New application of Chinese tamarisk twigs

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and provides novel medical application of Chinese tamarisk twigs. The Chinese tamarisk twigs can be used for preparing medicines for preventing and treating hyperuricemia, and medicines for treating uric acid kidney stone, tophus or uric acid nephropathy caused by hyperuricemia. The invention also provides a preparation method of the Chinese tamarisk twig extract, which adopts water or / and ethanol for extraction. The new medical application of the Chinese tamarisk twigs is provided, the medical value of the Chinese tamarisk twigs is expanded, and compared with an existing uric acid reducing medicine allopurinol, the obtained extract can reduce the uric acid level of hyperuricemia, can inhibit rising of renal function indexes such as serum creatinine and urea nitrogen induced by hyperuricemia, improves liver and kidney tissue injuries, and has the advantages of being free of toxic and side effects, safe and reliable. The traditional Chinese medicine composition has the effect of improving liver and kidney functions and has good safety.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a new medicinal use of Xihe Liuxin. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Uric acid is the final metabolite of purine compounds in humans, and purine metabolism disorders lead to hyperuricemia. Hyperuricemia (HUA) refers to the fasting blood uric acid level of more than 420 μmol / L in adults, regardless of gender, on two different days under a normal purine diet. With the development of social economy and changes in people's lifestyle and dietary structure, the prevalence of hyperuricemia in my country has increased year by year, and is showing a trend of younger age. It has become the second largest metabolic disease after diabetes. Hyperuricemia is the pathological basis for the occurrence of gout, and is also an independent risk factor for diseases such as diabetes, cardiovascular and cerebrovascular diseases, chronic kidney disease, erectile dysfunction, and obstructive sleep apnea syndrome.

[0004] At present, the drug mechanisms of action for the treatment of HUA mainly include two modes: (1) inhibiting uric acid production. This type of drug reduces uric acid synthesis by inhibiting the activity of xanthine oxidase. Commonly used drugs include allopurinol and febuxostat. (2) promoting uric acid excretion. This type of drug inhibits renal tubular uric acid reabsorption and increases uric acid excretion. The representative drug is benzbromarone. However, the toxic and side effects of the above drugs are usually large. For example, allopurinol can cause skin allergic reactions and liver and kidney damage. In severe cases, fatal exfoliative dermatitis, severe erythema multiforme drug eruption, toxic epidermal necrolysis and other hypersensitivity syndromes may occur; febuxostat adverse reactions include liver damage, nausea, rash, etc.; benzbromarone has side effects such as gastrointestinal irritation, diarrhea, rash and liver damage; and the tolerance of the above drugs is generally low. In summary, these problems limit the clinical application of these drugs to a certain extent. Therefore, it is necessary to develop a drug for the treatment of hyperuricemia with no side effects or less side effects.

[0005] Tamarix Tamarix chinensisLour.) is a tree plant of the genus Tamarix in the family Tamarix. It is a characteristic salt-tolerant plant resource and is widely distributed in the Yellow River Basin. Tamarix branches and leaves are the main medicinal parts of Tamarix, known as Xiheliu in the field of traditional Chinese medicine. They have the effect of dispelling wind and dampness, and are clinically used to treat rheumatism and joint pain. Modern research shows that Tamarix branches and leaves mainly contain chemical components such as flavonoids, triterpenes, phenolic acids, and polysaccharides, and have pharmacological activities such as anti-cerebral ischemia, hypoglycemia, anti-inflammatory, analgesic, antibacterial, anti-tumor, and liver protection. For example, patent CN 114767666 A discloses the use of flavonoid component Tamarix lutein in the preparation of drugs for preventing or treating ischemic encephalopathy, and CN 111012808 A discloses the use of Tamarix extract in diabetes and its complications. However, there is no report on the use of Tamarix branch and leaf extract in the field of lowering uric acid. Summary of the invention

[0006] In view of the problem that current drugs for treating hyperuricemia have significant toxic and side effects, the present invention provides a use of Salix elata in inhibiting uric acid production and promoting uric acid excretion.

[0007] Another object of the present invention is to provide a Salix chelidonii extract, which has a good therapeutic effect on hyperuricemia and related diseases, especially can effectively improve liver and kidney function, and reduce the complications of liver and kidney damage caused by hyperuricemia.

[0008] Another object of the present invention is to provide a method for preparing the Salix chelidonii extract, which is simple and suitable for industrial application.

[0009] To achieve the above object, the present invention adopts the following technical solution.

[0010] The invention discloses an application of Salix elata in preparing a drug for preventing and treating hyperuricemia, and a drug for treating uric acid kidney stones, tophi or uric acid nephropathy caused by hyperuricemia.

[0011] The Xihe willow is Tamarix chinensis ( Tamarix chinensis Lour.) The drying method may be drying, airing, shade drying, or freeze drying. A typical method for preparing Xihe Liu is to pick the tender branches and leaves of Tamarix chinensis before the flowers bloom in spring and shade drying. The growth environment of Tamarix chinensis is preferably saline-alkali land; after comparing the content of some active ingredients and anti-inflammatory effects from different origins, the Tamarix chinensis planted in Dongying, Shandong was selected.

[0012] The salix elata is a salix elata medicinal material or extract. In order to improve the efficacy, the salix elata is a water extract or an ethanol extract.

[0013] The preparation method of the above-mentioned Salix elata extract comprises the following steps: extracting the Salix elata with a solvent to obtain an extract.

[0014] The solvent is water or an ethanol-water solution in any proportion. In order to increase the extraction rate, the sedge can be crushed, and the extraction can be performed multiple times. The crushing fineness and the number of extractions can be adjusted and selected by monitoring the biological activity of the extract in reducing uric acid.

[0015] In order to facilitate storage and transportation and subsequent drug preparation, the extract can also be concentrated or the solvent removed to obtain a solid extract.

[0016] Specifically, the above preparation method comprises the following steps: (1) crushing the Salix oxyphylla to obtain powder; (2) Extract the powder with a solvent 1-2 times and separate the solid and liquid to obtain an extract.

[0017] The fineness of the powder is that it can pass through a No. 2 sieve.

[0018] The solvent is water or a 30% w / v-70% w / v ethanol solution.

[0019] The mass volume ratio of powder and solvent is 1g:(6-15)mL.

[0020] The extraction temperature is 50°C-100°C.

[0021] The extraction method can be decoction, reflux extraction, and ultrasonic extraction.

[0022] Furthermore, the method further comprises step (3): concentrating and drying the extract to obtain an extract. The concentration and drying methods may be those commonly used in the preparation of food or medicine, such as vacuum distillation concentration, freeze concentration, microwave vacuum concentration, spray drying, freeze drying, oven drying, etc.

[0023] The characteristic spectrum of the extract of Salix oxyphylla obtained by the above preparation method includes 8 characteristic peaks, which are chlorogenic acid, gallic acid, ellagic acid, rutin, quercetin, isorhamnetin, kaempferol and rhamnocitric acid. t R =49.5 min) as the reference peak, and the relative elution times of the other peaks were 0.0689, 0.1231, 0.6409, 0.7084, 0.9281, 0.9562, and 1.0828, respectively; the mass spectrometry data were 353.0862 [MH] - , 169.0136 [MH] - , 300.9982[MH] - , 609.1457 [MH] - ,303.0493 [M+H] + , 315.0333 [MH] -,287.0545 [M+H] + ,301.0706[M+H] + .

[0024] The liquid chromatography conditions in the above characteristic spectrum are: octadecylsilane bonded silica gel column, 0.1% formic acid-water as mobile phase A, acetonitrile as mobile phase B; gradient elution: initial concentration is 95% A and 5% B; to 10 minutes, 90% A and 10% B; to 20 minutes, 85% A and 15% B; to 33 minutes, 80% A and 20% B; to 40 minutes, 70% A and 30% B; to 50 minutes, 52% A and 48% B; to 60 minutes, 5% A and 95% B; to 65 minutes, 5% A and 95% B.

[0025] The mass spectrometry conditions in the above characteristic spectrum are: electrospray ion source (ESI), positive and negative ion detection mode; scanning range m / z 100-1500; sheath gas volume flow rate 1132.67 L / h; auxiliary gas volume flow rate 283.17 L / h; capillary temperature 350℃; auxiliary gas temperature 320℃; spray voltage is +3.50 kV (positive ion mode) and -3.00 kV (negative ion mode); scanning mode is FullMs / dd-Ms 2 , FullMs resolution 70 000, dd-Ms 2 Resolution 17500.

[0026] The salix chelidonii extract contains chlorogenic acid, gallic acid, ellagic acid, rutin, quercetin, isorhamnetin, kaempferol and rhamnocitric acid, and the contents of the above compounds are 1.53-4.12 mg / g, 2.06-6.33 mg / g, 1.01-4.36 mg / g, 0.53-3.00 mg / g, 0.46-2.02 mg / g, 0.23-1.02 mg / g, 0.08-0.66 mg / g and 0.05-0.43 mg / g, respectively.

[0027] A drug for preventing and treating hyperuricemia, and a drug for treating uric acid kidney stones, tophi or uric acid nephropathy caused by hyperuricemia, comprising the above-mentioned Salix dasyphylla extract. In order to facilitate the dispersion of active ingredients, adjust the blood drug peak of active ingredients, reduce the decomposition of active ingredients, reduce adverse drug reactions, facilitate taking and processing preparations, etc., the above-mentioned drug also includes pharmaceutically acceptable excipients. The optional dosage forms can be tablets, capsules, granules, oral liquids, etc.

[0028] The present invention has the following advantages: The present invention provides a new medical use and extraction method of Salix xihelix, which expands the medicinal value of Salix xihelix. Compared with the existing uric acid-lowering drug allopurinol, the obtained extract can not only reduce the uric acid level of hyperuricemia, but also inhibit the increase of renal function indicators such as serum creatinine and urea nitrogen induced by high uric acid, reduce liver and kidney tissue damage, have the effect of improving liver and kidney function, and have good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Characteristic spectrum of chemical components of Salix dahurica extract, among which 1 is chlorogenic acid, 2. Gallic acid, 3 is ellagic acid, 4 is rutin, 5. Quercetin, 6. Isorhamnetin, 7. Kaempferol, 8. Rhamnolide; Figure 2 The effect of the extract of Salix elata on the changes in body weight of model mice; Figure 3 The effect of Salix elata extract on ankle swelling in model mice; Figure 4 The effect of Salix elata extract on the ankle diameter of model mice; Figure 5 The effect of the extract of Salix elata on the pathological morphology of ankle joint tissue in model mice; Figure 6 The effect of the extract of Salix elata on the levels of IL-6 and IL-1β in the serum of model mice; Figure 7 The effect of the extract of Salix elata on the uric acid content in model mice; Figure 8 The effect of Salix elata extract on the urea nitrogen content in model mice; Fig. 9 The effect of the extract of Salix elata on the creatinine content in model mice; Fig.10 The effect of the extract of Salix elata on the pathological morphology of renal tissue in model mice; Fig.11 The effect of the extract of Salix elata on the pathological morphology of liver tissue in model mice; Fig.12 Effect of Salix oxyphylla extract on xanthine oxidase content in liver of model mice. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0031] Example 1 Preparation of Salix oxyphylla Extract The harvested tender branches and leaves of Tamarix chinensis were placed in an open area with ventilation and light protection to dry in the shade for 72 hours, and then cut into sections of 1-5 cm in length to obtain Tamarix chinensis. The moisture, total ash and extract were tested according to the "Chinese Pharmacopoeia 2020 Edition". After passing the test, the extraction was carried out according to the following method: (1) crushing the sedge willow with a grinder and passing through a No. 2 sieve to obtain sedge willow powder; (2) Take 100 g of Eleutherodactyla serrata powder, add 8 times the amount of water, soak for 30 minutes, decoct for 1 hour, filter the residue, and filter again with filter paper to obtain extract A; Take 100g of Eleutherodactyla serrata powder, add 8 times the amount of water, soak for 30 minutes, decoct twice, each time for 1 hour, filter the residue and filter with filter paper to obtain extract B; Take 100g of Eleutherodactyla serrata powder, add 6 times the amount of water, soak for 30 minutes, decoct twice, each time for 1 hour, filter the residue and combine the extracts, filter with filter paper to obtain extract C; The above extracts were concentrated to a volume of 200-300 mL by rotary evaporation at a temperature of 40°C, a rotation speed of 100-140 rpm, and a vacuum degree of 40 mbar, and then dried by a freeze dryer for 24 hours to obtain Salix elata extracts A, B, and C.

[0032] The rutin and rhamnocitric acid in the above extracts were tested, and the contents were 1.82 mg / g and 0.10 mg / g; 1.95 mg / g and 0.15 mg / g; and 1.86 mg / kg and 0.13 mg / g, respectively.

[0033] Example 2 Preparation of Salix oxyphylla Extract The safflower prepared in Example 1 was extracted according to the following method: (1) crushing the sedge willow with a grinder and passing through a No. 2 sieve to obtain sedge willow powder; (2) Take 100 g of Eleutherodactyla serrata powder, add 8 times the amount of 70% ethanol, soak for 30 minutes, perform ultrasonic extraction at 80°C for 1 hour, filter the residue, and filter again with filter paper to obtain extract D; Take 100 g of Eleutherodactyla serrata powder, add 10 times the amount of 50% ethanol, soak for 30 minutes, perform ultrasonic extraction at 40°C for 1 hour, filter the residue, and obtain extract E again; Take 100g of Eleutherodactyla serrata powder, add 12 times the amount of 30% ethanol, soak for 30 minutes, perform ultrasonic extraction twice at 50°C, each time for 1 hour, filter the residue, and filter again with filter paper to obtain extract F; The above extracts were concentrated to a volume of 200-300 mL by rotary evaporation at a temperature of 40°C, a rotation speed of 100-140 rpm, and a vacuum degree of 40 mbar, and then dried by a freeze dryer for 24 hours to obtain Salix elata extracts D, E, and F.

[0034] According to the method in Example 1, the extracts were extracted with 70%, 50% and 30% ethanol, and the rutin and rhamnocitricin in the extracts were detected. The contents were 1.84 mg / g and 0.11 mg / g; 1.25 mg / g and 0.06 mg / g; and 1.46 mg / g and 0.08 mg / g, respectively.

[0035] Example 3 Effect of Salix elata extract on uric acid levels in hyperuricemic mice Fifty healthy male Balb / c mice aged 8 weeks, weighing 18-20g, were provided by Beijing Huafukang Biotechnology Co., Ltd. They were adaptively raised for 4 days in a GLP clean animal room at a temperature of (23±1.5)℃ and a humidity of 45%±15%. 48 mice with a concentrated weight were randomly divided into 6 groups according to their weight, with 8 mice in each group, namely blank control group, model control group, positive control group, and experimental groups 1-6.

[0036] After the adaptation period, the mice were gavaged once a day in the morning for 7 consecutive days. The experimental groups 1-6 were given extracts A, B, C, D, E, and F prepared in Example 1 and Example 2, respectively, with a dosage of 4.5 g / kg. They were suspended with 0.5% sodium carboxymethyl cellulose (CMC-Na) solution; the positive control group was given 10 mg / kg of allopurinol and suspended with 0.5% sodium carboxymethyl cellulose (CMC-Na) solution; the blank control group and the model control group were gavaged with 0.5% sodium carboxymethyl cellulose (CMC-Na) solution; each group was gavaged for 7 consecutive days.

[0037] 0.5 hours after intragastric administration on the morning of the 7th day, mice in each group were intraperitoneally injected to establish hyperuricemia models. The blank control group was intraperitoneally injected with 0.5% sodium carboxymethylcellulose (CMC-Na) solution; the model control group, positive control group, and experimental group were all injected with 250 mg / kg potassium oxonate (PO), which was dissolved with CMC-Na solution.

[0038] 1.5 hours after hyperuricemia modeling, the eyeballs of mice in each group were removed for blood collection, and the blood collection volume was not less than 0.5 mL. The blood samples were placed at room temperature for about 1 hour after collection. After the blood was completely coagulated, it was centrifuged at 3500 rpm at 4°C for 10 minutes. The serum was collected and re-centrifuged under the same conditions for 5 minutes, and then 0.2 mL of serum was taken to detect the UA value using a biochemical analyzer.

[0039] Table 1 Effects on serum uric acid levels in hyperuricemia mice (mean ± SD) Note: ## Compared with the blank control group, P < 0.01; Indicates P < 0.01 compared with the model control group; After 7 days of administration, the effects of each group on the serum uric acid level of hyperuricemia mice are shown in Table 1. It can be seen from Table 1 that compared with the blank control group, the uric acid in the serum of the mice in the model control group was significantly increased (P<0.01), which indicates that the hyperuricemia model was successfully established; compared with the model control group, the decrease in the uric acid level in the serum of the mice in the experimental groups 1-6 was significantly different (P<0.01).

[0040] Example 4 Quality Control of Salix chelidonii Extract The extracts of Salix elata obtained in Examples 1 and 2 were subjected to high performance liquid chromatography tandem mass spectrometry detection: Sample: Dissolve 0.1 g of extract in 1.0 mL as the sample solution; Reference substances: chlorogenic acid, gallic acid, ellagic acid, rutin, quercetin, isorhamnetin, kaempferol, rhamnocitric acid; Liquid chromatography conditions: chromatographic column: InfinityLab Poroshell 120 EC-C18 (4 μm, 4.6 × 150 mm), 0.1% by volume formic acid-water as mobile phase A, acetonitrile as mobile phase B, gradient elution as specified in Table 2, detection wavelength at 254 nm; flow rate 1.0 mL / min.

[0041] Table 2 HPLC gradient elution conditions Eight common chromatographic peaks were obtained, with peak 7 as the reference peak, and the relative retention times of the remaining components were 0.0689, 0.1231, 0.6409, 0.7084, 0.9281, 0.9562, and 1.0828, respectively; the standard reference fingerprint was generated as follows Figure 1 shown.

[0042] The eight common chromatographic peaks were detected by high performance liquid chromatography tandem mass spectrometry: The chromatographic conditions of the liquid chromatography were as shown above, and the mass spectrometry conditions were as follows: electrospray ion source (ESI), positive and negative ion detection modes; scanning range m / z 100-1500; sheath gas volume flow rate 1132.67 L / h; auxiliary gas volume flow rate 283.17 L / h; capillary temperature 350°C; auxiliary gas temperature 320°C; spray voltage was +3.50 kV (positive ion mode) and -3.00 kV (negative ion mode); scanning mode was FullMs / dd-Ms 2 , FullMs resolution 70 000, dd-Ms 2 Resolution 17500.

[0043] Table 3 Identification of common peaks The mass-to-nuclear ratios of the eight common chromatographic peaks are 353.0862 [MH] - ;169.0136 [MH] - ; 300.9982[MH] - ;609.1457 [MH] - ; 303.0493 [M+H] + ;315.0333 [MH] - ; 287.0545 [M+H] + ; 301.0706[M+H] + Compared with the literature records, they are chlorogenic acid, gallic acid, ellagic acid, rutin, quercetin, isorhamnetin, kaempferol and rhamnocitric acid.

[0044] Example 5 Effect of Salix oxyphylla extract on lowering uric acid A total of 30 8-week-old male SPF Balb / c mice weighing 18-20 g were purchased from Beijing Huafukang Biotechnology Co., Ltd. with certificate number SCXK (Beijing) 2019-0008 and were kept in a GLP clean animal room with a temperature of (23±1.5)°C and a humidity of 45%±15%.

[0045] After 7 days of adaptive feeding, the mice were randomly divided into the normal (Control) group, the hyperuricemia and gout model (Model) group, the positive drug allopurine treatment (ALL) group, the medium-dose Salix elata extract treatment (M-Dose, 4.5 g / kg) group, and the high-dose Salix elata extract treatment (H-Dose, 9.0 g / kg) group. The adaptive feeding was set as day 0. From day 0 to day 24, mice in all groups except the normal group were gavaged with 250 mg / kg potassium oxonate (PO) every day, and the normal group was gavaged with an equal volume of normal saline. On day 21, mice in all groups except the normal group were anesthetized with 2.5% isoflurane, and monosodium urate (MSU, 50 μL, 10 mg / kg) was injected into the right ankle joint of each mouse. During the modeling period from day 21 to day 24, mice in different groups were gavaged with allopurine or different concentrations of Salix elata extract, and mice in the normal group were gavaged with an equal volume of PBS.

[0046] 1. Influence of body mass From day 0 to day 24, the mice were weighed daily to monitor the effects of different extracts on the changes in body weight of hyperuricemia and gout model mice. Figure 2As shown in the results, the body weight of the mice in the model group decreased significantly during the PO injection modeling period, and allopurine and medium and high doses of Salix chelidonii extract could effectively improve the decreasing trend of body weight of the model mice.

[0047] 2. Ankle joint impact The ankle diameter of the mice was measured with a vernier caliper 12, 24, and 48 hours after the injection of monosodium urate. The diameter change index was expressed as the percentage between the right ankle diameter and the left ankle diameter. The gait behavior index of the mice was observed and recorded, and the gait behavior index of each group of mice was scored according to the Coderre gait grading and scoring standard (1 point: normal gait, fast crawling, no lameness; 2 points: relatively normal gait, relatively fast crawling, no lameness; 3 points: abnormal gait, slow crawling, lameness; 4 points: abnormal gait, very slow crawling, severe lameness). After the mice were killed, the right ankle joint was fixed in 4% paraformaldehyde for routine pathological sectioning and staining.

[0048] Table 4 Gait behavior index of mice (mean ± SD) Note: ## Compared with the blank control group, P < 0.01; Indicates P < 0.01 compared with the model control group; Compared with the model control group, P < 0.05 After injection of sodium urate, the changes in swelling and diameter of the right ankle joint of mice were as follows: Figure 3 and 4 The gait behavior index of mice is shown in Table 4, and the pathological morphology of the right ankle joint is shown in Figure 5 The results show: After the injection of MSU, the mice in each group showed varying degrees of ankle swelling at 12 hours, and the swelling in the model group was the most significant, indicating that the model was successfully established. Compared with the model group, medium and high doses of Salix elata extract and allopurinol could alleviate the swelling of the mice's feet to varying degrees.

[0049] 24 hours after modeling, compared with the blank group, the model group rats showed slow crawling, severe lameness, right lower limb detachment, and significantly reduced autonomous activity, indicating that the modeling was successful. Compared with the model group, medium and high doses of Salix elata extract and allopurine can improve the degree of lameness of mice, reduce gait behavior index, and the autonomous activity is close to normal.

[0050] The chondrocytes in the ankle joints of the mice in the normal group had normal morphology and were arranged neatly. The surface of the synovial tissue was smooth, and no congestion, edema or inflammatory infiltration was observed. The surface of the synovial tissue in the ankle joints of the mice in the model group was rough, with congestion and edema, and significant proliferation of synovial surface cells and subsynovial tissue, with multiple focal inflammatory infiltrations and focal aggregation of lymphocytes and monocytes. Compared with the model group, the degree of ankle joint lesions and cell proliferation in the allopurinol group were improved. The high-dose Salix chelidonii extract group had mild inflammatory infiltration, with neatly arranged chondrocytes and basically normal cell morphology. The medium-dose Salix chelidonii leaf extract group had improved local inflammatory infiltration and synovial hyperplasia in the ankle joints.

[0051] 3. Effects on inflammatory factors Blood was collected from the orbital venous plexus of mice, and serum was collected. ELISA kits were used to detect changes in the levels of inflammatory factors IL-6 and IL-1β. Figure 6 As shown: Compared with the blank group, the levels of IL-6 and IL-1β in the serum of the mice in the model group were significantly increased (P<0.01). Compared with the model group, the levels of IL-6 and IL-1β in the mice in the allopurinol group were significantly decreased (P<0.01, P<0.05); the IL-1β level in the mice in the high-dose Salix elata extract group was significantly decreased (P<0.05), and the IL-6 level was decreased but no significant difference was observed; the IL-6 and IL-1β levels in the mice in the medium-dose Salix elata extract group were decreased but no statistical difference was observed. It shows that different concentrations of Salix elata extract can alleviate the inflammatory response in the ankle joint of mice by inhibiting the release of related inflammatory factors.

[0052] 4. Effects on renal function On the 24th day after the administration, fresh urine samples were collected from mice using metabolic cages and centrifuged quickly. Blood was collected from the orbital venous plexus to collect serum and centrifuged. The supernatant was stored at -80°C. The detection kit was used to detect changes in uric acid, urea nitrogen, and creatinine in blood and urine samples. The results were as follows: Figure 7-9 As shown: Compared with the blank group, the levels of uric acid, urea nitrogen and creatinine in the serum of the mice in the model group were significantly increased (P < 0.01, P < 0.05, P < 0.01). Compared with the model group, the levels of uric acid in the serum of the mice in the allopurinol group, the medium-dose group and the high-dose group of Salix elata extract were significantly decreased (P < 0.01); the levels of urea nitrogen and creatinine in the serum were decreased to varying degrees, but no significant differences were observed; Compared with the blank group, the levels of uric acid, urea nitrogen and creatinine in the urine of the mice in the model group were significantly decreased (P<0.01, P<0.01, P<0.01). Compared with the model group, the levels of uric acid, urea nitrogen and creatinine in the urine of the mice in the allopurinol group were significantly increased (P<0.05); the levels of uric acid and creatinine in the urine of the mice in the medium-dose Salix elata extract group were increased to varying degrees but no significant difference was found, and the level of urea nitrogen in the urine was significantly increased (P<0.05); the levels of uric acid in the urine of the mice in the high-dose Salix elata extract group were increased to varying degrees but no significant difference was found, and the levels of urea nitrogen and creatinine were significantly increased (P<0.01 and P<0.01); This shows that different concentrations of Salix chelidonii extract can inhibit the production of uric acid and promote the excretion of uric acid; it can improve the formation and excretion of urea nitrogen and alleviate renal function damage.

[0053] The kidneys were obtained by dissection after the mice were killed and fixed in 4% paraformaldehyde for routine pathological sectioning and staining and observed under a 400× microscope. Fig.10 As shown, the changes in renal tissue morphology of mice in each group were observed under the microscope after HE staining. The overall staining degree of mice in the normal group was lighter, the cell morphology of glomeruli and renal tubules was normal, the nuclear membrane was intact, the chromatin structure was normal and the matrix density was uniform, and no obvious inflammatory infiltration and fibrosis were observed in the renal interstitium; the overall staining degree of renal tissue of mice in the model group was deepened, and cell proliferation was observed in the upper layer of the glomerular visceral cortex, proximal and distal renal tubules, and mononuclear cells and inflammatory infiltration and fibrosis were observed in the interstitium; compared with the model group, the cell structure of mice in the allopurinol group and the high-dose Salix elata extract group was significantly improved, but there were still many renal tubular cast cells, and the abnormal cell morphology in the upper layer of the glomerular visceral cortex, proximal and distal renal tubules of mice in the medium-dose Salix elata extract group was improved.

[0054] 5. Effects on liver function After blood was collected, the mice were anesthetized and killed, and the livers of the mice were taken. Part of the livers were fixed in 4% paraformaldehyde for routine pathological sectioning and staining, and observed under high-power field of view (400×). Another part of the liver tissue was taken, ground and centrifuged to obtain the supernatant, and the detection kit was used to detect the changes in the content of xanthine oxidase (XOD) in the liver tissues.

[0055] After HE staining, the morphological changes of liver tissues of mice in each group were observed under a microscope. Fig.11As shown in the figure, the liver cells of the mice in the normal group were arranged neatly, the structures such as the liver lobules were intact, no congestion and edema were observed around the hepatic sinusoids, there was no inflammatory infiltration in the liver interstitium, and no fibrous tissue hyperplasia in the portal area; the liver lobule structure of the mice in the model group was significantly damaged, the hepatic cords were disordered, the hepatocytes were deformed, the portal area and the central veins were significantly defibrous and connective, the cords became thicker and connected to each other to form fibrous septa, and the hepatic lobules had different degrees of degeneration and necrosis tendencies accompanied by inflammatory infiltration; the inflammatory infiltration of the liver tissue of the mice in the allopurinol group was significantly alleviated, the arrangement of the hepatocytes was improved, but there was still a tendency of fibrosis that gradually developed from the central area of ​​the hepatic lobule to the outside; the disorder of the liver fiber structure and the tendency of hepatocyte degeneration and necrosis in the mice in the medium-dose Salix elata extract group were improved to a certain extent; the liver fibrous connective hyperplasia of the mice in the high-dose Salix elata extract group was significantly reduced, the fibrous cords were narrowed, the fibrous septa and hepatocyte edema were significantly improved, and the degree of inflammatory infiltration was significantly alleviated.

[0056] Effect of xanthine oxidase content in mouse liver tissue Fig.12 As shown in the figure, compared with the blank group, the expression of xanthine oxidase in the liver tissue of mice in the model group was significantly increased (P<0.01). Compared with the model group, the expression of xanthine oxidase in the liver tissue of mice in the allopurinol group, medium-dose and high-dose Salix elata extract groups was significantly decreased (P<0.01); the expression of xanthine oxidase in the liver tissue of mice in the medium-dose Salix elata extract group was decreased but there was no statistical difference. This indicates that different concentrations of Salix elata extract may inhibit uric acid production by inhibiting the expression of the key enzyme XOD in the purine metabolism process.

[0057] Example 6 Preparation of a drug containing Salix chelidonii extract 1. Tablets The safflower prepared in Example 1 was extracted according to the following method: (1) crushing the sedge willow with a grinder and passing through a No. 2 sieve to obtain sedge willow powder; (2) Take 100 g of Eleutherodactyla serrata powder, add 6 times the amount of 43% ethanol, soak for 30 minutes, heat under reflux for extraction for 1 hour, remove the residue, and filter again with filter paper to obtain the extract; (3) The extract was concentrated to a volume of about 200 mL using a rotary evaporator at 40°C, a rotation speed of 100-140 rpm, and a vacuum degree of 40 mbar to obtain a concentrated solution.

[0058] The above concentrate is mixed with dextrin to form wet granules, and after drying, an appropriate amount of magnesium stearate is added, and after mixing evenly, the granules are pressed into tablets.

[0059] 2. Capsules The safflower prepared in Example 1 was extracted according to the following method: (1) crushing the sedge willow with a grinder and passing through a No. 2 sieve to obtain sedge willow powder; (2) Take 100 g of Eleutherodactyla serrata powder, add 15 times the amount of water, boil for 1 hour, filter to remove the residue, and filter the filtrate again with filter paper to obtain the extract; (3) The extract was concentrated to a volume of about 500 mL by rotary evaporation at a temperature of 40°C, a rotation speed of 100-140 rpm, and a vacuum degree of 40 mbar to obtain a concentrated solution; the concentrated solution was spray-dried to obtain a powdered extract.

[0060] The extract is filled into empty capsules to prepare capsules.

[0061] 3. Oral solution The safflower prepared in Example 1 was extracted according to the following method: (1) Take 100 g of Eleutherodactyla serrata powder, add 10 times the amount of 10% ethanol, soak for 30 minutes, perform ultrasonic extraction at 60°C for 1 hour, filter the residue and then filter with filter paper to obtain the extract; (2) The extract was concentrated to 200 mL using a rotary evaporator at 40 °C, 120 rpm, and 40 mbar to obtain a concentrated extract of the Salix elata extract.

[0062] The concentrate is added with flavoring agents and a proper amount of preservatives, homogenized, and finely filtered to obtain an oral solution.

[0063] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

[0064] Example 7 Comparison of anti-inflammatory activity of Salix elata extracts from different origins and harvesting periods The leaves and branches of Tamarix chinensis collected from saline-alkali land in different locations (Dongying, Shandong, Weifang, Shandong, and Hotan, Xinjiang) and different plant growth states (before flowering, flowering, and after leaf fall) were placed in an open area with ventilation and light protection to dry in the shade for 72 hours, and then cut into sections of 1-5 cm in length to obtain Tamarix chinensis. The moisture, total ash, and extract were tested according to the "Chinese Pharmacopoeia 2020 Edition". After passing the test, the extraction was performed according to the following method: (1) crushing the sedge willow with a grinder and passing through a No. 2 sieve to obtain sedge willow powder; (2) Take 5 g of Eleutherodactyla sibiricum powder, add 8 times the amount of water, soak for 30 minutes, decoct for 1 hour, filter the residue, and filter again with filter paper to obtain the extract; (3) The above extracts were concentrated to a volume of 200-300 mL by rotary evaporation at a temperature of 40°C, a rotation speed of 100-140 rpm, and a vacuum degree of 40 mbar, and then dried by freeze drying for 24 hours to obtain the Salix elata extracts XHL01-XHL06.

[0065] The contents of rutin and rhamnocitric acid in the extracts of Salix dahurica XHL01-XHL06 were determined respectively. LPS-induced mouse RAW 264.7 macrophages were used as an inflammatory model. The MTT method was used to detect the effects of XHL01-XHL06 on the activity of RAW264.7 cells at a concentration of 4-128 μg / mL. The Griess method was used to determine the content of NO at 20 μg / mL (Pang Daoran, Li Shanshan, Chen Xiaonan, et al. LC-MS-guided separation of two dihydrochalane cyclized trimers with anti-inflammatory activity from Dragon's Blood [J]. China Journal of Traditional Chinese Medicine, 2019, 44(13): 2675-2679.). Each treatment was repeated 3 times and the results are expressed as mean ± standard deviation.

[0066] Table 5 Contents of rutin and rhamnocitric acid in Salix cylindrica harvested from different origins and seasons Table 6 Inhibition rate of LPS-induced NO production in RAW264.7 cells by Salix sylvestris harvested from different origins and seasons Note: Indicates P < 0.01 compared with the model control group; Compared with the model control group, P < 0.05) The contents of rutin and rhamnocitin in different Salix oxyphylla extracts are shown in Table 5. The content of rhamnocitin in Salix oxyphylla collected in spring before blooming in various places is higher.

[0067] The results of the cell survival rate experiment showed that XHL01-XHL06 could promote cell growth to varying degrees at a concentration of 4-128 μg / mL. At a concentration of 20 μg / mL, XHL01-XHL06 had a significant inhibitory effect on NO production, with inhibition rates of 70.1%, 35.1%, 40.2%, 46.8%, 43.1% and 49.8%, respectively (Table 6). At the same time, a pairwise comparison of the samples in each group showed that XHL01 had the best activity (P<0.05). Therefore, XHL01-XHL06 all have potential anti-inflammatory activity, among which the anti-inflammatory activity of Salix sylvestris collected before flowering in Dongying, Shandong was the best.

Claims

1. A use of Salix elata in the preparation of a drug for preventing and treating hyperuricemia, and a drug for treating uric acid kidney stones, tophi or uric acid nephropathy caused by hyperuricemia.

2. The use according to claim 1, characterized in that: The Xihe willow is Tamarix chinensis ( Tamarix chinensis Lour.); preferably, the dried tender branches and leaves of Tamarix chinensis picked before the flowers bloom in spring, and the growth environment of Tamarix chinensis is saline-alkali land; the more preferred collection location is Dongying, Shandong; the said tamarisk is a medicinal material or extract of tamarisk; the said tamarisk is a water extract or an ethanol extract.

3. A method for preparing a Salix oxyphylla extract, characterized in that: The following steps are involved: After the elm of Salix xiheenensis is extracted with a solvent, an extract is obtained; the solvent is water or an ethanol-water solution in any proportion.

4. The preparation method according to claim 3, characterized in that: The method also includes the steps of crushing the sedge before extraction; performing extraction multiple times; and concentrating the extract or removing the solvent to obtain a solid extract.

5. The preparation method according to claim 3, characterized in that: The following steps are involved: (1) crushing the Salix oxyphylla to obtain powder; (2) extracting the powder with a solvent 1-2 times, and separating the solid and liquid to obtain an extract; The powder has a fineness that can pass through a No. 2 sieve; the solvent is water or a 30% w / v-70% w / v ethanol solution; the mass volume ratio of the powder to the solvent is 1 g:(6-15) mL; the extraction temperature is 50° C.-100° C.; and the extraction method is selected from decoction, reflux extraction, and ultrasonic extraction.

6. The preparation method according to claim 5, characterized in that: The preparation method also includes step (3): concentrating the extract and drying it to obtain an extract; the concentration and drying are selected from the group consisting of reduced pressure distillation concentration, freeze concentration, microwave vacuum concentration, spray drying, freeze drying, and oven drying.

7. A Salix chelidonii extract according to any one of claims 3 to 6, characterized in that Its characteristic spectrum It includes 8 characteristic peaks, namely chlorogenic acid, gallic acid, ellagic acid, rutin, quercetin, isorhamnetin, kaempferol and rhamnocitric acid. Taking the peak 7 kaempferol as the reference peak, the relative elution times of the other peaks are 0.0689, 0.1231, 0.6409, 0.7084, 0.9281, 0.9562 and 1.0828 respectively. The mass spectrometry data are 353.0862 [MH] - , 169.0136 [MH] - , 300.9982[MH] - , 609.1457 [MH] - ,303.0493 [M+H] + , 315.0333 [MH] - ,287.0545 [M+H] + ,301.0706[M+H] + . The liquid chromatography conditions in the above characteristic spectrum are: octadecylsilane bonded silica gel column, 0.1% formic acid-water as mobile phase A, acetonitrile as mobile phase B; gradient elution: initial concentration is 95% A and 5% B; after 10 minutes, 90% A and 10% B; after 20 minutes, 85% A and 15% B; By 33 min, 80% A and 20% B; By 40 min, 70% A and 30% B; by 50 min, 52% A and 48% B; by 60 min, 5% A and 95% B; by 65 min, 5% A and 95% B. The mass spectrometry conditions in the above characteristic spectrum are: electrospray ion source (ESI), positive and negative ion detection mode; scanning range m / z 100-1500; sheath gas volume flow rate 1132.67 L / h; auxiliary gas volume flow rate 283.17 L / h; capillary temperature 350℃; auxiliary gas temperature 320℃; spray voltage is +3.50 kV (positive ion mode) and -3.00 kV (negative ion mode); scanning mode is FullMs / dd-Ms 2 , FullMs resolution 70 000, dd-Ms 2 Resolution 17500.

8. A Salix chelidonii extract according to any one of claims 3 to 6, characterized in that It contains chlorogenic acid, gallic acid, ellagic acid, rutin, quercetin, isorhamnetin, kaempferol and rhamnocitric acid, and their contents are 1.53-4.12 mg / g, 2.06-6.33 mg / g, 1.01-4.36 mg / g, 0.53-3.00 mg / g, 0.46-2.02 mg / g, 0.23-1.02 mg / g, 0.08-0.66 mg / g and 0.05-0.43 mg / g, respectively.

9. A drug for preventing and treating hyperuricemia, and a drug for treating uric acid kidney stones, tophi or uric acid nephropathy caused by hyperuricemia, comprising Salix philadelphica or the Salix philadelphica extract as described in claim 7 or 8.

10. The drug according to claim 9, characterized in that It also includes pharmaceutically acceptable excipients; the drug preparation is selected from tablets, capsules, granules or oral liquids.

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