and it was statistically significant ( Figure 2 C, D, E, F, P < 0.01 for all), indicating that it could effectively inhibit the synthesis of UA in the body. In addition, starting from the 7th day, there was no significant statistical difference in the effect of inhibiting XOD activity among the three XAN dose groups and the ALLO group ( Figure 2 D, E, F). Thus, it can be seen that XAN can significantly inhibit XOD activity, reduce UA synthesis, and thus play a role in reducing UA.

[0035] 3. Effects of xanthohumol on renal function indexes.

[0036] The CRE and BUN levels of rats in each XAN treatment group all decreased to varying degrees ( Figure 3 , P < 0.01; Figure 4 , P < 0.05); and the effect of reducing CRE level in the XAN-H group was equivalent to that in the ALLO group ( Figure 3 F, P > 0.05), and there was no significant statistical difference from the CON group ( Figure 3 C, D, E, F, P > 0.05); while its effect of reducing BUN level was more stable and persistent ( Figure 4 , P < 0.01 for all). All these indicate that XAN can effectively reduce the CRE and BUN levels in HUA rats, prevent kidney damage induced by HUA, and have a certain renal protective effect. Thus, it can be seen that XAN can reduce the UA level in HUA rats by protecting and repairing the renal function of HUA rats with renal insufficiency and promoting the excretion of accumulated UA in the body.

[0037] 4. Effects of xanthohumol on bone metabolism indexes.

[0038] The ALP activities of rats in each XAN treatment group ( Figure 5 A, P < 0.01, P < 0.001) and Runx2 levels ( Figure 5 B, P < 0.01) were significantly up-regulated. In addition, as shown in Figure 5 C, each XAN treatment group could down-regulate the expression of CTSK (P < 0.01, P < 0.001), and the reversed CTSK levels in the medium and high XAN dose groups were significantly lower than those in the CON group (P < 0.05, P < 0.001). These indicate that XAN can not only effectively regulate the ALP activity in HUA rats, up-regulate the expression of the bone transcription factor Runx2, and promote bone formation and bone repair; but also down-regulate the expression of the bone destruction protein CTSK, inhibit bone loss, and prevent bone damage induced by HUA, and have a good bone protective effect.

[0039] 5. Effects of xanthohumol on bone metabolism signaling pathways.

[0040] Each XAN treatment group could significantly inhibit the secretion of RANKL ( Figure 6 A, P < 0.001), and the high-dose XAN group could significantly improve the expression of OPG ( Figure 6 B, P < 0.01), thereby inhibiting RANKL-induced osteoclast differentiation and effectively promoting bone repair. In addition, the XAN treatment groups could downregulate the RANKL / OPG ratio to varying degrees ( Figure 6 C, P < 0.05, P < 0.01, P < 0.001), and there was no significant statistical difference in the reversed ratio between the medium- and high-dose XAN groups and the CON group ( Figure 6 C, P > 0.05). Thus, it can be seen that XAN can not only effectively regulate the secretion of RANKL and the expression of OPG in HUA rats, correct the bone metabolism imbalance caused by HUA, but also reverse the RANKL / OPG ratio, intervene in the RANKL / OPG signaling pathway, and effectively regulate bone homeostasis.

[0041] The above shows that this example confirms that xanthohumol is a multi-target and multi-path anti-hyperuricemia drug, which can not only be used to prepare relatively ideal and safe uric acid-lowering drugs, but also be used as a bone metabolism regulator or renal function protector to prepare drugs for treating acute and chronic hyperuricemia with renal insufficiency and bone metabolism disorders.

[0042] Example 2 - Study on the effect of xanthohumol against gouty arthritis (GA) in rats.

[0043] In the embodiments of the present invention, the positive drug etoricoxib (ETO) used was purchased from Merck Sharp (batch number: H20110036). The experimental animals were SPF-grade 8-week-old male Wistar rats, weighing 170±10 g, provided by Shanghai Slake Experimental Animal Co., Ltd., with the certificate number: 20220004024235; the animal production license number was SCXK (Shanghai) 2022-0004. Fifty rats were randomly divided into 5 groups: blank control group (CON), model group (MOD), positive drug (ETO) group, xanthohumol low-dose group (XAN-L), and xanthohumol high-dose group (XAN-H), with 10 rats in each group. MSU was repeatedly injected into the ankle joint cavity for modeling, twice a week, and after each modeling, the positive drug ETO was given by gavage at a dose of 18 mg / kg, while the xanthohumol groups at each dose were given by gavage at 15 mg / kg and 45 mg / kg respectively every day for continuous intervention for 10 weeks. During the experiment, the ankle joint swelling degree was measured weekly. After the experiment, serum samples were collected to detect inflammatory factors, and the diseased ankle joints were collected for Micro-CT scanning to observe the changes in adjacent bone tissues and bone microstructure. HE, safranin-fast green (SO), and TRAP staining were used for histological evaluation, and immunohistochemistry and immunofluorescence double staining were used to investigate bone metabolism-related proteins and signal pathway-related proteins for analysis.

[0044] 1. Effect of xanthohumol on ankle joint swelling degree.

[0045] As Figure 7 shown, after repeated injection of MSU into the joint cavity, the average level of ankle joint swelling in the MOD group gradually increased from the baseline to 14.07% over weeks, while it was 2.63% in the ETO group, 8.43% in the XAN-L group, and 5.36% in the XAN-H group. The results showed that XAN could significantly reduce ankle joint swelling (P<0.05). In addition, the improvement degree of ankle joint swelling in the XAN-H group was better than that in the XAN-L group (P<0.05), and it was comparable to the effect of the positive drug.

[0046] 2. Effect of xanthohumol on the levels of inflammatory factors.

[0047] As Figure 8 shown, compared with the CON group, the levels of inflammatory cytokines such as IL-1β, IL-6, TNF-α, and PGE2 in the serum of the MOD group were significantly increased (P<0.01 for all). The results showed that the inflammatory response was activated after injection of MSU into the ankle joint cavity. At the same time, the results showed that after gavage with XAN, the levels of inflammatory cytokines such as IL-1β, IL-6, TNF-α, and PGE2 could be significantly reduced (P<0.01 for all); and there was no significant difference between the XAN group and the ETO group. Therefore, we believe that XAN can effectively reduce the inflammatory response caused by MSU and is comparable to the effect of the ETO positive drug.

[0048] 3. Effects of xanthohumol on the morphology of bone tissue adjacent to joints and bone microstructural parameters.

[0049] As Figure 9 shown, XAN significantly improved the bone mass loss and bone microstructure damage induced by MSU ( Figure 9 A). Compared with the MOD group, the XAN-H treatment group significantly increased BMD ( Figure 9 B, P<0.01), BS / TV ( Figure 9 C, P<0.05), BV / TV ( Figure 9 D, P<0.05), Tb.N. ( Figure 9 E, P<0.05) and Conn.Dn ( Figure 9 H, P<0.05), while Tb.Sp. ( Figure 9 F, P<0.05), Tb.Pf. ( Figure 9 G, P<0.01) and SMI ( Figure 9 I, P<0.05) were significantly decreased. It can be seen that XAN can effectively correct the bone damage caused by MSU, improve the bone microstructural parameters of the bone adjacent to the ankle joint of GA rats to varying degrees, significantly increase bone density, bone surface area density and bone volume fraction; improve the bone microstructure changes of trabecular bone, and increase the number and connection density of trabecular bone. In addition, the improvement of the XAN-H (45 mg / kg) group was better than that of the XAN-L (15 mg / kg) group, and there was no significant difference from the ETO group.

[0050] 4. Effects of xanthohumol on the morphological evaluation of joint pathological tissues.

[0051] As Figure 10 shown in A, HE staining showed that XAN could reduce synovial tissue hyperplasia and reduce inflammatory cell infiltration; SO staining showed that XAN could effectively repair the cartilage damage caused by MSU, and the subchondral bone density was increased; TRAP staining confirmed that XAN could significantly reduce the osteoclast differentiation induced by MSU, reduce the generation of osteoclasts, achieve the effect of inhibiting bone resorption activity, and the empty bone lacunae were significantly fewer and smaller. And with the increase of the dose, the improvement effect of XAN was significantly enhanced ( Figure 10 B, P<0.01). It can be seen that xanthohumol can effectively reduce the body's inflammation level and significantly improve the inflammatory bone damage caused by MSU. The results of Markin's scoring showed that XAN could also repair the cartilage damage caused by MSU (Table 1, P<0.01). The results are shown in Table 1.

[0052] Table 1. Markin's scoring table (n = 10)

[0053]

[0054] # P < 0.05, ## P < 0.01 vs. CON; * P < 0.05, ** P < 0.01 vs. MOD.

[0055] 5. Effects of xanthohumol on the expression of proteins related to bone metabolism.

[0056] As Figure 11 shown in A, compared with the MOD group, the number and density of positive cells in the XAN treatment group were significantly reversed, indicating that XAN can effectively intervene and repair the bone metabolism imbalance induced by MSU. In addition, the significant differences between the XAN-H (45 mg / kg) group and the MOD group were as follows: CTSK ( Figure 11 B, P < 0.01), MMP-9 ( Figure 11 C, P < 0.01), MMP-13 ( Figure 11 D, P < 0.01) and Runx2 ( Figure 11 E, P < 0.05), indicating that oral administration of XAN at a dose of 45 mg / kg can effectively regulate the expression of these four bone metabolism-related proteins, and there was no significant difference between the high-dose group and the positive drug group. Thus, XAN can not only up-regulate the expression of Runx2 to promote bone formation, but also down-regulate the expression of CTSK, MMP-9 and MMP-13 to inhibit bone destruction, and can effectively intervene and regulate the bone metabolism imbalance in the GA model induced by MSU.

[0057] 6. Effects of xanthohumol on the expression of proteins related to bone metabolism signaling pathways.

[0058] As Figure 12 shown, compared with the MOD group, the number of positive cells in the XAN group was significantly reduced ( Figure 12 A), and the RANKL / OPG ratio was significantly decreased ( Figure 12 B, P < 0.01). This may be because XAN down-regulates the expression of RANKL, effectively corrects the RANKL / OPG ratio, and reverses the transformation of osteoblasts to osteoclasts, thereby inhibiting bone destruction and promoting bone formation. There was no significant difference between the ETO group and the XAN-H (45 mg / kg) group. These indicate that XAN may regulate the metabolic balance between bone destruction and bone formation by down-regulating the RANKL / OPG ratio and reversing the transformation of OB to OC, and correct the bone metabolism imbalance induced by MSU by intervening in the RANKL / RANK / OPG bone metabolism signaling pathway.

[0059] The above shows that this example confirms that XAN can be used to prepare anti-gouty arthritis drugs, which can be used not only for anti-gout inflammatory reactions but also for anti-gout bone injuries.

[0060] Example 3 - Study on the effect of xanthohumol on MSU - damaged osteoblasts.

[0061] In the examples of the present invention, the osteoblasts used were primary osteoblasts extracted from the calvaria of male Wistar Han rats within 24 hours after birth (Slac, Shanghai, certificate number: 20220004024235, animal production license number: SCXK(Shanghai)2022 - 0004). Osteoblasts of passages 3 - 5 with good growth status were used for subsequent experimental studies. After trypsin digestion, single - cell suspensions were collected for plating and drug administration. The effects of XAN on the proliferation and differentiation of MSU - damaged osteoblasts, BCIP / NBT alkaline phosphatase staining, alkaline phosphatase (ALP) activity, bone nodule staining, and mineralization level were detected respectively.

[0062] 1. Effect of xanthohumol on osteoblast proliferation.

[0063] First, the cells extracted and isolated from the calvaria of neonatal rats and then cultured were identified. As Figure 13 shown in A, under the microscope, the cells were spindle - shaped, polygonal or irregular in shape, with obvious nuclei and often accompanied by pseudopodia, which was consistent with the morphological identification of osteoblasts. At the same time, after BCIP / NBT alkaline phosphatase staining, insoluble dark blue to blue - violet NBT - formazan was formed in the cells, which was consistent with the staining identification of osteoblasts. This identification result confirmed that these cells were osteoblasts and could be used for subsequent experiments.

[0064] Secondly, XAN and MSU were used on osteoblasts under different conditions, and by observing their effects on the proliferation activity of osteoblasts, the optimal drug concentration was screened to provide a reference for the next step of research. As Figure 13 shown in B, when the concentration of MSU was 500 μg / mL and the action time was 3 days, it had a continuous and stable inhibitory effect on the proliferation activity of osteoblasts, and the inhibition rate was about 44.8%. Therefore, this action condition was selected as the MSU - damaged osteoblast model for subsequent experimental studies. Figure 13 As shown in C, within the concentration range of 100 μM, XAN had no obvious inhibitory effect on the proliferation of osteoblasts (P > 0.05). Therefore, XAN < 100 μM was the safe drug concentration. Figure 13 As shown in D, compared with the MSU group, when the concentrations were 3.125 μM and 6.25 μM, XAN could significantly reverse the damage caused by MSU to the proliferation activity of osteoblasts (P < 0.01 for both).

[0065] Finally, MSU at a concentration of 500 μg / mL was selected as the modeling concentration, and XAN at 3.125 μM and 6.25 μM were used as the administration concentrations. The animals were grouped into a blank group (CON), a model group (MOD), a low-dose xanthohumol group (XAN-L), and a high-dose xanthohumol group (XAN-H) and administered drugs for 3 days, and then the subsequent experiments were continued.

[0066] 2. Effects of xanthohumol on BCIP / NBT staining and ALP activity of osteoblasts.

[0067] ALP activity is an important indicator for measuring the early differentiation of osteoblasts. As Figure 14 shown in A, compared with the CON group, the staining color in the MOD group was significantly lighter, and the ALP activity of MSU-damaged osteoblasts was significantly decreased (P<0.001). Compared with the MOD group, the colors in the low- and high-dose XAN groups were significantly darker than that in the MOD group, and the ALP activity of osteoblasts was significantly enhanced ( Figure 14 B, P<0.01, P<0.001). This indicates that XAN can significantly enhance ALP activity and promote the differentiation level of osteoblasts.

[0068] 3. Effects of xanthohumol on osteoblast bone nodule staining and mineralization level.

[0069] As Figure 15 shown in A, compared with the CON group, MSU could significantly inhibit the formation of osteoblast bone nodules; after dissolving the bone nodules, the absorbance value was measured and quantified analysis found that MSU-damaged osteoblasts caused a significant decrease in their bone mineralization level ( Figure 15 B, P<0.001). Compared with the MOD group, XAN significantly promoted the formation of osteoblast bone nodules and significantly increased the bone mineralization level of damaged osteoblasts.

[0070] 4. Effects of xanthohumol on the expression of proteins related to the osteoblast bone metabolism pathway.

[0071] Figure 16 The target protein bands are shown in A. Compared with the MSU group, 6.25 μM XAN significantly increased the expression of Runx2 ( Figure 16 E, P<0.05); both high- and low-dose XAN (3.125 μM and 6.25 μM) could effectively inhibit the excessive secretion of RANKL ( Figure 16 B, P<0.05 for both), and significantly reversed the RANKL / OPG ratio ( Figure 16 D, P<0.001 for both), and the reversal effect was positively correlated with the dose ( Figure 16 D, P<0.01); and 6.25 μM XAN could also significantly up-regulate the expression of OPG ( Figure 16C, P < 0.01). The above indicates that XAN regulates downstream bone transcription factors by mediating the RANKL / OPG signaling pathway, significantly improving the proliferation and differentiation levels of OB cells, enhancing osteogenic mineralization function, and effectively promoting bone formation.

[0072] The above indicates that this example confirms that XAN can be used as a bone metabolism regulator for anti-gouty bone damage (bone erosion, osteoporosis, etc.).

[0073] Example 4 - Study on the effect of xanthohumol on MSU-stimulated osteoclasts.

[0074] In the examples of the present invention, the osteoclasts used were differentiated from mouse mononuclear macrophage cell line RAW264.7 cells induced by RANKL (purchased from MCE, HY-P73388) (purchased from Shanghai Fuheng Biotechnology Co., Ltd., FH0328). Take RAW 264.7 cells with good growth state and a density of about 70%, and gently blow them into a single-cell suspension with DMEM complete medium for plating. After the cells adhere overnight, add α-MEM complete medium containing 30 ng / mL RANKL according to the recommended dose of the reagent manufacturer, and place it in the cell culture incubator for induction. Replace the α-MEM complete medium containing 30 ng / mL RANKL every 48 h, and observe the cell morphology changes every day. After about 6 days, a large number of mature and differentiated multinucleated cells can be seen. According to the instructions of the TRAP staining kit, perform staining identification. Then carry out drug administration culture, and detect the effects of XAN on the proliferation and differentiation of MSU-stimulated osteoclasts, TRAP staining and the number of positive cells, and TRAP activity respectively.

[0075] 1. Effect of xanthohumol on the proliferation and differentiation of osteoclasts.

[0076] First, identify the cells cultured after RAW264.7 cells are induced by RANKL. As Figure 17 shown in A, under the microscope, the cells are in a multinucleated mass, thick, round, oval or irregular shape, of different sizes, and there are multiple cell nuclei inside the cells. The cell nuclei are counterstained blue, and the cell nucleus mass is clearly visible, which is completely consistent with the phenotypic characteristics of osteoclasts. At the same time, after TRAP staining, insoluble red azo dyes are formed inside the cells, which are the active sites of TRAP enzyme, and the color depth is closely related to the enzyme content in the cytoplasm, which is consistent with the staining identification of osteoclasts. Therefore, after identification, these cells are osteoclasts, and this method can successfully induce osteoclasts for subsequent experimental research.

[0077] Secondly, select XAN and MSU to act on osteoclasts under different conditions, and screen the optimal drug concentration by observing their effects on the proliferation activity of osteoclasts, providing a reference for the next experiment. As Figure 17As shown in Figure B, when the MSU concentration was 15.625 μg / mL, it could significantly stimulate the differentiation and maturation of osteoclasts (P < 0.001). When the concentration exceeded 31.25 μg / mL, it had obvious cytotoxic effects (P < 0.001). Therefore, the optimal concentration for establishing the osteoclast model was 15.625 μg / mL. In addition, Figure 17 As shown in Figure C, when the XAN concentration exceeded 3.125 μM, XAN had obvious cytotoxic effects on the proliferative activity of osteoclasts (P < 0.001). Therefore, the safe drug concentration range of XAN was within 3.125 μM. Figure 17 As shown in Figure D, compared with the MSU group, when the XAN concentrations were 0.5 μM, 1.0 μM, and 2.0 μM, XAN could significantly reverse the proliferative activity of osteoclasts stimulated by MSU (P < 0.001 for all).

[0078] Finally, 15.625 μg / mL of MSU was selected as the modeling concentration, and 0.5 μM, 1.0 μM, and 2.0 μM of XAN were selected as the dosing concentrations. The subsequent experiments were designed according to the blank control group (CON), model group (MOD), xanthohumol low-dose group (XAN-L), xanthohumol medium-dose group (XAN-M), and xanthohumol high-dose group (XAN-H).

[0079] 2. Effects of xanthohumol on TRAP staining and activity of osteoclasts.

[0080] TRAP staining was mainly used to observe osteoclasts with obvious differentiation. Compared with the MOD group, Figure 18 As shown in Figure A, after xanthohumol treatment, the TRAP staining became lighter, indicating that the number of osteoclasts in each xanthohumol dose group decreased significantly ( Figure 18 Figure C, P < 0.001), which indicated that xanthohumol could significantly inhibit the differentiation and maturation of osteoclasts. In addition, TRAP activity was a characteristic marker for evaluating the function of osteoclasts, and xanthohumol could also significantly down-regulate TRAP activity ( Figure 18 Figure B, P < 0.001), thereby inhibiting bone resorption and having the effect of preventing bone destruction.

[0081] 3. Effects of xanthohumol on the expression of proteins related to the osteoclast bone metabolism pathway.

[0082] Figure 19 Figure A was the target protein band. Compared with the MSU group, the low- and high-dose xanthohumol groups (0.5 μM and 1.0 μM) could significantly down-regulate RANK ( Figure 19 Figure B, P < 0.01, P < 0.001), TRAF6 ( Figure 19 Figure C, P < 0.001 for both), NFATc1 ( Figure 19 Figure D, P < 0.001 for both), and CTSK ( Figure 19The expression of bone resorption-related proteins such as E, P < 0.05, P < 0.01), and when XAN inhibited the expression of TRAF6 ( Figure 19 C, P < 0.05) and NFATc1 ( Figure 19 D, P < 0.01), the effect of the high-dose group was significantly better than that of the low-dose group. The above results confirmed that XAN reduced the proliferation and differentiation levels of OC cells, decreased their TRAP activity, and down-regulated the expression of bone resorption-related proteins by inhibiting the RANK / TRAF6 / NFATc1 / CTSK signaling pathway, thereby effectively improving the osteoclastic resorption function of OC cells. The results showed that XAN could be used to prepare bone metabolism regulators for regulating the functional activities related to gout bone destruction.

[0083] In summary, the present invention confirmed that XAN has definite effects of reducing uric acid, anti-inflammatory, and anti-gouty bone damage, and the existing literature reported that its safety is also relatively high. It can be used to prepare both uric acid-lowering drugs and anti-gout drugs, which not only solves the pain points of clinical medication but also provides a new solution and scientific support for the transformation and application of traditional Chinese medicine.

Claims

1. Use of xanthohumol in the preparation of a drug for treating hyperuricemia.

2. Use of xanthohumol in the preparation of a drug for treating hyperuricemia accompanied by renal insufficiency and bone metabolism disorders.

3. Use of xanthohumol in the preparation of a drug for treating gouty arthritis.

4. Use of xanthohumol in the preparation of a drug for treating gout.

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