Application of Amomum villosum extract in preparing UA-lowering and anti-gouty arthritis drugs

Abstract

The invention belongs to the technical field of application of traditional Chinese medicines, and specifically relates to an application of an Amomum villosum extract in preparing a UA-lowering and anti-gouty arthritis drug. The Amomum villosum extract is extracted to obtain the Amomum villosum extract. A hyperuricemia mouse model and a gouty arthritis mouse model are established, and the results show that the Amomum villosum ethanol extract reduces the UA level by synergistically reducing excessive UA generation and promoting UA excretion, and comprehensively exerts an anti-gout effect by inhibiting the release of inflammatory factors such as IL-6, TNF-α and IL-1β, and has the characteristics of better drug efficacy and better drug safety. Therefore, the Amomum villosum ethanol extract can be applied to drugs and health foods for gout patients, and has better development and utilization value as well as huge social and economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of application of traditional Chinese medicines, and particularly relates to application of an Amomum villosum extract in preparing UA-lowering and anti-gouty arthritis medicines. Background Art

[0002] Gout is the second largest metabolic disease in my country after diabetes. It is mainly caused by purine metabolism disorder or abnormal uric acid excretion, which increases blood uric acid (UA) and causes hyperuricemia. Excessive UA causes sodium urate crystals to deposit in joints and soft tissues. It is an inflammatory and organic disease. The occurrence and development of gout are closely related to the high level of UA in the body. Its course can be divided into four periods: asymptomatic gout period (i.e. hyperuricemia), acute gout attack period, gout interval period and chronic gout attack period (i.e. gouty arthritis). In recent years, with the changes in human lifestyle and dietary structure, the incidence of gout has increased year by year. According to the data of the current status report of gout in my country, the number of gout patients in my country has reached 170 million, and is increasing rapidly at an annual growth rate of 9.7%. The onset of gout is often accompanied by a variety of complications, including kidney disease, cardiovascular disease and metabolic syndrome, which seriously threatens human health.

[0003] Hyperuricemia is the first stage of gout attack. During this period, UA levels increase, but there are no symptoms of gouty arthritis or sodium urate crystals. Its occurrence is mainly related to the excessive production and insufficient excretion of UA. When the UA concentration is too high, gout enters the acute stage. After several attacks, it enters an asymptomatic intermittent period, and then becomes more frequent, resulting in more joints affected and longer symptoms. Undissolved UA can form uric acid crystals in the kidneys, joints and other tissues, and then transform into chronic gouty arthritis, thereby causing gout and its complications. Therefore, early prevention and treatment of hyperuricemia is of great significance for the prevention of gout and its complications. In the human body, endogenous UA is the main metabolite of purine catalyzed by xanthine oxidase (XOD). In this pathway, XOD is the key enzyme in the body to metabolize hypoxanthine and xanthine to produce uric acid. Excessive expression of XOD leads to increased UA levels and hyperuricemia. The current clinical criteria for hyperuricemia are: fasting blood UA levels of >420 μmol / L for men and >360 μmol / L for women on two different days under a normal purine diet. 65%-75% of UA is excreted by the kidneys, and the remaining 25%-35% is excreted through the gastrointestinal tract. The excretion and reabsorption of UA are mediated by transporters such as the main ATP binding family (ABCG2), multidrug resistance protein (MRP4), uric acid transporter 1 (URAT1), glucose transporter (GLUT9), organic anion transporter 1 (OAT1) and organic cation transporter 1 (OCT1). It is reported that 90% of hyperuricemia is caused by impaired renal reabsorption and excretion.

[0004] Gouty arthritis is an inflammatory reaction caused by elevated uric acid in the body due to purine metabolism disorders and obstructed uric acid excretion, and the deposition of sodium urate crystals in tissues such as joint capsules and synovium. Typical clinical manifestations in the acute phase are sudden onset at night, severe joint pain, general weakness, fever, headache, etc. The intermittent period is several months or years. With frequent attacks and an increase in diseased joints, the condition gradually worsens and turns into chronic arthritis. Chronic arthritis will cause joint stiffness and deformity, limited movement, etc., and is usually accompanied by metabolic syndromes such as diabetes, hyperglycemia, hyperlipidemia, and renal damage, which seriously affect people's health and quality of life.

[0005] At present, the treatment of hyperuricemia mainly focuses on two aspects: one is to reduce the excessive production of UA, mainly by inhibiting the activity of XOD to reduce the UA content, such as the use of XOD inhibitors - allopurinol, febuxostat, etc.; the other is to promote UA excretion drugs - such as benzbromarone, probenecid, etc. However, due to their many side effects, such as nausea, joint pain, "hypersensitivity syndrome", rash, severe liver and kidney toxicity, etc., their clinical application is greatly limited. Therefore, finding and developing new anti-hyperuricemia drugs with better efficacy and safety is still an urgent problem to be solved.

[0006] In recent years, studies have found that Chinese medicine has a long history of treating gout. It exerts its efficacy by reducing UA, anti-inflammatory, anti-oxidation, and protecting the kidneys. It has the advantages of multiple targets and few toxic side effects. Therefore, finding safe and effective drugs from the rich resources of Chinese medicine has become a research hotspot for the development of drugs for the treatment of gout.

[0007] Amomum villosum Lour., Amomum villosum Lour.var.xanthioides TLWu et Senjen or Amomum longiligulare TLWu, all of the ginger family. It is one of the four traditional southern medicines in my country. It has been used as medicine and food for more than 1,300 years and has been included in the list of medicine and food homology by the National Health Commission. It is pungent in taste and warm in nature. It has the effects of removing dampness and stimulating appetite, warming the spleen and stopping diarrhea, regulating qi and calming the fetus. It is used for dampness and oil obstruction, abdominal distension without hunger, spleen hyperuricemia, stomach deficiency and coldness, vomiting and diarrhea, nausea during pregnancy, and restless fetus. Modern research shows that the chemical components of Amomum villosum are mainly volatile oil components such as acetylborneol, camphor, borneol, caryophyllene, etc., and contain flavonoid components such as quercetin, catechin, quercetin, isoquercetin, coumarins such as flavancoumarins and isoflavancoumarins, and organic acids such as vanillic acid, stearic acid, palmitic acid, etc., which have the effects of anti-platelet aggregation, anti-oxidation, vasodilating, inhibiting pepsin activity, inhibiting gastric acid secretion, promoting intestinal motility, analgesia, antibacterial, etc. So far, the research on the anti-gout effect of Amomum villosum is basically blank, and there is no report on the material basis and specific mechanism of action of this activity.

[0008] The currently disclosed technologies basically use Amomum villosum and other various medicinal materials to form a Chinese medicine prescription to prevent and treat gout; for example, the patent document with the announcement number CN103585561B discloses a Chinese medicine composition for treating gout, which is composed of Sophora flavescens, Epimedium, Aconite, Chuanxiong, Pyrola, Chaenomeles sinensis and Amomum villosum, and proves that the composition has a good effect of reducing uric acid in the hyperuricemia model of mice caused by xanthine, and has a good detumescence effect in the auricle swelling model of mice caused by xylene. However, since the Chinese medicine composition is composed of 7 medicinal materials, the corresponding technical effect is achieved. Although Amomum villosum is contained in the composition formula, Amomum villosum is the lowest in weight fraction in the composition, so it is impossible to know the specific role played by the single Amomum villosum medicinal material in the composition, and the combination of various medicinal materials may have toxic and side effects on the gastrointestinal tract, liver, kidney, etc. The patent document with the announcement number CN102940762B discloses a Chinese medicine for treating gouty arthritis, which is composed of Rhizoma Anemarrhenae, Cortex Moutan, Fructus Kochiae, Semen Plantaginis, Stamen Nelumbinis, Da Yun, Fructus Amomum, Gymnema, Dioscorea opposita, Honey, Fructus Laevigatae, Semen Strychnos nux vomica, Ganoderma lucidum, Cortex Cinnamomi, Cynomorium songaricum, Morinda officinalis, Semen Cuscutae, Eucommia ulmoides, Gecko, Flos Lonicerae and Herba Prunellae. The Fructus Amomum in the document is the stroma of Hypocrella bambusae (Berk. et Br.) Sacc. of Hypocrellaceae, but our research object Fructus Amomum is the dried mature fruit of Amomum villosum Lour. of Zingiberaceae, so the two are Chinese medicinal materials of completely different families and genera, and are not in conflict or overlap with our research object Fructus Amomum.

[0009] In the previous basic work, the applicant established a method for determining the in vitro inhibitory activity of XOD using xanthine as substrate and XOD inhibitor as reaction enzyme, and investigated the inhibitory effects of 27 Chinese herbal medicine extracts on XOD. The results showed that the inhibitory activity of Amomum villosum 95% ethanol extract (EA) was the strongest (IC 50 Therefore, we further established a hyperuricemia mouse model by intraperitoneal injection of potassium oxonate combined with oral administration of hypoxanthine, and a gouty arthritis mouse model by using sodium urate crystals, to investigate the anti-gout effect of EA and explore its mechanism, so as to provide a scientific basis for the application of Amomum villosum in anti-gout drugs, health foods, etc. Summary of the invention

[0010] In order to solve the above problems, the present invention provides an application of an Amomum villosum extract in preparing a UA-lowering and anti-gouty arthritis drug.

[0011] This is achieved specifically through the following technical solutions:

[0012] 1. Application of an Amomum villosum extract in the preparation of UA-lowering drugs.

[0013] Furthermore, the preparation of the UA-lowering drug is to combine the Amomum villosum extract with acceptable auxiliary materials in pharmacy and health food to prepare the desired preparation.

[0014] 2. Application of an Amomum villosum extract in the preparation of anti-gouty arthritis drugs.

[0015] Furthermore, the preparation of the anti-gouty arthritis drug is to combine the Amomum villosum extract with acceptable auxiliary materials in pharmacy and health food to prepare the desired preparation.

[0016] 3. The above-mentioned Amomum villosum extract is prepared by grinding dried Amomum villosum, adding 95% ethanol in an amount of 6.5-8 times the mass of Amomum villosum, and reflux extraction for 3-4 times, each extraction time for 1 hour, and combining the extracts to prepare an extract.

[0017] In summary, the beneficial effects of the present invention are as follows: the present invention extracts Amomum villosum to obtain Amomum villosum extract; by establishing a hyperuricemia mouse model and a gouty arthritis mouse model, the anti-gout effect of Amomum villosum alcohol extract is investigated, and its mechanism of action is discussed. The study found that EA mainly reduces the production of UA by inhibiting the activity of XOD, and increases the excretion of UA by regulating the protein and mRNA expression of related transporters such as OAT1, ABCG2, URAT1 and GLUT9, thereby achieving the purpose of reducing UA, thereby playing an anti-gout role, and EA has a good relief and protective effect on liver and kidney damage caused by hyperuricemia. At the same time, EA has a significant improvement effect on gouty arthritis, can alleviate the damage of mouse paw periarthritis caused by sodium urate crystals, reduce inflammatory cell infiltration, reduce the swelling of paw periarthritis, inhibit the release of inflammatory factors such as IL-6, TNF-α and IL-1β, and has a significant anti-gouty arthritis effect.

[0018] Compared with existing anti-gout drugs, EA reduces UA levels by reducing excessive UA production and promoting UA excretion in multiple ways, and exerts a comprehensive anti-gout effect by inhibiting the release of inflammatory factors such as IL-6, TNF-α and IL-1β. It has better efficacy and better drug safety. Therefore, EA can be used in drugs, health foods, etc. for gout patients, and has better development and utilization value as well as huge social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the total ion chromatogram of EA sample by UPLC-LTQ-Qrbitrap-MS (ESI).

[0020] Figure 2 The inhibitory effect of Amomum villosum extract on XOD; wherein, (A) is EA, and (B) is Amomum villosum water extract.

[0021] Figure 3 The inhibitory effects of Amomum villosum extract on XOD; wherein (A) is EA, and (B) is Amomum villosum water extract.

[0022] Figure 4 Effects of EA on body weight and organ coefficients in hyperuricemia mice Among them, (A) is body weight, (B) is spleen coefficient, (C) is liver coefficient, and (D) is kidney coefficient. # P<0.05, ## P<0.01, compared with the normal control group; *P<0.05, **P<0.01 compared with the model group.

[0023] Figure 5 Effects of EA on serum and urine UA, Creatinine, BUN and FEUA in hyperuricemia mice Among them, (A) is UA, (B) is Creatinine, (C) is BUN, and (D) is FEUA. # P<0.05, ## P<0.01, compared with the normal control group; *P<0.05, **P<0.01 compared with the model group.

[0024] Figure 6 Effects of EA on serum and liver XOD, ALT and AST activities in hyperuricemia mice Among them, (A) is serum XOD, (B) is liver XOD, (C) serum ALT, (D) liver ALT, (E) serum AST, and (F) liver AST. # P<0.05, ## P<0.01, compared with the normal control group; *P<0.05, **P<0.01 compared with the model group.

[0025] Figure 7 Effects of EA on SOD, MDA activities and IL-1β, TNF-α levels in hyperuricemia mice Among them, (A) is serum SOD, (B) is liver SOD, (C) is kidney SOD, (D) is serum MDA, (E) is liver MDA, (F) is kidney MDA, (G) is liver IL-1β, (H) is liver TNF-α, (I) is kidney IL-1β, and (J) is kidney TNF-α. # P<0.05, ## P<0.01, compared with the normal control group; *P<0.05, **P<0.01 compared with the model group.

[0026] Figure 8The effect of EA on the pathological morphology of renal tissue in hyperuricemia mice; (A) is the normal group, (B) is the hyperuricemia model group, (C) is the positive control group, (D) is the EA low-dose group, (E) is the EA medium-dose group, and (F) is the EA high-dose group.

[0027] Fig. 9 Effects of EA on the expression of XOD protein in liver and related transporter proteins and mRNA in kidney of hyperuricemia mice Among them, (A) is the Western blot electrophoresis of liver XOD protein, (B) is the relative expression of liver XOD protein, (C) is the Western blot electrophoresis of kidney GLUT9, URAT1, OAT1 and ABCG2 proteins, (D)-(G) are the relative protein expressions of kidney GLUT9, URAT1, OAT1 and ABCG2, and (H)-(K) are the relative mRNA expressions of kidney GLUT9, URAT1, OAT1 and ABCG2. # P<0.05, ## P<0.01, compared with the normal control group; *P<0.05, **P<0.01 compared with the model group.

[0028] Fig.10 Effect of EA on body weight of mice with gouty arthritis

[0029] Fig.11 Effect of EA on the swelling of the paw tissues in mice with gouty arthritis

[0030] Fig.12 Effects of EA on the levels of IL-6 (A), TNF-α (B) and IL-1β (C) in mice with gouty arthritis ## P<0.01, compared with the normal control group; *P<0.05, **P<0.01, compared with the model group.

[0031] Fig.13 The effects of EA on the pathological morphology of the paw periarticular tissues of model mice; (A) is the normal group, (B) is the model group, (C) is the positive control group, (D) is the low-dose EA group, (E) is the medium-dose EA group, and (F) is the high-dose EA group. DETAILED DESCRIPTION

[0032] The specific embodiments of the present invention are further described in detail below, but the present invention is not limited to these embodiments, and any improvement or substitution based on the basic spirit of the present embodiment still falls within the scope of protection required by the claims of the present invention.

[0033] Example 1

[0034] Preparation of 95% ethanol extract (EA) of Amomum villosum:

[0035] Weigh 300g of dried Amomum villosum, crush it and add 3L 95% ethanol respectively, reflux extraction 3 times, each time for 1h, combine the extracts, vacuum filter, concentrate and freeze-dry the obtained liquid to obtain 28.16g of Amomum villosum water extract, with an extraction rate of 9.39%.

[0036] Screening Example 1

[0037] Preparation of Amomum villosum aqueous extract:

[0038] Weigh 300g of dried Amomum villosum, crush it and add 3L of water respectively, decoct it 3 times, each time for 1h, combine the extracts, and vacuum filter, concentrate and freeze-dry the resulting liquid to obtain 36.84g of Amomum villosum water extract, with an extraction rate of 12.28%.

[0039] Screening Example 2

[0040] Preparation of Amomum villosum methanol extract:

[0041] Weigh 300 g of dried Amomum villosum, crush it and add 3 L of methanol respectively, reflux extraction 3 times, each time for 1 hour, combine the extracts, and the resulting liquid is vacuum filtered, concentrated, and freeze-dried to obtain 43.11 g of Amomum villosum water extract, with an extraction rate of 14.37%.

[0042] The in vitro XOD inhibitory activity assay was used to screen the extracts of Amomum villosum extracted with different solvents (water, methanol, and 95% ethanol). Among them, the 95% ethanol extract of Amomum villosum (EA) had the best in vitro XOD inhibitory activity (IC 50 was 54.65 μg / mL), so 95% ethanol was selected as the extraction solvent, the extraction times were 3 times, and the extraction time was selected as 1 h.

[0043] 1. Determination of in vitro inhibitory activity of the extract obtained by the method of Example 1 on XOD and investigation of enzyme kinetics

[0044] 1.1 Study on XOD inhibitory activity

[0045] The total volume of the reaction system was 200 μL. First, 50 μL of sample solution and enzyme solution (final concentration of reaction was 0.05 U / mL) were added and mixed. After incubation at 25°C for 15 min, 50 μL of xanthine substrate solution (final concentration of reaction was 150 μmol / L) was added. After incubation for 20 min, 50 μL of 1 mol / L HCl solution was added to terminate the reaction. The OD value was measured at a wavelength of 290 nm using a multifunctional microplate reader to calculate the inhibition rate. The IC50 of the in vitro inhibitory activity of Amomum villosum methanol extract, water extract, and EA on XOD were 328.49 μg / mL, 254.00 μg / mL, and 54.65 μg / mL, respectively. Therefore, EA and water extract were selected for enzyme kinetics investigation.

[0046] 2.2 Enzyme kinetics of XOD inhibitory activity

[0047] The concentration of substrate xanthine (final concentration of 150 μmol / L) was fixed, and different concentrations of Amomum villosum extracts were added (EA final concentrations of 0, 6.875, 13.75, 27.5, 55 μg / mL; water extract final concentrations of 0, 31.75, 63.5, 127, 254 μg / mL), and the effect on enzyme activity at different concentrations of XOD (final concentrations of 0.00625, 0.0125, 0.025, 0.05, 0.1 U / mL) was determined to determine whether the inhibitory effect of Amomum villosum extract on XOD was reversible. The enzyme reaction rate kinetic equation under different concentrations of Amomum villosum extract was as follows: Figure 1 As shown, the straight lines basically pass through the origin, and with the continuous increase in the mass concentration of Amomum villosum extract, the reaction rate decreases accordingly, indicating that the inhibitory effects of Amomum villosum water extract and EA on XOD are reversible inhibition.

[0048] The enzyme concentration was fixed (0.05U / ml), and different masses of Amomum villosum extracts were added (the final concentrations of EA were 0, 6.875, 13.75, 27.5, and 55μg / mL; the final concentrations of water extracts were 0, 31.75, 63.5, 127, and 254μg / mL). The effects on enzyme activity at different substrate concentrations (the final reaction concentrations were 37.5, 75, 150, 300, and 600μmol / L) were determined to determine the competition type of Amomum villosum extracts on XOD. Figure 2 The results showed that the Km value increased with the increase of the concentration of Amomum villosum extract, and all the straight lines intersected in the second quadrant, indicating that the inhibition of Amomum villosum water extract and EA on XOD belonged to the competitive-noncompetitive inhibition type of mixed inhibition.

[0049] Since EA has the strongest inhibitory activity against XOD in vitro (IC 50 is 54.65μg / mL), so EA was selected for the following experimental study on lowering UA and preventing gouty arthritis.

[0050] Chemical composition characterization of 2EA

[0051] EA samples were dissolved in methanol and filtered through a 0.45 μm microporous membrane. The EA samples were chemically characterized and analyzed by liquid chromatography-linear ion trap-orbitrap (LC-LTQ-Qrbitrap, ThermoFisher Scientific, Bremen, Germany). Liquid phase conditions: the chromatographic column was ACQUITY UPLC C18 (2.10 mm × 150 mm, 1.7 μm), the mobile phase was (A) methanol water and (B) 0.1% formic acid water (v / v), the gradient elution started from 5% A and increased to 100% within 45 min; the flow rate was 0.3 mL / min, the column temperature was 40°C, and the detection wavelength was 325 nm. Mass spectrometry conditions: Electrospray ionization source positive ion (ESI) mode, electrospray voltage 2.5KV, desolvation gas temperature 380°C, sheath gas flow 35arb, auxiliary gas flow 10arb, mass spectrometry scanning range of mass-to-charge ratio m / z 80-1200, resolution 70000 (primary mass spectrometry) and 17500 (secondary mass spectrometry). The experimental results are shown in Figure 3 shown.

[0052] The results show that each chromatographic peak in the EA sample has a good response in the positive ion mode ( Figure 3 ), the molecular weight, molecular formula, UV absorption diagram and secondary mass spectrometry data of each chromatographic peak were obtained, and the structures of 6 relatively high chromatographic peaks in EA were preliminarily determined by analyzing the data. By comparing with the mass spectrum, retention time and other data of the reference substance, the 6 peaks were determined to be quercetin, magnoshinin, eudesobovatol A, diisobutyl phthalate, oleamide and 13E-Docosenamide.

[0053] Determination of the maximum tolerated oral dose of 3EA in mice

[0054] Ten Kunming mice, half male and half female, weighing 18-22g, were fasted for 12 hours and given the maximum concentration (77.5mg / kg) by gavage at a volume of 0.4mL / 10g. The mice were fed normally and their survival was observed after 14 days. No mice died after 14 days of gavage. The maximum oral tolerance of EA in mice was calculated to be 3100mg / kg.

[0055] Study on the uric acid-lowering effect and mechanism of 4EA on hyperuricemia mice

[0056] 60 Kunming male mice were randomly divided into normal group, model group, allopurinol group (5mg / kg), EA low (97.5mg / kg), medium (195mg / kg), and high dose group (390mg / kg). The model was established by intraperitoneal injection of potassium oxonate (300mg / kg) combined with oral gavage of hypoxanthine (600mg / kg). The drug group was gavaged with the corresponding drug 1 hour after model establishment, and the drug was administered continuously for 14 days. After administration on the 13th day, the mice were placed in a metabolic cage, and the urine of the mice was collected within 24 hours, and blood was collected 1 hour after the last administration. After centrifugation of whole blood and urine at 3000rpm for ten minutes, the supernatant was aspirated and stored at -20℃; and liver, spleen, and kidney tissues were taken. Except for the right kidney fixed in 4% paraformaldehyde, the rest of the organs were stored at -80℃.

[0057] 4.1 Effects of EA on body weight and organ coefficients in hyperuricemia mice

[0058] like Figure 4 As shown in A, compared with the normal group, there was no significant difference in the body weight of mice in each EA dose group (P>0.05), but the body weight of mice in the allopurinol group continued to decrease after continuous administration for 10 days and 14 days (P<0.05, P<0.01). Figure 4 As shown in B, compared with the normal group, there was no significant difference in the liver, spleen, and kidney coefficients of mice in each EA dose group (P>0.05), but the spleen index of mice in the model group and allopurinol group was significantly decreased (P<0.05), and the kidney index was significantly increased (P<0.01), and the liver index of mice in the allopurinol group was significantly increased (P<0.05). The results suggest that EA has no obvious toxic side effects on the liver, spleen, and kidneys of hyperuricemia mice, while allopurinol may have certain toxic side effects on the liver, spleen, and kidneys of hyperuricemia mice.

[0059] Effects of EA on serum and urine UA, Creatinine, BUN, and FEUA in hyperuricemia mice

[0060] like Figure 5 As shown in A, compared with the normal group, the serum UA level of mice in the model group increased significantly to 442.6 μmol / L (P<0.01), and the urine UA level decreased significantly (P<0.01). Compared with the model group, EA dose-dependently reduced the serum UA level of mice (P<0.01), and the serum UA level of mice in the high-dose group decreased to 144.49 μmol / L.

[0061] like Figure 5As shown in B-5D, compared with the normal group, the serum Creatinine and BUN levels of mice in the model group and allopurinol group were significantly increased (P<0.01), and the urinary Creatinine, BUN and FEUA were significantly decreased (P<0.01). Compared with the model group, the serum Creatinine in the high-dose EA group was significantly decreased (P<0.01), and the urinary Creatinine, BUN and FEUA were significantly increased (P<0.01). The results suggest that EA has a certain protective effect on the kidneys of hyperuricemia mice.

[0062] Effects of EA on serum and liver XOD activity in hyperuricemia mice

[0063] like Figure 6 As shown in A and B, compared with the normal group, the serum and liver XOD activities of the mice in the model group were significantly increased (P<0.01). Compared with the model group, the serum and liver XOD activities of the mice in each EA dose group were significantly decreased (P<0.01), especially the serum and liver XOD activities of the mice in the high-dose group were 6.84U / L and 37.30U / g protein, which were basically close to the serum and liver XOD activities of the mice in the normal group. The results suggest that EA exerts its anti-gout effect by inhibiting the activity of XOD.

[0064] Effects of EA on ALT and AST activities in serum and liver of hyperuricemia mice

[0065] like Figure 6 As shown in CF, compared with the normal group, the serum and liver ALT and AST activities of the model group mice were significantly increased (P<0.01), which were 6.84U / L and 396.40U / g protein, 28.35U / L and 355.87U / g protein, respectively. Compared with the model group, the serum and liver ALT and AST activities of the mice in the EA medium and high dose groups were significantly decreased (P<0.05, P<0.01), especially the serum and liver ALT and AST activities of the mice in the high dose group were 4.54U / L and 251.54U / g protein, 20.39U / L and 340.41U / g protein, which were basically close to the serum and liver ALT and AST activities of the mice in the normal group. The results suggest that EA has a good protective effect on liver damage in hyperuricemia mice.

[0066] Effects of 4.5EA on SOD and MAD activities in serum, liver and kidney of hyperuricemia mice

[0067] like Figure 7As shown in AF, compared with the model group, the SOD activity in serum, liver and kidney of mice in the high-dose EA group was significantly increased (P<0.05, P<0.01), and the MDA activity in serum, liver and kidney of mice in the medium and high-dose groups was significantly decreased (P<0.01). The results showed that EA has a good antioxidant effect on hyperuricemia mice, suggesting that the anti-gout mechanism of EA may be related to its antioxidant capacity.

[0068] Effects of 4.6EA on IL-1β and TNF-α levels in liver and kidney of hyperuricemia mice

[0069] like Figure 7 As shown in GJ, compared with the normal group, the IL-1β and TNF-α contents in the liver and kidney of the model group mice increased significantly (P<0.01), indicating that the hyperuricemia mice had certain liver and kidney inflammatory damage. Compared with the model group, the IL-1β and TNF-α contents in the liver and kidney of the mice in each EA dose group decreased, especially in the high dose group (P<0.05, P<0.01), and the L-1β content in the kidney was 99.84pg / μg protein, which was close to the L-1β content in the kidney of the normal group mice. The results suggest that EA may play an anti-gout role by reducing the inflammatory response in the hyperuricemia mice.

[0070] Effects of 4.7EA on renal pathological morphology in hyperuricemia mice

[0071] like Figure 8 As shown in the figure, the epithelial cells of mice in the normal group were arranged neatly, while the renal tissue morphology of mice in the model group was abnormal, with increased glomerular cells, mesangial hyperplasia, inflammatory cell infiltration, mild edema and hyperplasia of the renal tubules. In the allopurinol group, the glomerular capillaries of mice were dilated and hemorrhaged, with a large number of inflammatory cells infiltrating, and the renal tubules were significantly dilated and hyperplastic, accompanied by the shedding of epithelial cells and cell fragments in the lumen. The lesions of the glomeruli and renal tubules of mice in each dose group of EA were improved to varying degrees, and the number of inflammatory cells was significantly reduced. The results suggest that EA has a certain protective effect on renal damage in hyperuricemia mice.

[0072] Effects of EA on the expression of XOD protein in liver and the expression of related transporter proteins and mRNA in kidney of hyperuricemia mice

[0073] like Fig. 9 As shown in A and B, compared with the model group, the expression of XOD protein in the liver of mice in each EA dose group was significantly downregulated (P<0.01), and these results were consistent with the results of XOD activity determination ( Figure 6 A and B), which once again confirmed that EA exerts its anti-gout effect by inhibiting the activity of XOD.

[0074] like Fig. 9As shown by CK, compared with the normal group, the mRNA and protein expressions of GLUT9 and URAT1 in the model group mice kidney were significantly increased (P<0.01), while the mRNA and protein expressions of OAT1 and ABCG2 were significantly decreased (P<0.01). Compared with the model group, the mRNA and protein expressions of GLUT9 and URAT1 in the EA medium and high dose groups were significantly decreased (P<0.05, P<0.01), while the mRNA and protein expressions of OAT1 and ABCG2 were significantly increased (P<0.01). Results The anti-hyperuricemia effect of EA may be related to its regulation of the expression of uric acid transporters such as GLUT9, URAT1, OAT1 and ABCG2.

[0075] Study on the effect and mechanism of 5EA on gouty arthritis in mice

[0076] Sixty Kunming male mice were randomly divided into normal group (CON), model group (MODEL), positive control group (COLCHICINE), EA low (97.5mg / kg), medium (195mg / kg), and high (390mg / kg) dose groups, with 10 mice in each group. Except for the normal group and the model group, the other groups were gavaged with the corresponding drugs once a day for 10 consecutive days. One hour after the last administration on the tenth day, 0.1ml of MSU crystals were subcutaneously injected into the right sole of the mouse to induce acute gouty arthritis. The thickness of the mouse paw was measured with a vernier caliper before injection and at 0h, 1h, 4h, 12h, and 24h after injection to calculate the degree of foot swelling. 24h after the injection of MSU crystals, the mice were anesthetized and the right periarthritis tissue was removed and fixed in 4% paraformaldehyde.

[0077] Effect of EA on body weight of mice with gouty arthritis

[0078] Depend on Fig.10 It can be shown that, compared with the normal group, there was no significant difference in the body weight of mice in each experimental group (P>0.05), indicating that EA had no significant effect on the body weight of mice with gouty arthritis.

[0079] 5.2 Effect of EA on the swelling of the paw tissues in mice with gouty arthritis

[0080] Depend on Fig.11 It can be seen that compared with the normal group, the swelling of the model group mice at the same time point (0, 1, 4, 12, 24h) was significantly increased compared with the normal group (P<0.01). Except for the normal group, the swelling of the mice in other groups continued to increase from 0 to 1h after modeling, reached a peak after 1h, and decreased after 4h, indicating that the modeling was successful. Compared with the model group, the swelling of the toes in the medium and high dose EA groups was significantly reduced at 12 hours and 24 hours after modeling (P<0.01), and the swelling of the toes of the high dose group mice at 24 hours was almost the same as that of the colchicine group.

[0081] 5.3 Effect of EA on IL-6, TNF-α and IL-1β levels in mice with gouty arthritis

[0082] Fig.12 The results showed that compared with the normal group, the levels of IL-6, TNF-α and IL-1β in the paw tissues of mice in the model group were significantly increased (P<0.01). Compared with the model group, the levels of IL-6, TNF-α and IL-1β in the paw tissues of mice in the medium and high dose EA groups were significantly decreased (P<0.01), suggesting that EA exerts its anti-gouty arthritis effect by reducing the inflammatory response in the paw tissues of mice with gouty arthritis.

[0083] Effects of EA on pathological morphology of paw tissues in mice with gouty arthritis

[0084] like Fig.13 As shown in the figure, the connective tissue around the paws of mice in the normal group contained abundant capillaries, adipocytes and nerves, and the muscle fibers were neatly arranged, without obvious fibrous tissue hyperplasia or inflammatory cell infiltration and obvious degeneration and necrosis. In the model group, a large amount of homogeneous strong basophilic substances accumulated locally, with obvious inflammatory cell infiltration and fibrous tissue hyperplasia. The lesions of the paw tissues of mice in each dose group of EA were improved to varying degrees, as shown by reduced inflammatory cell infiltration and less fibrous tissue hyperplasia. The results suggest that EA has a certain protective effect on the damage of the paw tissues of mice with gouty arthritis.

[0085] 6 Comparative Experiments

[0086] Preparation of a formula for extract: (Formula source: a composition in a patent document with announcement number CN103585561B)

[0087] Weigh 8g of dried Sophora flavescens, 12g of Epimedium, 7g of Aconite, 6g of Ligusticum chuanxiong, 10g of Pyrola, 7g of Papaya, and 5g of Amomum villosum, crush them into powder and decoct them in water twice, combine the extracts, concentrate them, and freeze-dry them to obtain the extract of Formula 1.

[0088] Preparation of formula II extract: (Formula source: Xiaotong Decoction No. Ⅱ from "Secret Recipe for Gout Effectiveness". Wang Maohong. Secret Recipe for Gout Effectiveness [M]. Beijing: China Medical Science and Technology Press, 2014: 250)

[0089] Weigh 30g of dried Astragalus, 30g of Smilax glabra, 20g of Saposhnikovia divaricata, 20g of Coix seed, 15g of Atractylodes macrocephala, 15g of Plantago, 15g of Amomum villosum, 12g of Citrus reticulata, 12g of Cyathula capitata, and 10g of Licorice, crush them into powder and decoct them in water twice, combine the extracts, concentrate and freeze-dry them to obtain the extract of formula 2.

[0090] Preparation of formula three extracts: (Formula source: Huanglian Jiedu Decoction combined with Shengjiang Powder in "Secret Recipe for Gout Effectiveness". Wang Maohong. Secret Recipe for Gout Effectiveness [M]. Beijing: China Medical Science and Technology Press, 2014: 211)

[0091] Weigh 6g of dried Scutellaria baicalensis, 6g of Coptis chinensis, 12g of Phellodendron chinense, 6g of Gardenia jasminoides, 30g of Lonicera japonica, 6g of Rhubarb, 15g of Bombyx batryticatus, 15g of Periostracum cicadae, 15g of Curcuma longa, 15g of Plantago seed, 6g of Amomum villosum, 6g of Corydalis yanhusuo, and 3g of Licorice, crush them into powder and decoct them in water twice. Combine the extracts, concentrate and freeze-dry them to obtain the extract of composition three.

[0092] Study on the inhibitory activity of different prescriptions on XOD:

[0093] The total volume of the reaction system was 200 μL. First, 50 μL of sample solution and enzyme solution (final reaction concentration was 0.05 U / mL) were added and mixed. After incubation at 25°C for 15 min, 50 μL of xanthine substrate solution (final reaction concentration was 150 μmol / L) was added. After further incubation for 20 min, 50 μL of 1 mol / L HCl solution was added to terminate the reaction. The OD value was measured at a wavelength of 290 nm using a multifunctional microplate reader to calculate the inhibition rate.

[0094] Table 1 Inhibitory effects of different prescriptions on XOD

[0095]

[0096] The results of XOD inhibition activity are shown in Table 1. At a concentration of 500 μg / mL, the in vitro inhibitory activities of the above three prescriptions against XOD were 38.35%, 29.57%, and 45.39%, respectively. 50 It was 54.65 μg / mL. Compared with the above three prescriptions, EA showed a very obvious ability to inhibit XOD.

Claims

1. An application of an Amomum villosum extract in the preparation of a UA-lowering drug, characterized in that: The Amomum villosum extract is a 95% ethanol extract EA of Amomum villosum; The EA is prepared by grinding dried Amomum villosum, adding 95% ethanol in an amount of 6.5-8 times the mass of Amomum villosum, refluxing and extracting for 3-4 times, each time for 1 hour, and combining the extracts to prepare an extract. The EA was subjected to chemical characterization analysis, and six active ingredients with relatively high contents were quercetin, magnoshinin, eudesobovatol A, diisobutyl phthalate, oleamide, and 13E-Docosenamide.

2. The use of the Amomum villosum extract in the preparation of a UA-lowering drug as claimed in claim 1, characterized in that: The UA lowering also includes the relief and protection of liver and kidney damage caused by hyperuricemia.

3. The use of the Amomum villosum extract in the preparation of a UA-lowering drug as claimed in claim 1, characterized in that: The Amomum villosum extract reduces UA by reducing excessive UA production and promoting UA excretion through multiple pathways to reduce UA levels.

4. The use of the Amomum villosum extract in the preparation of a UA-lowering drug as claimed in claim 3, characterized in that: The Amomum villosum extract reduces UA by inhibiting the activity of XOD to reduce the production of UA, and regulating the protein and mRNA expressions of OAT1, ABCG2, URAT1 or GLUT9 related transporters to increase the excretion of UA, thereby comprehensively achieving the purpose of reducing UA.

5. Use of an Amomum villosum extract in the preparation of an anti-gouty arthritis drug, characterized in that: The Amomum villosum extract is a 95% ethanol extract EA of Amomum villosum; The EA is prepared by grinding dried Amomum villosum, adding 95% ethanol in an amount of 6.5-8 times the mass of Amomum villosum, refluxing and extracting for 3-4 times, each time for 1 hour, and combining the extracts to prepare an extract. The EA was subjected to chemical characterization analysis, and six active ingredients with relatively high contents were quercetin, magnoshinin, eudesobovatol A, diisobutyl phthalate, oleamide, and 13E-Docosenamide.

6. The use of the Amomum villosum extract in the preparation of an anti-gouty arthritis drug as claimed in claim 5, characterized in that: The Amomum villosum extract EA is used to fight gouty arthritis by relieving tissue damage caused by sodium urate crystals, reducing inflammatory cell infiltration, reducing tissue swelling, and inhibiting the release of IL-6, TNF-α or IL-1β inflammatory factors to achieve the anti-inflammatory purpose.

7. Use of the Amomum villosum extract according to any one of claims 1 to 2 in the preparation of a UA-lowering drug, characterized in that: The method for preparing the UA-lowering drug is to combine the 95% ethanol extract EA of Amomum villosum with an adjuvant acceptable in pharmacy and health food to prepare the desired preparation.

8. Use of the Amomum villosum extract according to any one of claims 5 to 6 in the preparation of an anti-gouty arthritis drug, characterized in that: The preparation of the anti-gouty arthritis drug is to combine the 95% ethanol extract EA of Amomum villosum with acceptable auxiliary materials in pharmacy and health food to prepare the required preparation.

9. An extract of Amomum villosum for reducing UA and resisting gouty arthritis, characterized in that: The Amomum villosum extract is a 95% ethanol extract EA of Amomum villosum; The EA was subjected to chemical characterization analysis, and six active ingredients with relatively high contents were quercetin, magnoshinin, eudesobovatol A, diisobutyl phthalate, oleamide, and 13E-Docosenamide.

10. The Amomum villosum extract for reducing UA and resisting gouty arthritis according to claim 9, characterized in that: The EA is prepared by grinding dried Amomum villosum, adding 95% ethanol in an amount of 6.5-8 times the mass of Amomum villosum, refluxing and extracting for 3-4 times, each time for 1 hour, and combining the extracts to prepare an extract.

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