CN116211978A - Application of Amomum extract in preparation of UA-lowering and anti-gouty arthritis medicine - Google Patents

Abstract

The invention belongs to the technical field of traditional Chinese medicine application, and particularly relates to application of fructus amomi extract in preparation of UA-degrading and anti-gout arthritis medicines; the invention extracts fructus amomi to obtain fructus amomi extract; through establishing a hyperuricemia mouse model and a gouty arthritis mouse model, researches show that the fructus amomi ethanol extract is synergistic from two aspects of reducing excessive generation of UA and promoting excretion of UA to reduce UA level, and comprehensively plays the anti-gout role by inhibiting the release of inflammatory factors such as IL-6, TNF-alpha, IL-1 beta and the like, and has the characteristics of better drug effect and better drug safety, so that the fructus amomi ethanol extract can be applied to the aspects of drugs and health foods of gouty patients, and has better development and utilization values and huge social and economic benefits.

Description

Application of Amomum extract in preparation of UA-lowering and anti-gout arthritis medicine

technical field

The invention belongs to the technical field of application of traditional Chinese medicines, and in particular relates to the application of an amomum extract in the preparation of UA-reducing and anti-gout arthritis drugs.

Background technique

Gout is the second largest metabolic disease in my country after diabetes. It is mainly caused by the increase of blood uric acid (Uric acid, UA) due to purine metabolism disorder or abnormal excretion of uric acid, which causes hyperuricemia, and excessive UA causes uric acid. An inflammatory and organic disorder in which sodium crystals deposit in the joints and soft tissues. The occurrence and development of gout are closely related to the high level of UA in the body, and its course can be divided into four periods: asymptomatic gout period (ie, hyperuricemia), acute gout attack period, gout intermittent period, and chronic gout attack period (ie, gout Arthritis). In recent years, with the change of human life style and dietary structure, the incidence of gout has been increasing year by year. According to the data of my country's gout status report, the number of gout patients in my country has reached 170 million, and it is increasing rapidly at an annual growth rate of 9.7%. , and the onset of gout is often accompanied by a variety of complications, including kidney disease, cardiovascular disease and metabolic syndrome, which seriously threaten human health.

Hyperuricemia is the first stage of gout attack. During this period, the level of UA increases, but there are no symptoms of gouty arthritis and sodium urate crystals. The occurrence of hyperuricemia is mainly related to the excessive production and insufficient excretion of UA. When the UA concentration is too high, it enters the acute stage of gout. After several attacks, it enters the intermittent period of asymptomatic, and then becomes more frequent, so that the more joints are involved, the longer the duration of symptoms. Undissolved UA can be in the kidneys, joints and Uric acid crystals form in other tissues, which turns into chronic gouty arthritis, triggering gout and its complications. Therefore, early prevention and treatment of hyperuricemia is of great significance to the prevention of gout and its complications. In the human body, endogenous UA is the main metabolite of purine under the catalysis of xanthine oxidase (Xanthine oxidase, XOD). In this pathway, XOD is the key enzyme for the metabolism of hypoxanthine and xanthine in the human body to produce uric acid , the overexpression of XOD leads to the increase of UA level and the formation of hyperuricemia. The current clinical judgment standard for hyperuricemia is: under normal purine diet, UA levels in blood on an empty stomach > 420 μmol/L for males and > 360 μmol/L for females twice on different days. 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 mainly ATP-binding family (ABCG2), multidrug resistance protein (MRP4), Transporters such as transporter protein 1 (URAT1), glucose transporter protein (GLUT9), organic anion transporter 1 (OAT1), and organic cation transporter 1 (OCT1) are mediated. It has been reported that 90% of hyperuricemia is due to impaired renal reabsorption and excretion.

Gouty arthritis is an inflammatory reaction caused by the increase of uric acid in the body caused by the disorder of purine metabolism and the obstruction of uric acid excretion, and the deposition of sodium urate crystals in the joint capsule, synovium and other tissues. The typical clinical manifestations in the acute phase are sudden onset at night, severe joint pain, general weakness, fever, and headache. The intermission period is several months or several years. With the frequent outbreaks of the disease, the number of diseased joints increases, and the disease gradually worsens and turns into chronic arthritis. In the chronic arthritis stage, there will be joint stiffness and deformity, limited movement, etc., usually accompanied by metabolic syndrome such as diabetes, hyperglycemia, hyperlipidemia and renal function damage, which seriously affects people's life, health and quality of life.

At present, the treatment of hyperuricemia mainly starts from two aspects. One is to reduce the excessive production of UA, mainly by inhibiting the activity of XOD to reduce the content of UA, such as using XOD inhibitors—allopurinol, febuxostat, etc.; Is to promote the excretion of UA drugs - such as benzbromarone, probenecid and so on. However, due to their many side effects, such as nausea, arthralgia, "hypersensitivity syndrome", skin rash, severe liver and kidney toxicity, etc., their clinical application is greatly limited. Therefore, finding and developing new antihyperuricemia drugs with better efficacy and safety is still an urgent problem to be solved.

In recent years, studies have found that traditional Chinese medicine has a long history of treating gout. It exerts curative effects by reducing UA, anti-inflammation, anti-oxidation, and protecting the kidneys. It has the advantages of multiple targets and less toxic and side effects. Therefore, searching for safe and efficient drugs from the abundant resources of traditional Chinese medicine has become a research hotspot in the development of drugs for the treatment of gout.

Amomum villosum is the dry mature fruit of Amomum villosum Lour., Amomum villosum Lour.var.xanthioides T.L.Wu et Senjen or Amomum longiligulare T.L.Wu. It is one of the four traditional southern medicines in my country. Its medicinal and edible history has been more than 1,300 years, and it has been included in the list of homologous medicine and food by the National Health Commission. It is pungent in taste and warm in nature, and has the effects of dispelling dampness and appetizing, warming the spleen and stopping diarrhea, regulating qi and preventing miscarriage. It is used for resistance to damp oil, abdominal distension without hunger, spleen hyperuricemia, stomach deficiency and cold, vomiting and diarrhea, pregnancy nausea, and fetal restlessness. Modern studies have shown that the chemical components of amomum are mainly volatile oil components such as acetylborneol, camphor, borneol, and caryophyllene, and contain flavonoids such as quercetin, catechin, quercetin, and isoquercetin. Coumarins such as flavan coumarin and isoflavan coumarin, as well as organic acids such as vanillic acid, stearic acid, palmitic acid, etc., have anti-platelet aggregation, anti-oxidation, dilating blood vessels, inhibiting pepsin activity, Inhibit gastric acid secretion, promote intestinal motility, analgesic, antibacterial and other effects. So far, the anti-gout research of amomum is basically blank, and there is no report on the material basis and specific mechanism of this activity.

In the currently disclosed technology, Amomum and other various medicinal materials are basically used to form a traditional Chinese medicine prescription to prevent and treat gout; as the patent document with the notification number CN103585561B discloses a Chinese medicine composition for the treatment of gout, which is composed of Sophora flavescens , epimedium, aconite, Chuanxiong, Shiwei, papaya and amomum, and proved that the composition has a good effect of reducing uric acid on xanthine-induced mouse hyperuricemia model, p-xylene-induced mice The auricle swelling model has a good detumescence effect. However, since the traditional Chinese medicine composition is composed of seven medicinal materials, so as to achieve corresponding technical effects, although the composition formula contains amomum, amomum has the lowest weight fraction in the composition, so it is impossible to know the single Amomum medicinal materials play a specific role in the composition, and the combination of various medicinal materials may have toxic side effects such as gastrointestinal tract, liver, and kidney. The patent document with the notification number CN102940762B discloses a Chinese medicine for treating gouty arthritis, which is composed of Anemarrhena, Cortex Moutan, Kochia, Semen Plantaginis, Lianxu, Dayun, Amomum japonicus, Wuxueteng, Composition of yam, honey, rosa chinensis, chrysanthemum seed, ganoderma lucidum, cinnamon, Cynomorium cynomorium, Morinda officinalis, dodder seed, eucommia ulmoides, gecko, honeysuckle and prunella vulgaris. Amomum villosum in this document is a sub-seat of Hypocrella bambusae (Berk. et Br.) Sacc., a fungus of the family Hypocrenaceae, but Amomum villosum Lour. Ripe fruit, so the two are Chinese medicinal materials of completely different families and genera, and there is no conflict or duplication with our research object Amomum.

In the basic work in the early stage, the applicant established a method for measuring the in vitro inhibitory activity of XOD with xanthine as the substrate and XOD inhibitor as the reaction enzyme, investigated the inhibitory effect of 27 kinds of Chinese herbal extracts on XOD, and found that Amomum 95% ethanol extract (EA) had the strongest inhibitory activity (IC ₅₀ was 54.65μg/mL). Therefore, we further established a mouse model of hyperuricemia by intraperitoneal injection of potassium oxonate combined with intragastric administration of hypoxanthine, and a mouse model of gouty arthritis by using sodium urate crystals to investigate the anti-gout effect of EA and explore its mechanism. Provide scientific basis for the application of amomum in anti-gout drugs, health food and other aspects.

Contents of the invention

In order to solve the above problems, the present invention provides an application of the amomum extract in the preparation of UA-reducing and anti-gout arthritis drugs.

Specifically, it is realized through the following technical solutions:

1. The application of Amomum extract in the preparation of UA-lowering medicine.

Further, the preparation of the UA-lowering drug is to combine the extract of Amomum chinensis with the acceptable excipients in pharmacy and health food to prepare the desired preparation.

2. The application of an amomum extract in the preparation of anti-gout arthritis medicine.

Furthermore, the preparation of the anti-gout arthritis drug is to combine the extract of Amomum chinensis with the acceptable auxiliary materials in pharmacy and health food to prepare the desired preparation.

3. The above-mentioned amomum extract is made by using dry Amomum sativa, adding 95% ethanol which is 6.5-8 times the weight of amomum after crushing, refluxing extraction for 3-4 times, each extraction time is 1h, and combining the extracts Extract can be.

In summary, the beneficial effect of the present invention lies in: the present invention extracts Amomum amomum to obtain Amomum extract; by establishing a mouse model of hyperuricemia and a mouse model of gouty arthritis, the alcohol extraction of amomum is investigated. The anti-gout effect of the drug and its mechanism of action were discussed. Studies have 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, so as to achieve the purpose of reducing UA, so as to exert It has the effect of anti-gout, and EA can relieve and protect the liver and kidney damage caused by hyperuricemia. At the same time, EA has a significant improvement effect on gouty arthritis, can relieve the injury of mouse paw tissue caused by sodium urate crystals, reduce inflammatory cell infiltration, reduce the swelling of paw tissue, and inhibit IL-6, TNF-α and IL-6. The release of inflammatory factors such as -1β has a significant anti-gouty arthritis effect.

Compared with the existing anti-gout drugs, EA can reduce the level of UA by reducing the excessive production of UA and promoting the excretion of UA, and by inhibiting the release of inflammatory factors such as IL-6, TNF-α and IL-1β. And comprehensively play the role of anti-gout, it has the characteristics of better efficacy and better drug safety, therefore, EA can be applied to the medicine and health food for patients with gout, and has better development and utilization value and huge potential. social and economic benefits.

Description of drawings

Fig. 1 is the UPLC-LTQ-Qrbitrap-MS (ESI) total ion chromatogram of EA sample.

Figure 2 is the inhibitory effect of Amomum extract on XOD; wherein, (A) is EA, and (B) is the aqueous extract of Amomum.

Figure 3 is the effect of Amomum extract on the type of inhibition of XOD; where (A) is EA, (B) is the water extract of Amomum.

其中，(A)为体重，(B)为脾脏系数，(C)为肝脏系数，(D)为肾脏系数。^#P<0.05,^##P<0.01,与正常对照组比较；*P<0.05,**P<0.01与模型组比较。Figure 4 shows the effect of EA on the body weight and organ coefficient of mice with hyperuricemia

Among them, (A) is body weight, (B) is spleen coefficient, (C) is liver coefficient, (D) is kidney coefficient. ^# P<0.05, ^## P<0.01, compared with normal control group; *P<0.05, **P<0.01 compared with model group.

其中，(A)为UA,(B)为Creatinine,(C)为BUN,(D)为FEUA。^#P<0.05,^##P<0.01,与正常对照组比较；*P<0.05,**P<0.01与模型组比较。Figure 5 shows the effect 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 normal control group; *P<0.05, **P<0.01 compared with model group.

其中，(A)为血清XOD,(B)为肝XOD,(C)血清ALT,(D)肝ALT,(E)血清AST,(F)肝AST。^#P<0.05,^##P<0.01,与正常对照组比较；*P<0.05,**P<0.01与模型组比较。Figure 6 shows the effect of EA on serum and liver XOD, ALT and AST activities of hyperuricemia mice

Among them, (A) is serum XOD, (B) is liver XOD, (C) serum ALT, (D) liver ALT, (E) serum AST, (F) liver AST. ^# P<0.05, ^## P<0.01, compared with normal control group; *P<0.05, **P<0.01 compared with model group.

其中，(A)为血清SOD,(B)为肝SOD,(C)为肾SOD,(D)为血清MDA,(E)为肝MDA,(F)为肾MDA，(G)为肝IL-1β,(H)为肝TNF-α，(I)为肾IL-1β,(J)为肾TNF-α。^#P<0.05,^##P<0.01,与正常对照组比较；*P<0.05,**P<0.01与模型组比较。Figure 7 shows the effect of EA on the activity of SOD and MDA and the levels of IL-1β and TNF-α 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, (J) is kidney TNF-α. ^# P<0.05, ^## P<0.01, compared with normal control group; *P<0.05, **P<0.01 compared with model group.

Figure 8 is the effect of EA on the pathological morphology of kidney tissue in hyperuricemia mice; where (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 middle-dose group, (F) is the EA high-dose group.

其中，(A)为肝脏XOD蛋白Western blot电泳图,(B)为肝脏XOD蛋白相对表达量,(C)为肾脏GLUT9、URAT1、OAT1和ABCG2蛋白Western blot电泳图,(D)-(G)分别为肾脏GLUT9、URAT1、OAT1和ABCG2的蛋白相对表达，(H)-(K)分别为肾脏GLUT9、URAT1、OAT1和ABCG2的mRNA相对表达。^#P<0.05,^##P<0.01,与正常对照组比较；*P<0.05,**P<0.01与模型组比较。Figure 9 shows the effect of EA on the expression of liver XOD protein, kidney-related transporter protein and mRNA expression in 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 protein, (D)-(G) are the relative protein expressions of GLUT9, URAT1, OAT1 and ABCG2 in the kidney, respectively, and (H)-(K) are the relative mRNA expressions of GLUT9, URAT1, OAT1 and ABCG2 in the kidney, respectively. ^# P<0.05, ^## P<0.01, compared with normal control group; *P<0.05, **P<0.01 compared with model group.

Figure 10 is the effect of EA on body weight of gouty arthritis mice

Figure 11 is the effect of EA on the paw tissue swelling degree of gouty arthritis mice

^##P<0.01,与正常对照组比较；*P<0.05,**P<0.01,与模型组比较。Figure 12 is the effect of EA on the contents of IL-6 (A), TNF-α (B) and IL-1β (C) in gouty arthritis mice

^## P<0.01, compared with the normal control group; *P<0.05, **P<0.01, compared with the model group.

Figure 13 is the impact of EA on the pathological morphology of the paw tissue of model mice; where (A) is the normal group, (B) is the model group, (C) is the positive control group, (D) is the EA low-dose group, ( E) is the middle dose group of EA (F) is the high dose group of EA.

Detailed ways

The specific embodiments of the present invention will be described in further detail below, but the present invention is not limited to these embodiments, and any improvement or replacement on the basic spirit of this embodiment still belongs to the scope of protection claimed by the claims of the present invention.

Example 1

Preparation of Amomum 95% ethanol extract (EA):

Weigh 300g of dried Amomum sativa, add 3L of 95% ethanol respectively after pulverization, reflux extraction 3 times, each time for 1h, and combine the extracts, the obtained medicinal solution is vacuum filtered, concentrated, and freeze-dried to obtain the water-extracted amomum. Extract 28.16g, the extraction rate is 9.39%.

Screening Example 1

Preparation of amomum water extract:

Weigh 300g of dried Amomum sativa, add 3L of water after crushing, decoct 3 times, 1h each time, combine the extracts, the obtained medicinal solution is vacuum filtered, concentrated, and freeze-dried to obtain the water extract of Amomum leaching. The paste is 36.84g, and the extraction rate is 12.28%.

Screening example 2

Preparation of amomum methanol extract:

Weigh 300g of dried Amomum sativa, add 3L methanol respectively after pulverization, reflux extraction 3 times, each time for 1h, combine the extracts, the obtained medicinal solution is vacuum filtered, concentrated, and freeze-dried to obtain the water extract of Amomum leaching. The paste is 43.11g, and the extraction rate is 14.37%.

Different solvents (water, methyl alcohol, 95% ethanol) extracted Amomum extracts adopt the in vitro inhibitory activity assay method of XOD to screen the activity of each extract, wherein Amomum 95% ethanol extract (EA) has an inhibitory effect on XOD The in vitro inhibitory activity was the best (IC ₅₀ was 54.65 μg/mL), so 95% ethanol was selected as the extraction solvent, the number of extractions was 3 times, and the extraction time was selected as 1 h.

The extract that 1 embodiment 1 method obtains is to XOD in vitro inhibitory activity assay and enzyme kinetics investigation

1.1 Study on XOD inhibitory activity

The total volume of the reaction system is 200 μL, first add 50 μL of sample solution and enzyme solution (final reaction concentration: 0.05 U/mL) and mix, incubate at 25°C for 15 minutes, then add 50 μL of xanthine substrate solution (final reaction concentration: 150 μmol/L), continue After incubation for 20 min, 50 μL of 1 mol/L HCl solution was added to terminate the reaction, and the OD value was measured at a wavelength of 290 nm by a multifunctional microplate reader, and the inhibition rate was calculated. The IC50 of methanol extract, water extract and EA on XOD in vitro inhibitory activity were 328.49μg/mL, 254.00μg/mL, 54.65μg/mL respectively. Therefore, EA and water extracts were chosen to investigate the enzyme kinetics.

2.2 Enzyme kinetics study on XOD inhibitory activity

The concentration of the substrate xanthine (final concentration of 150 μmol/L) was fixed, and different concentrations of amomum extracts were added (final concentrations of EA were 0, 6.875, 13.75, 27.5, 55 μg/mL; final concentrations of water extracts were 0, 31.75 , 63.5, 127, 254μg/mL), and determine the effect on enzyme activity under different concentrations of XOD (final concentration is 0.00625, 0.0125, 0.025, 0.05, 0.1U/mL), to determine whether the inhibitory effect of Amomum extract on XOD reversible. The kinetic equation of the enzyme reaction rate under different concentrations of Amomum extract, the results are shown in Figure 1, the straight line basically passes through the origin, and with the continuous increase of the concentration of Amomum extract, the reaction rate decreases, indicating that Amomum The inhibitory effects of kernel water extract and EA on XOD were reversible.

Fix the enzyme concentration (0.05U/ml), add different quality amomum extract (EA final concentration is 0, 6.875, 13.75, 27.5, 55μg/mL; water extract final concentration is 0, 31.75, 63.5, 127, 254μg /mL). The effects of different substrate concentrations (reaction final concentrations of 37.5, 75, 150, 300, and 600 μmol/L) on enzyme activity were determined to determine the competition type of Amomum extract for XOD. As shown in the results in Figure 2, the Km value increases with the increase of the concentration of Amomum extract, and all the straight lines intersect in the second quadrant, indicating that the water extract of Amomum and EA belong to the competition-non-competition in the mixed inhibition type of XOD. competitive inhibition.

Since EA has the strongest inhibitory activity on XOD in vitro (IC ₅₀ is 54.65 μg/mL), EA is selected for the experimental research of lowering UA and anti-gouty arthritis.

Characterization of chemical composition of 2EA

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

The results show that each chromatographic peak in the EA sample has a better response in the positive ion mode (Figure 3), and the molecular weight, molecular formula, UV absorption figure and secondary mass spectrometry data of each chromatographic peak are obtained, and through the analysis of the data , preliminarily determined the structure of 6 relatively high chromatographic peaks in EA, and compared with the mass spectrum, retention time and other data of the reference substance, it was determined that the 6 peaks were Quercetin, Magnolin (Magnoshinin), Eudesobovatol A, Diisobutyl phthalate, Oleamide, 13E-Docosenamide.

Determination of oral maximum tolerated dose of 3EA in mice

10 Kunming mice, half male and half female, weighing 18-22g, fasted for 12 hours, given the maximum concentration (77.5mg/kg) to the stomach according to the volume of 0.4mL/10g, fed normally and observed the mice after 14 days Survival situation. No mice died after intragastric administration for 14 days, and the maximum tolerated oral dose of EA in mice was calculated to be 3100 mg/kg.

Effect of 4EA on reducing uric acid in mice with hyperuricemia and its mechanism

60 Kunming male mice were randomly divided into normal group, model group, allopurinol group (5mg/kg), EA low (97.5mg/kg), medium (195mg/kg), high dose group (390mg/kg) . The model was established by intraperitoneal injection of potassium oxonate (300 mg/kg) combined with intragastric administration of hypoxanthine (600 mg/kg). The drug group was intragastrically administered the corresponding drug 1 hour after the establishment of the model for 14 consecutive days. After administration on the 13th day, the mice were put into metabolic cages, the urine of the mice was collected within 24 hours, and the blood was collected 1 hour after the last administration. Whole blood and urine were centrifuged at 3000rpm for 10 minutes, and the supernatant was absorbed and stored at -20°C; liver, spleen, and kidney tissues were collected, except for the right kidney, which was fixed in 4% paraformaldehyde, and other organs were stored at -80°C save.

4.1 Effect of EA on body weight and organ coefficient of hyperuricemia mice

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

4.2 Effect of EA on serum and urine UA, Creatinine, BUN and FEUA in hyperuricemia mice

As shown in Figure 5A, compared with the normal group, the serum UA level of mice in the model group was significantly increased to 442.6 μmol/L (P<0.01), and the urinary UA level was significantly decreased (P<0.01). Compared with the model group, EA dose-dependently significantly 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.

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

4.3 Effect of EA on XOD activity in serum and liver of hyperuricemia mice

As shown in Figure 6A and B, compared with the normal group, the serum and liver XOD activities of mice in the model group were significantly increased (P<0.01). Compared with the model group, the serum and liver XOD activities of mice in each dose group of EA were significantly reduced (P<0.01), especially the serum and liver XOD activities of mice in the high dose group were 6.84U/L and 37.30U/g, respectively The proteins were basically close to the serum and liver XOD activities of normal mice. The results suggest that EA plays an anti-gout role by inhibiting the activity of XOD.

4.4 Effect of EA on serum and liver ALT and AST activities of hyperuricemia mice

As shown in Figure 6C-F, compared with the normal group, the serum and liver ALT and AST activities of the mice in the model group were significantly increased (P<0.01), which were 6.84U/L and 396.40U/g protein, 28.35U /L and 355.87U/g protein. Compared with the model group, the serum and liver ALT and AST activities of the mice in the middle and high dose groups of EA 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 respectively They 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 normal mice. The results suggest that EA has a better protective effect on liver injury in hyperuricemia mice.

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

As shown in Figure 7A-F, compared with the model group, the serum, liver, and kidney SOD activities of mice in the high-dose EA group were significantly increased (P<0.05, P<0.01), and the serum levels of mice in the middle and high-dose groups , liver, and kidney MDA activity were significantly reduced (P<0.01), the results showed that EA had a good antioxidant effect on hyperuricemia mice, suggesting that the mechanism of EA's anti-gout may be related to its antioxidant capacity.

4.6 Effect of EA on IL-1β and TNF-α contents in liver and kidney of mice with hyperuricemia

As shown in Figure 7G-J, compared with the normal group, the IL-1β and TNF-α levels in the liver and kidney of the mice in the model group were significantly increased (P<0.01), suggesting that hyperuricemia mice have certain liver and kidney inflammation damage. Compared with the model group, the contents of IL-1β and TNF-α in the liver and kidney of mice in each dose group of EA decreased, especially in the high-dose group (P<0.05, P<0.01). The content of 99.84pg/μg protein is close to the content of L-1β in the normal mouse kidney. The results suggest that EA may play an anti-gout effect by reducing the inflammatory response in mice with hyperuricemia.

4.7 Effects of EA on renal histopathological morphology of hyperuricemia mice

As shown in Figure 8, the epithelial cells of the mice in the normal group were arranged neatly, and the renal tissue morphology of the mice in the model group was abnormal, with increased glomerular cells, mesangial hyperplasia, inflammatory cell infiltration, and mild tubular edema and hyperplasia. In the allopurinol group, the glomerular telangiectasia and hemorrhage were observed, a large number of inflammatory cells infiltrated, the renal tubules were obviously dilated and proliferated, and the epithelial cells and cell debris in the lumen were shed. The lesions of the glomeruli and renal tubules of the 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 kidney injury in hyperuricemia mice.

4.8 Effect of EA on liver XOD protein expression, kidney-related transporter protein and mRNA expression in hyperuricemia mice

As shown in Figure 9A and B, compared with the model group, the expression of XOD protein in the liver of mice in each dose group of EA was significantly down-regulated (P<0.01), and these results were consistent with the results of XOD activity assay (Figure 6A and B), confirming again that EA plays an anti-gout role by inhibiting the activity of XOD.

As shown in Figure 9C-K, compared with the normal group, the mRNA and protein expressions of GLUT9 and URAT1 in the model group were significantly increased (P<0.01), while the mRNA and protein expressions of OAT1 and ABCG2 were significantly decreased (P<0.01). 0.01). Compared with the model group, the mRNA and protein expressions of GLUT9 and URAT1 in the middle and high dose groups of EA 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). 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.

Study on the effect and mechanism of 5EA on anti-gouty arthritis mice

60 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), high (390mg/kg ) dose group, 10 rats in each group, except the normal group and the model group, the other groups were intragastrically administered corresponding drugs once a day for 10 consecutive days. One hour after the last administration on the tenth day, 0.1 ml of MSU crystals were subcutaneously injected into the right sole of the mice to induce acute gouty arthritis. Before injection, after injection 0h, 1h, 4h, 12h, 24h, the mouse paw thickness was measured with a vernier caliper, which was used to calculate the paw swelling degree. After 24 hours of injection of MSU crystals, the mice were anesthetized, and the right grasping tissue was removed and fixed in 4% paraformaldehyde.

5.1 Effect of EA on body weight of gouty arthritis mice

It can be seen from Figure 10 that, compared with the normal group, there was no significant difference in the body weight of mice in each experimental group (P>0.05), suggesting that EA had no significant effect on the body weight of mice with gouty arthritis.

5.2 Effect of EA on paw tissue swelling in gouty arthritis mice

As can be seen from Figure 11, compared with the normal group, the mice in the model group significantly increased (P<0.01) at the same time point (0, 1, 4, 12, 24h) compared with the normal group. The swelling degree of the mice continued to increase 0-1h after modeling, reached the peak after 1h, and decreased after 4h, indicating that the modeling was successful. Compared with the model group, the middle and high dose groups of EA significantly reduced the degree of toe swelling at 12 hours and 24 hours after modeling (P<0.01), and the degree of swelling of the toes of mice in the high dose group at 24 hours was almost the same as that of the model group. The colchicine group was the same.

5.3 Effects of EA on the content of IL-6, TNF-α and IL-1β in gouty arthritis mice

Figure 12 shows that compared with the normal group, the levels of IL-6, TNF-α and IL-1β in the mouse paw tissues of 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 tissue of the mice in the middle and high dose groups of EA were significantly decreased (P<0.01), suggesting that EA can reduce the inflammation of the paw tissue of mice with gouty arthritis. The inflammatory response in the tissue thus plays an anti-gouty arthritis effect.

5.4 Effect of EA on the pathological morphology of paw tissue in mice with gouty arthritis

As shown in Figure 13, the connective tissue around the paws of mice in the normal group contained abundant capillaries, fat cells and nerves, and the muscle fibers were arranged neatly. In the model group, a large number of homogeneous strong basophilic substances accumulated locally, and obvious inflammatory cell infiltration was seen, accompanied by fibrous tissue hyperplasia. The tissue lesions around the paws of the mice in each dose group of EA were improved to varying degrees, and the infiltration of inflammatory cells was reduced, and the proliferation of fibrous tissue was rare. The results suggest that EA has a certain protective effect on the injury of paw tissue in mice with gouty arthritis.

6 comparative experiments

Preparing prescription one extract: (prescription source: the composition in the patent document whose announcement number is CN103585561B)

Weigh 8g of dried Sophora flavescens, 12g of Epimedium, 7g of Aconite, 6g of Rhizoma Chuanxiong, 10g of Shiwei, 7g of Papaya, and 5g of Amomum, decoct it twice with water, combine the extracts, concentrate and freeze-dry to obtain the composition Fang Yi Extract.

Preparation of the second extract of the prescription: (the source of the prescription: No. Ⅱ prescription of Xiaotong Decoction in "Gout Effectiveness Secret Prescription". Wang Maohong. Gout Effectiveness Secret Recipe [M]. Beijing: China Medical Science and Technology Press, 2014: 250)

Weigh 30g dried astragalus, 30g Smilax, 20g Fangfeng, 20g coix seed, 15g Atractylodes macrocephala, 15g plantain, 15g amomum, 12g tangerine peel, 12g Sichuan achyranthes bidentata, 10g licorice, decoct twice with water, combine The extract is concentrated and freeze-dried to obtain the extract of the second prescription.

Preparation of the three extracts of the prescription: (The source of the prescription: Huanglian Jiedu Decoction and Shengjiang Powder in "Gout Effects and Experiences Secret Recipe". Wang Maohong. Gout Effects and Experiences Secret Recipe [M]. Beijing: China Medical Science and Technology Press, 2014: 211)

Weigh 6g of dried Scutellaria baicalensis, 6g of Coptis chinensis, 12g of Cortex Phellodendri, 6g of Gardenia, 30g of Lonicerae vine, 6g of Rhubarb, 15g of silkworm, 15g of cicada, 15g of turmeric, 15g of Plantago, 6g of amomum, 6g of Rhizoma Rhizoma, 3g of licorice, After pulverization, add water and decoct twice, combine the extracts, concentrate and freeze-dry to obtain the extract of the third prescription.

Research on XOD inhibitory activity of different prescriptions:

The total volume of the reaction system is 200 μL, first add 50 μL of sample solution and enzyme solution (final reaction concentration: 0.05 U/mL) and mix, incubate at 25°C for 15 minutes, then add 50 μL of xanthine substrate solution (final reaction concentration: 150 μmol/L), continue After incubation for 20 min, 50 μL of 1 mol/L HCl solution was added to terminate the reaction, and the OD value was measured at a wavelength of 290 nm by a multifunctional microplate reader, and the inhibition rate was calculated.

Table 1 The inhibitory effects of different prescriptions on XOD

The results of XOD inhibitory activity are shown in Table 1. When the concentration of the above three formulations was 500 μg/mL, the in vitro inhibitory activities against XOD were 38.35%, 29.57%, and 45.39%, respectively. The IC ₅₀ of EA's in vitro inhibitory activity on XOD was 54.65 μg/mL. Compared with the above three formulations, EA showed a very obvious ability to inhibit XOD.

Claims (5)

1. An application of fructus Amomi extract in preparing medicine for reducing UA is provided.

2. An application of fructus Amomi extract in preparing medicine for treating gout and arthritis is provided.

3. The use of the fructus Amomi extract according to claim 1 or 2, wherein the fructus Amomi extract is prepared by pulverizing fructus Amomi, adding 95% ethanol with a mass of 6.5-8 times of that of fructus Amomi, reflux-extracting for 3-4 times, each for 1 hr, mixing the extractive solutions, and making into extract.

4. The use of the extract of amomum villosum in the preparation of a drug for reducing UA according to claim 1, wherein the preparation of the drug for reducing UA is to combine the extract of amomum villosum with pharmaceutically acceptable auxiliary materials in health-care food to prepare the required preparation.

5. The use of the fructus Amomi extract according to claim 2 for preparing anti-gouty arthritis drugs, wherein the preparation of anti-gouty arthritis drugs is to combine fructus Amomi extract with pharmaceutically acceptable auxiliary materials in health care foods to prepare the required preparation.

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