A mulberry bark extract with uric acid-lowering and renal-protective activities, its preparation method, and applications.

Abstract

This invention belongs to the field of active compound extraction technology, specifically relating to a mulberry bark extract with uric acid-lowering and renal-protective activities, its preparation method, and applications. The invention involves extracting mulberry bark slices by heating and reflux with an ethanol solution. The extract is then filtered and concentrated under reduced pressure to obtain an extract paste. The paste is subsequently extracted with petroleum ether, dichloromethane, ethyl acetate, and n-butanol. The extracted portion is then post-processed to obtain the mulberry bark extract. This invention provides an in-depth and systematic study of the chemical composition of mulberry bark and its in vitro xanthine oxidase inhibitory activity, in vivo uric acid-lowering activity, and renal-protective activity, providing detailed data support for the clinical application of the traditional Chinese medicine mulberry bark. The preparation method provided by this invention can effectively extract the effective active components from mulberry bark, and the extracted extract exhibits xanthine oxidase inhibitory activity, in vivo uric acid-lowering activity, and renal-protective activity.

Description

Technical Field

[0001] This invention belongs to the field of active compound extraction technology, specifically relating to mulberry bark extract with uric acid-lowering and kidney-protective activities, its preparation method, and its application. Background Technology

[0002] Uric acid is the end product of purine metabolism in the human body. Under normal circumstances, the production and excretion of uric acid in the body are balanced and stable. When purine metabolism is disordered and / or uric acid excretion is impaired, the uric acid level in the blood increases (at 37°C, serum uric acid is greater than 416 mg / L). μ (mol / L), causing hyperuricemia. Hyperuricemia is a group of chronic metabolic diseases caused by purine metabolism disorders. Its clinical features include long-term hyperuricemia and recurrent attacks of acute gouty arthritis, tophi deposition, tophaceous chronic arthritis and joint deformities caused by it. It often affects the kidneys, causing chronic interstitial nephritis and the formation of uric acid kidney stones.

[0003] About two-thirds of uric acid in the human body is excreted in urine via the kidneys. When uric acid enters the glomeruli through blood circulation, more than 98% of it is filtered by the glomeruli, with most of it being reabsorbed by the proximal tubules and then excreted in urine by the distal convoluted tubules. Patients with hyperuricemia are in a state of chronic uric acid supersaturation, increasing the burden on the kidneys. Over time, this leads to the formation of urate crystals and uric acid stones in the kidney tissue, triggering a strong inflammatory response and ultimately causing and developing uric acid kidney damage. Therefore, treating hyperuricemia requires simultaneously addressing inflammation and protecting the kidneys.

[0004] Currently, commonly used clinical drugs are allopurinol (which inhibits uric acid production) and benzbromarone (which promotes uric acid excretion), but both have significant side effects. Allopurinol easily causes hypersensitivity syndrome, and the mortality rate once a hypersensitivity reaction occurs is as high as 30%. HLA-B5801 and other related gene tests should be performed before using allopurinol; those who test positive are contraindicated. Therefore, the use of allopurinol is somewhat limited. Benzbromarone has serious hepatotoxicity; it was withdrawn from the European market in 2003 for this reason, and the State Drug Administration of China also warned of its liver damage risk in 2004. Hyperuricemia is a chronic metabolic disease, and patients must take medication for a long time to control the concentration of uric acid in their bodies; therefore, the toxic side effects of these drugs cannot be ignored. In view of this, there is an urgent clinical need for safer and more effective new uric acid-lowering drugs.

[0005] As of September 2019, among the 1,881 new drugs related to human diseases, excluding vaccines and biologics, approximately 62% of the small molecule drugs were directly derived from or related to natural products. Therefore, exploring active ingredients with uric acid-lowering and kidney-protective effects from the treasure trove of traditional Chinese medicine resources is an important direction for developing new uric acid-lowering drugs.

[0006] Xanthine oxidase (XOD) is a flavoprotein enzyme. XOD is the rate-limiting enzyme in the purine metabolism pathway and plays a decisive role in uric acid production. Inhibiting XOD activity reduces uric acid production, thereby lowering the concentration of uric acid in the body. Allopurinol lowers uric acid based on this principle, making XOD inhibitors an important direction in the development of drugs for hyperuricemia.

[0007] Mulberry bark ( Mori Cortex (This refers to the mulberry tree, a plant belonging to the genus *Morus* in the family Moraceae.) Morus alba The dried root bark of *L.* is mainly distributed in China, Korea, Japan, Mongolia, Central Asian countries, Russia, Europe, India, and Vietnam. Mulberry bark is a traditional Chinese medicine, included in the *Chinese Pharmacopoeia*, primarily used to clear lung heat, relieve asthma, and promote diuresis and reduce edema. Currently, there are almost no reports on research regarding the effects of mulberry bark extract on hyperuricemia and kidney protection, representing a research gap. Summary of the Invention

[0008] To address the technological gaps in the existing technology, this invention provides a mulberry bark extract with uric acid-lowering and kidney-protective activities.

[0009] The present invention also provides a method for preparing the above-mentioned mulberry bark extract.

[0010] Another object of the present invention is to provide the application of the above-mentioned mulberry bark extract.

[0011] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a mulberry bark extract with uric acid-lowering and kidney-protective activities, wherein the mulberry bark extract is composed of the following compounds: .

[0012] This invention also provides a method for preparing the above-mentioned mulberry bark extract with uric acid-lowering and renal-protective activities, comprising the following steps: (1) Extract the mulberry bark slices by heating and reflux with ethanol solution, filter the extract and concentrate under reduced pressure to obtain the extract paste; (2) The extract was dissolved in distilled water and extracted sequentially with petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract, n-butanol extract, and aqueous extract, respectively. The ethyl acetate extract, exhibiting the best activity, was obtained through in vitro XOD enzyme activity targeting. (3) The ethyl acetate extract was separated by silica gel column chromatography with DCM / MeOH gradient elution, each gradient elution for 5 column volumes to obtain 5 fractions Fr A−Fr E; Fr. A was separated by silica gel column chromatography with PE / EA elution to obtain 6 subsegments Fr. A1-Fr. A6; (4) Fr. A3 was separated by Sephadex LH-20 column and eluted with MeOH; then separated by silica gel column to obtain compounds morusin and kuwanon C; Fr. A4 was separated twice by Sephadex LH-20 column and then separated by silica gel column to obtain compound nigrasin C; Fr. A5 was separated by Sephadex LH-20 column and eluted with MeOH; separated by silica gel column (200-300 mesh silica gel), and finally purified by reversed-phase semi-preparative HPLC to obtain compounds moracin P and moracin O; (5) Fr. B was separated by silica gel column chromatography to obtain three components Fr. B1-Fr. B3: Fr. B1 was separated by Sephadex LH-20, silica gel column chromatography, and purified by reversed-phase semi-preparative HPLC to obtain compounds 4-hydroxyderricin and heyneanachalcone; Fr. B2 was separated by Sephadex LH-20, silica gel column chromatography, to obtain compound moracin N; Fr. B3 was separated by Sephadex LH-20, followed by reversed-phase MPLC, and finally by silica gel column chromatography to obtain compound 2. ' ,3,4 ' ,5,5 ' -pentahydroxy- cis -stilbene and the compound moracin P; (6) Fr. C was separated by silica gel column chromatography to obtain three components: Fr. C1-Fr. C3. Fr. C1 was separated by Sephadex LH-20 column chromatography to obtain compound moracin M. Fr. C3 was separated by silica gel column chromatography to obtain four subcomponents: Fr. C3-1-Fr. C3-4. Fr. C3-3 was separated by Sephadex LH-20 column chromatography with DCM / MeOH = 1 / 1 elution. Subsequently, it was separated by silica gel column chromatography to obtain compounds moracin B, moracin C, and 2-(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran. Fr. C3-4 was separated by silica gel column chromatography, and then separated by silica gel column chromatography and reversed-phase semi-preparative HPLC to obtain compounds 7,2 ' 4 '-trihydroxyflavanone and the compound 2,4-dihydroxybenzoic acid; (7) Fr. D was separated by silica gel column chromatography and gradient elution to obtain four components Fr. D1 - Fr. D4: Fr. D1 was separated by silica gel column chromatography to obtain three subcomponents Fr. D1-1 - Fr. D1-3; Fr. D1-2 was separated by Sephadex LH-20 column chromatography and silica gel column chromatography to obtain compound albanol B; Fr. D2 was separated by silica gel column chromatography and Sephadex LH-20 column chromatography, followed by silica gel column chromatography to obtain compound mulberrofuran G; Fr. D3 was separated by Sephadex LH-20 column chromatography and silica gel column chromatography to obtain compound cathayanin B.

[0013] Furthermore, in step (1), the ratio of the mulberry bark slices to the ethanol solution is 1 kg : 4 L; the concentration of the ethanol solution is 80%; the reflux extraction is performed 3 times, and the extraction is carried out at 80-90℃ for 5 h.

[0014] Furthermore, in step (2), the ratio of the mulberry bark slices to distilled water is 10 kg : 3 L; the amount of petroleum ether, dichloromethane, ethyl acetate, and n-butanol added is the same as the volume of distilled water; each solvent is used for extraction three times, each time for 2 hours.

[0015] Furthermore, in step (3), the DCM / MeOH gradient is 50 / 1→30 / 1→20 / 1→10 / 1→5 / 1→0 / 1; and the PE / EA = 30 / 1.

[0016] Furthermore, in step (4), the specific separation process is as follows: Fr. A3 was separated by Sephadex LH-20 column elution with MeOH; elution was then performed on a Sephadex LH-20 column with PE / MeOH / DCM = 2 / 1 / 1; followed by silica gel column elution with PE / EA = 3 / 1 to obtain compounds morusin and kuwanon C. Fr. A4 was separated by Sephadex LH-20 column elution with DCM / MeOH = 1 / 1; elution was then performed on a Sephadex LH-20 column with MeOH; followed by silica gel column elution with PE / EA = 5 / 1 to obtain compound nigrasin C. Fr. A5 was separated by Sephadex LH-20 column elution with MeOH; elution was then performed on a silica gel column with PE / EA = 4 / 1; finally, compounds moracin P and moracin O were purified by reversed-phase semi-preparative HPLC. The parameters of the reversed-phase semi-preparative HPLC were: YMC-Pack ODS-A, 250 × 10 mm, 5 μ m; H2O / MeOH = 40 / 60, λ = 210-385 nm, 2.5 mL / min.

[0017] Further, in step (5), Fr. B is separated using a silica gel column (200-300 mesh silica gel) with PE / EA = 8 / 1 elution to obtain three components Fr. B1-Fr. B3; Fr. B1 is separated using Sephadex LH-20 with DCM / MeOH = 1 / 1 elution; then separated using a silica gel column (200-300 mesh silica gel) with PE / EA = 8 / 1 elution; finally, the compounds 4-hydroxyderricin and heyneanachalcone are obtained by reversed-phase semi-preparative HPLC purification; Fr. B2 is separated using Sephadex LH-20 with DCM / MeOH = 1 / 1 elution; then separated using a silica gel column (200-300 mesh silica gel) with PE / EA = 6 / 1 elution to obtain the compound moracinN; Fr. B3 ... purified using Sephadex LH-20 with DCM / MeOH = 1 / 1 elution; then separated using a silica gel column (200-300 mesh silica gel) with PE / EA = 6 / 1 elution to obtain the compound moracinN; and Fr. B3 is purified using Sephadex LH-20 with DCM / MeOH = 1 / 1 elution; then separated using a silica gel column (200-300 mesh silica gel) with PE / EA = 6 / 1 elution. LH-20 separation, eluted with MeOH; followed by reversed-phase MPLC (RP-C18) separation, eluted with H2O / MeOH = 60 / 40; finally separated by silica gel column (200-300 mesh silica gel), eluted with PE / EA = 5 / 1 to obtain compound 2. ' ,3,4 ' ,5,5 ' -pentahydroxy- cis-stilbene and compound moracin P; the parameters of the reversed-phase semi-preparative HPLC were YMC-Pack ODS-A, 250 × 10 mm, 5 μ m; H2O / MeOH = 15 / 75, λ = 210-385 nm, 2.5 mL / min.

[0018] Further, in step (6), Fr. C is separated by a silica gel column (200-300 mesh silica gel) with PE / EA = 4 / 1 elution to obtain three components Fr. C1-Fr. C3; Fr. C1 is separated by a Sephadex LH-20 column with DCM / MeOH = 1 / 1 elution to obtain compound moracin M; Fr. C3 is separated by a silica gel column (200-300 mesh silica gel) with PE / EA = 3 / 1 elution to obtain four subcomponents Fr. C3-1-Fr. C3-4; Fr. C3-3 is separated by a Sephadex LH-20 column with DCM / MeOH = 1 / 1 elution; subsequently separated by a silica gel column (200-300 mesh) with DCM / MeOH = 30 / 1 elution to obtain compound moracin B; PE / EA = 3 / 1 elution to obtain compound moracin C; PE / AC = Elution with a 2 / 1 ratio yielded compound 2-(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran; Fr. C3-4 was separated by silica gel column chromatography (200-300 mesh) with PE / EA = 3 / 1 elution; subsequently, compounds 7,2 were separated by silica gel column chromatography with mobile phase DCM / MeOH = 20 / 1 and reversed-phase semi-preparative HPLC. ' 4 ' -trihydroxyflavanone and compound 2,4-dihydroxybenzoic acid; the parameters of the reversed-phase semi-preparative HPLC were YMC-Pack ODS-A, 250 × 10 mm, 5 μ m; H2O / ACN = 60 / 40, λ= 210-385 nm, 2.5 mL / min.

[0019] Further, in step (7), Fr. D is separated by silica gel column chromatography, eluted with a DCM / MeOH gradient of 50 / 1→30 / 1→20 / 1→10 / 1→5 / 1→0 / 1 to obtain four components Fr. D1-Fr. D4; Fr. D1 is separated by silica gel column chromatography, eluted with DCM / MeOH = 30 / 1 to obtain three subcomponents Fr. D1-1-Fr. D1-3; Fr. D1-2 is separated by Sephadex LH-20 column chromatography, mobile phase: DCM / MeOH = 1 / 1 and silica gel column chromatography, PE / EA = 4 / 1 to obtain compound albanol B; Fr. D2 is separated by silica gel column chromatography, mobile phase: DCM / MeOH = 30 / 1 and Sephadex LH-20 column chromatography, mobile phase: DCM / MeOH = 1 / 1, and then separated by silica gel column chromatography, DCM / MeOH = Elution with 25 / 1 yielded compound mulberrofuran G; Fr. D3 was separated by Sephadex LH-20 column with mobile phase: DCM / MeOH = 1 / 1, and silica gel column with mobile phase: DCM / MeOH = 6 / 1, yielding compound cathayanin B.

[0020] The present invention also provides the application of the above-mentioned mulberry bark extract in the preparation of a drug with uric acid-lowering and renal-protective activities.

[0021] The beneficial effects of this invention are as follows: (1) This invention has conducted in-depth and systematic research on the chemical composition of mulberry bark and its in vitro xanthine oxidase inhibitory activity, in vivo uric acid-lowering activity and renal protective activity, providing detailed data support for the clinical application of the traditional Chinese medicine mulberry bark.

[0022] (2) The preparation method provided by the present invention can effectively extract the effective active ingredients from mulberry bark. The extracted extract has xanthine oxidase inhibitory activity, in vivo uric acid lowering activity and kidney protective activity. Attached Figure Description

[0023] Figure 1 This refers to the inhibitory activity of allopurinol against XOD; Figure 2 The inhibitory activity of total extract of mulberry bark on XOD; Figure 3 The inhibitory activity of the dichloromethane extract fraction against XOD; Figure 4 The inhibitory activity of the ethyl acetate extract against XOD; Figure 5 The inhibitory activity of the n-butanol extract on XOD; Figure 6The inhibitory activity of the desaccharified aqueous extract against XOD; Figure 7 A timeline of the animal experiment process; Figure 8 Graphs showing the uric acid-lowering and kidney-protective indicators of different extraction fractions; Figure 9 Anatomical diagrams of the kidneys of mice in each group; Figure 10 Pathological sections of kidney tissue from each group of mice; Figure 11 Masson staining images of kidney tissue from mice in each group. Detailed Implementation

[0024] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0025] Example 1 (1) 10 kg of mulberry bark slices were extracted three times by reflux with 80% ethanol (40 L × 5 hours) at 85°C. The extract was filtered and concentrated under reduced pressure to obtain 1.4 kg of extract. (2) Dissolve the extract in 3 L of distilled water and extract it sequentially with 3 L of petroleum ether, dichloromethane, ethyl acetate and n-butanol. Each solvent is used for extraction 3 times (after shaking and extraction each time, the extract is separated and allowed to stand for 2 hours) to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract, n-butanol extract and aqueous extract. The petroleum ether extract, dichloromethane extract, ethyl acetate extract and n-butanol extract are concentrated and dried under reduced pressure.

[0026] (3) The aqueous extract was desugared by D101 macroporous adsorption resin, eluted with distilled water until the eluent was colorless, and the methanol eluent was collected, concentrated and dried under reduced pressure to obtain the desugared aqueous extract.

[0027] The yields of extracts obtained from each extraction fraction are shown in Table 1.

[0028] Table 1 Effect Example (a) The in vitro XOD inhibitory activity of petroleum ether, dichloromethane, ethyl acetate, n-butanol, and the aqueous extract after sugar removal was evaluated. Allopurinol (AP) was used as a positive control. The inhibition rate and IC50 were calculated. 50 The inhibitory activity of different extracts of mulberry bark on XOD was evaluated, as shown in Table 2.

[0029] Table 2 Note: - indicates not detected.

[0030] like Figures 1-6 As shown, the IC of MA-EA is calculated. 50 The value was 0.1160 mg / mL, indicating that MA-EA is a potentially effective XOD inhibitor.

[0031] (ii) The ethyl acetate extract of mulberry bark, which has the best XOD inhibitory activity, was used to verify the in vivo effects in mice with hyperuricemia.

[0032] ① Establishment and administration of hyperuricemia mouse model: like Figure 7 As shown: Kunming mice (male, SPF, 20±2 g) were acclimatized to the laboratory environment for one week, during which time they were fed normally and their body weight was monitored. All mice were randomly divided into two groups: a normal control group (n=6) and a model group (n=44). Except for the normal control group, the other groups were administered 500 mg / kg / day of potassium oxonate and 300 mg / kg / day of adenine (prepared by grinding in a mortar and pestle and adding to 0.5% CMC-Na) daily by gavage to induce hyperuricemia. The normal control group was administered 0.5% CMC-Na by gavage. All mice received the drugs once daily. After two weeks of drug administration, two mice were randomly selected from the model group to measure their serum uric acid concentration and kidney damage to determine whether the hyperuricemia model was successfully established. Successful establishment (serum uric acid exceeding 300 mg / kg / day) was considered a model. µ (mol / L) for subsequent drug administration.

[0033] The mice in the established model group were randomly divided into 7 groups (model group, allopurinol positive group, benzbromarone positive group, low-dose administration group, medium-dose administration group, high-dose administration group, and allopurinol and medium-dose combined administration group, with 6 mice in each group). The model group continued to be administered potassium oxonate at 500 mg / kg / d and adenine at 300 mg / kg / d by gavage daily. The normal control group was fed with normal feed and was treated by gavage with an equal volume of physiological saline to prepare 0.5% CMC-Na. One hour after inducing mouse modeling with adenosine and potassium oxonate in combination, mice in the low, medium, and high dose groups were administered different concentrations of ethyl acetate extract of mulberry bark (MA-EA) solution (100 mg / kg / d, 200 mg / kg / d, and 400 mg / kg / d) by gavage. The positive control group was administered allopurinol (0.5% CMC-Na) solution (10 mg / kg / d) by gavage. The MA-EA and allopurinol combined treatment group was administered a medium-dose mixture of allopurinol (10 mg / kg / d) and MA-EA (200 mg / kg / d) by gavage. Administered once daily at the same time for two weeks, with body weight recorded daily. Mice had free access to food and water during the experiment.

[0034] ②Collection of laboratory animal tissue samples: 1) Blood sample collection After 28 days of continuous administration, the bedding was changed one day in advance, and the mice were fasted for more than 12 hours. One hour after administration on the 28th day, the mice in each group were anesthetized by injection of 10% chloral hydrate. Blood samples were collected from the eyes of the mice in each group by enucleation. The blood samples were collected in 1.5 mL EP tubes containing the anticoagulant sodium heparin. After standing at 4 ℃ for 15 min, the samples were centrifuged at 3000 rpm at 4 ℃ for 10 min. The supernatant was collected and placed in 1.5 mL ordinary EP tubes and stored at -20 ℃ for later use.

[0035] 2) Harvesting of liver, kidney, and intestinal tissues after dissection After blood was collected from the mice, they were euthanized by cervical dislocation, fixed in a supine position, and their skin was disinfected with 75% alcohol. Sterilized surgical scissors and forceps were used to open the mice layer by layer along the linea alba. The abdominal cavity was rinsed with physiological saline, and the liver lobe was carefully cut off and placed in a 2 mL cryovial at -20°C. Both kidneys were carefully separated from the retroperitoneal position, and the kidney capsule was carefully removed. The left kidney was removed and placed in a 2 mL cryovial at -20°C. The small intestine from the stomach to the ileocecal junction was carefully combed out with forceps, and the duodenum, jejunum, and ileum were separated into three thirds. After rinsing the intestines with pre-cooled physiological saline, approximately 10 cm of the ileum was cut off starting 20 cm above the cecum and placed in a 5 mL centrifuge tube at -20°C. Once the liver, left kidney, and ileum tissues were collected, they were transferred to a -80°C freezer for storage until use. The right kidney and part of the ileum were fixed in a 4% paraformaldehyde ethanol solution, which was about 10 times the volume of the tissue, and stored at 4°C. The organs and tissues were used for subsequent experiments.

[0036] ③ Experimental results and analysis: like Figure 8 As shown in Figure A, compared with the normal control group (33.76 ± 5.09), μ Compared to the control group (mol / L), the serum uric acid (SUA) level in mice in the hyperuricemia model (HUA) group was significantly increased to 141.27 ± 38.33 mol / L. μ The result was mol / L (p<0.001), indicating that the hyperuricemia model had been successfully established. Compared with the HUA group, the SUA levels in mice in the positive control groups (allopurinol and benzbromarone) were significantly reduced to 28.49 ± 8.74 mol / L (p<0.001), indicating that the hyperuricemia model had been successfully established. μ mol / L and 87.94 ± 18.37 μ The SUA level was mol / L (p<0.001); however, the SUA levels in the low, medium, and high dose groups of the ethyl acetate fraction of mulberry bark (MA-EA) were significantly reduced to 68.36 ± 9.79 mol / L (p<0.001); μ mol / L, 65.69 ± 5.96 μmol / L, 60.90 ± 15.41 μ The concentration of uric acid in the group was 20.09 ± 4.23 μmol / L (p<0.001), showing a certain dose-dependent effect. Furthermore, the combination therapy group (20.09 ± 4.23 μmol / L) significantly reduced SUA levels (p<0.001), demonstrating a better uric acid-lowering effect than either drug alone. These data indicate that MA-EA has significant uric acid-lowering activity and is a potential drug for treating hyperuricemia.

[0037] Serum creatinine (SCr) and blood urea nitrogen (BUN) are important indicators for diagnosing renal function. To further evaluate the renal repair capacity of MA-EA, we measured and analyzed the SCr and BUN levels in hyperuricemic mice administered different doses of MA-EA. As shown in Figures B and C, compared with the Control group, the SCr and BUN levels in the HUA group were significantly increased (p<0.001). The positive control drugs allopurinol and benzbromarone had no significant effect on the SCr and BUN levels in hyperuricemic mice (p>0.05). However, compared with the HUA group, the high-dose MA-EA group significantly reduced SCr levels (p<0.01). Furthermore, different doses of MA-EA (100, 200, 400 mg / kg) also significantly reduced BUN levels in hyperuricemic mice (p<0.001). The Combination group significantly reduced SCr and BUN levels in hyperuricemic mice (p<0.001). This indicates that MA-EA has a certain reparative capacity for renal damage.

[0038] XOD is the main enzyme driving uric acid synthesis, and hyperuricemia is usually associated with upregulation of hepatic XOD activity. We investigated whether MA-EA inhibited XOD activity in hyperuricemic mice. As shown in Figures D and E, compared with the Control group, the serum and hepatic XOD activities of mice in the HUA group were significantly increased (p<0.001, p<0.05). Allopurinol (10 mg / kg), a commonly used XOD inhibitor in clinical practice, significantly reduced serum and hepatic XOD activity in hyperuricemic mice (p<0.001, p<0.01). Compared with the HUA group, the low, medium, and high dose groups of MA-EA (100, 200, 400 mg / kg) all significantly reduced serum and hepatic XOD activity in hyperuricemic mice (p<0.001), showing a dose-dependent effect. Furthermore, the combination group (10 mg / kg AP + 200 mg / kg MA-EA) also significantly reduced serum and liver XOD activity in hyperuricemic mice (p<0.001), even showing superior XOD inhibitory activity compared to allopurinol. These results suggest that MA-EA may exert its uric acid-lowering effect by inhibiting XOD activity and reducing uric acid production.

[0039] ④ Anatomical photographs, pathological sections, and analysis of the kidneys Hyperuricemia often progresses with kidney damage; therefore, we observed the effect of MA-EA on the appearance of the kidneys in hyperuricemic mice. The results showed that, as Figure 9 As shown: The kidneys of mice in the HUA group were enlarged, pale, and uneven in appearance. However, after MA-EA treatment, the kidney surface became smooth and returned to its reddish-brown color.

[0040] To further investigate the effects of MA-EA on the pathological changes of the kidneys in mice with hyperuricemia, histological examinations were performed on the kidneys of mice in different drug groups. Kidney sections were stained with hematoxylin and eosin (H&E) to observe morphology and perform pathological analysis.

[0041] like Figure 10 As shown, in the normal control group, the glomeruli of mice had normal morphology, clear tubular structures, and tightly packed tubular epithelial cells. In the hyperuricemia group, extensive renal tubular dilation was observed, cytoplasmic vacuolation of renal tubular epithelial cells was more common, the renal tubular structure was unclear, and connective tissue hyperplasia, neutrophil infiltration, and interstitial fibrosis were also present. Allopurinol (10 mg / kg) and benzbromarone (10 mg / kg) had no significant effect on the repair of kidney damage. Compared with the HUA group, different doses of MA-EA (100, 200, 400 mg / kg) and the Combination group improved kidney damage, and the MA-EAH and Combination groups showed significantly better improvement in kidney damage than the AP and Ben groups.

[0042] The above experimental data and histopathological results confirm that MA-EA has an anti-hyperuricemia effect and can effectively improve kidney damage caused by hyperuricemia.

[0043] Masson staining was used on kidney tissue sections to observe the collagen structure. For example... Figure 11 As shown, Masson staining results revealed significant renal tubular dilation, focal inflammatory cell infiltration, and marked renal fibrosis in the interstitial region of hyperuricemic mice. In the allopurinol group, interstitial fibrosis was significant, with numerous tissue vacuoles and multiple inflammatory manifestations in the kidneys. Compared to the HUA group, different doses of MA-EA (100, 200, 400 mg / kg) and the Combination group improved kidney damage, with MA-EAH and the Combination group showing significantly better improvement in kidney damage than the AP and Ben groups. Furthermore, the kidney tissues of mice in the MA-EA group and the treated group showed uniform staining, normal glomerular morphology and structure, clear tubular structure, tightly packed tubular epithelial cells, and numerous focal lymphocyte infiltrations around local blood vessels, indicating that MA-EAH and the Combination group both have a certain protective effect against kidney damage caused by hyperuricemia.

[0044] (3) To elucidate the material basis of MA-EA’s action, the part of MA-EA with the best XOD inhibitory activity was separated by silica gel (200-300 mesh) column and eluted by DCM / MeOH gradient (50 / 1→30 / 1→20 / 1→10 / 1→5 / 1→0 / 1), with each gradient eluting for 5 column volumes, to obtain 5 components Fr A−Fr E; (4) Fr. A was separated by a silica gel column (200-300 mesh silica gel) with petroleum ether / ethyl acetate = 30 / 1 elution to obtain 6 subfractions Fr. A1-Fr. A6. Fr. A3 was separated by a Sephadex LH-20 column with MeOH elution; then separated by a Sephadex LH-20 column with PE / MeOH / DCM = 2 / 1 / 1 elution; then separated by a silica gel column (200-300 mesh silica gel) with PE / EA = 3 / 1 elution to obtain the compound morusin (15.6 mg) and the compound kuwanon C (8.2 mg). Fr. A4 was separated by a Sephadex LH-20 column with DCM / MeOH = 1 / 1 elution; then separated by a Sephadex LH-20 column with MeOH elution; then separated by a silica gel column (200-300 mesh silica gel) with PE / EA = 5 / 1 elution to obtain the compound nigrasin C (7.9 mg). Fr. A5 was separated using a Sephadex LH-20 column with MeOH elution; followed by silica gel column separation (200-300 mesh silica gel) with PE / EA = 4 / 1 elution; finally, it was separated by reversed-phase semi-preparative HPLC (YMC-Pack ODS-A, 250 × 10 mm, 5...). μ m; H2O / MeOH = 40 / 60, λ The compound moracin P (5.3 mg, t) was purified by filtration at 210-385 nm (2.5 mL / min). R = 16.3 min) and compound moracin O (9.2 mg, t R = 18.2 min).

[0045] (5) Fr. B was separated using a silica gel column (200-300 mesh silica gel) with PE / EA = 8 / 1 elution to obtain three components (B1-B3). Fr. B1 was separated using a Sephadex LH-20 HPLC system with DCM / MeOH = 1 / 1 elution; followed by silica gel column separation (200-300 mesh silica gel) with PE / EA = 8 / 1 elution; finally, it was separated by reversed-phase semi-preparative HPLC (YMC-Pack ODS-A, 250 × 10 mm, 5 μ m; H2O / MeOH = 15 / 75, λ = 210-385 nm, 2.5 mL / min) purification yielded compound 4-hydroxyderricin (4.2 mg, t R = 4.4 min) and compound heyneanachalcone (3.8 mg, t) R= 17.7 min). Fr. B2 was separated by Sephadex LH-20 elution with DCM / MeOH = 1 / 1; followed by silica gel column separation (200-300 mesh silica gel) with PE / EA = 6 / 1 to give compound moracin N (5.8 mg). Fr. B3 was separated by Sephadex LH-20 elution with MeOH; then by reversed-phase MPLC (RP-C18) with H2O / MeOH = 60 / 40; finally, by silica gel column separation (200-300 mesh silica gel) with PE / EA = 5 / 1 to give compound 2. ' ,3,4 ' ,5,5 ' -pentahydroxy- cis -stilbene (4.5 mg) and compound moracin P (9.6 mg).

[0046] (6) Fr. C was separated by silica gel column chromatography (200-300 mesh silica gel), eluting with PE / EA = 4 / 1, yielding 3 components (C1-C3). Fr. C1 was separated by Sephadex LH-20 column chromatography, eluting with DCM / MeOH = 1 / 1, yielding compound moracin M (28.3 mg). Fr. C3 was separated by silica gel column chromatography (200-300 mesh silica gel), eluting with PE / EA = 3 / 1, yielding 4 subcomponents (C3-1 - C3-4). Fr. C3-3 was separated using a Sephadex LH-20 column with DCM / MeOH = 1 / 1 elution; subsequently separated using a silica gel column (200-300 mesh) with DCM / MeOH = 30 / 1 elution to give compound moracin B (14.8 mg); PE / EA = 3 / 1 elution to give compound moracin C (7 mg); PE / AC = 2 / 1 elution to give compound 2-(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran (8 mg). Fr. C3-4 was separated using a silica gel column (200-300 mesh) with PE / EA = 3 / 1 elution; subsequently separated using a silica gel column (mobile phase DCM / MeOH = 20 / 1) and a reversed-phase semi-preparative HPLC (YMC-Pack ODS-A, 250 × 10 mm, 5...). μ m; H2O / ACN = 60 / 40, λ Compound 7,2 was isolated by separation at 210-385 nm (2.5 mL / min). ' 4 '-trihydroxyflavanone (7 mg) and compound 2,4-dihydroxybenzoic acid (4.2 mg, t R = 7.3 min).

[0047] (7) Fr. D was separated on a silica gel column with a gradient elution of DCM / MeOH (50 / 1→30 / 1→20 / 1→10 / 1→5 / 1→0 / 1) to obtain four components (D1-D4). Fr. D1 was separated on a silica gel column with a DCM / MeOH = 30 / 1 elution to obtain three subcomponents (D1-1-D1-3). Fr. D1-2 was separated on a Sephadex LH-20 column (mobile phase: DCM / MeOH = 1 / 1) and a silica gel column (mobile phase: PE / EA = 4 / 1) to obtain compound albanol B (7.7 mg). Fr. D2 was separated by silica gel column chromatography (mobile phase: DCM / MeOH = 30 / 1) and Sephadex LH-20 column chromatography (mobile phase: DCM / MeOH = 1 / 1), followed by silica gel column chromatography with elution at DCM / MeOH = 25 / 1, yielding compound mulberrofuran G (10.7 mg). Fr. D3 was separated by Sephadex LH-20 column chromatography (mobile phase: DCM / MeOH = 1 / 1) and silica gel column chromatography (mobile phase: DCM / MeOH = 6 / 1), yielding compound cathayanin B (8.1 mg).

Claims

1. A method for preparing a mulberry bark extract with uric acid-lowering and renal-protective activities, characterized in that, Includes the following steps: (1) Extract the mulberry bark slices by heating and reflux with ethanol solution, filter the extract and concentrate under reduced pressure to obtain the extract paste; (2) The extract was dissolved in distilled water and extracted sequentially with petroleum ether, dichloromethane, ethyl acetate and n-butanol to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract, n-butanol extract and aqueous extract, respectively. The ethyl acetate extract with the best activity was obtained by in vitro XOD enzyme activity targeting. (3) The ethyl acetate extract was separated by silica gel column chromatography with DCM / MeOH gradient elution, each gradient elution for 5 column volumes to obtain 5 fractions Fr A-Fr E; Fr. A was separated by silica gel column chromatography with PE / EA elution to obtain 6 subsegments Fr. A1 - Fr. A6; (4) Fr. A3 was separated by Sephadex LH-20 column, eluted with MeOH, and then separated by silica gel column to obtain compounds morusin and kuwanon C; Fr. A4 was separated twice using Sephadex LH-20 and then by silica gel column separation to obtain compound nigrasin C; Fr. A5 was separated using Sephadex LH-20, eluted with MeOH, separated by silica gel column separation, and finally purified by reversed-phase semi-preparative HPLC to obtain compounds moracin P and moracin O; (5) Fr. B was separated by silica gel column chromatography to obtain three components: Fr. B1 - Fr. B3. Fr. B1 was separated by Sephadex LH-20 chromatography, followed by silica gel column chromatography, and purified by reversed-phase semi-preparative HPLC to obtain compounds 4-hydroxyderricin and Heyneanachalcone. Fr. B2 was separated by Sephadex LH-20 chromatography, followed by silica gel column chromatography to obtain compound moracin N. Fr. B3 was separated by Sephadex LH-20 chromatography, followed by reversed-phase MPLC, and finally by silica gel column chromatography to obtain compound 2',3,4',5,5'-pentahydroxy- cis -stilbene and the compound moracin P; (6) Fr. C was separated by silica gel column chromatography to obtain three components: Fr. C1 - Fr. C3; Fr. C1 was separated by Sephadex LH-20 column chromatography to obtain compound moracin M; Fr. C3 was separated by silica gel column chromatography to obtain four subcomponents: Fr. C3-1 - Fr. C3-4; Fr. C3-3 was separated by Sephadex LH-20 column chromatography with DCM / MeOH = 1 / 1 elution, followed by silica gel column chromatography to obtain compounds moracin B, moracin C, and 2-(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran; Fr. C3-4 was separated by silica gel column chromatography, followed by silica gel column chromatography and reversed-phase semi-preparative HPLC to obtain compounds 7,2 ' 4 ' -trihydroxyflavanone and the compound 2,4-dihydroxybenzoic acid; (7) Fr. D was separated on a silica gel column and eluted by gradient to obtain four components Fr. D1 - Fr. D4; Fr. D1 was separated on a silica gel column to obtain three subcomponents Fr. D1-1 - Fr. D1-3; Fr. D1-2 was separated by a Sephadex LH-20 column and a silica gel column to obtain compound albanol B; Fr. D2 was separated by a silica gel column and a Sephadex LH-20 column, and then separated by a silica gel column to obtain compound mulberrofuran G; Fr. D3 was separated by Sephadex LH-20 column and silica gel column to obtain the compound cathayanin B; The mulberry bark extract is composed of the following compounds: 。 2. The preparation method according to claim 1, characterized in that, In step (1), the ratio of mulberry bark slices to ethanol solution is 1 kg : 4 L; the concentration of ethanol solution is 80%; the reflux extraction is performed 3 times, and the extraction is carried out at 80-90℃ for 5 h.

3. The preparation method according to claim 1 or 2, characterized in that, In step (3), the DCM / MeOH gradient is 50 / 1→30 / 1→20 / 1→10 / 1→5 / 1→0 / 1; and PE / EA=30 / 1.

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