A total alkaloid extract of Corydalis dwarfi for preventing and treating hyperuricemia, and its preparation method and application

Abstract

The present invention belongs to the technical field of active compound extraction, and specifically relates to a total alkaloid extract of Corydalis dwarf with activity in preventing and treating hyperuricemia, as well as a preparation method and application thereof. The present invention comprises the following steps: extracting the medicinal material of Corydalis dwarf with an ethanol solution through cold soaking, filtering the extract and concentrating it under reduced pressure to obtain an extract concentrate; dissolving the extract concentrate in a dilute hydrochloric acid aqueous solution, extracting it with ethyl acetate while shaking and dropwise adding concentrated ammonia water, and then post-processing the extracted portion to obtain a total alkaloid extract of Corydalis dwarf. The present invention conducts in-depth and systematic research on the secondary metabolites of the total alkaloids of Corydalis dwarf and their in vitro xanthine oxidase inhibitory activity, in vivo uric acid-lowering activity and kidney-protective activity, providing detailed data support for the clinical application of the Tibetan medicine Corydalis dwarf. The preparation method provided by the present invention can effectively extract the effective active ingredients in Corydalis dwarf, and the extracted extract has xanthine oxidase inhibitory activity, in vivo uric acid-lowering activity and kidney-protective activity.

Description

Technical Field

[0001] The invention belongs to the technical field of active compound extraction, and particularly relates to a Corydalis dwarfi total alkaloid extract having activity in preventing and treating hyperuricemia, and a preparation method and application thereof. Background Art

[0002] Hyperuricemia is a chronic metabolic disease caused by excessive uric acid production or decreased excretion. The kidneys are key regulators of the uric acid cycle, and approximately 70% of the body's uric acid is excreted in the urine via the kidneys. When uric acid flows into the glomeruli through the bloodstream, over 98% of it is filtered through the glomeruli, with most of it being reabsorbed by the proximal renal tubules and then secreted by the distal convoluted tubules for excretion in the urine. Hyperuricemia patients are in a state of chronic uric acid oversaturation, which increases the burden on the kidneys. Approximately 90% of hyperuricemia patients experience renal excretion disorders, leading to kidney damage.

[0003] The first drug treatment strategy for hyperuricemia is to control blood uric acid. Currently, there are two main types of representative first-line clinical drugs: allopurinol, febuxostat (inhibit uric acid production) and benzbromarone (promote uric acid excretion). However, all of the above drugs have obvious side effects on the kidneys and liver. Hyperuricemia is a chronic metabolic disease, and patients must take medication for a long time to control the uric acid concentration in the body. However, there are currently few available drugs for this disease, and the toxic side effects of the drugs cannot be ignored. Therefore, there is an urgent need for new, safer and more effective uric acid-lowering drugs in clinical practice.

[0004] Xanthine oxidase (XOD) is the rate-limiting enzyme in the purine metabolic pathway and plays a crucial role in the production of uric acid. Allopurinol works by inhibiting XOD activity to reduce uric acid production and, consequently, lower uric acid concentrations in the body. Therefore, XOD inhibitors are an important area of ​​drug development for hyperuricemia.

[0005] The uric acid transport system comprises reabsorptive and secretory proteins that coordinately regulate uric acid excretion and maintain serum uric acid homeostasis. Key reabsorptive proteins, such as URAT1 and GLUT9, mediate the transfer of uric acid from the renal tubular lumen back into the blood, thereby regulating reabsorption through the renal apical membrane. Meanwhile, uric acid excretion is primarily promoted by the secretory protein ABCG2, which actively transports uric acid from the renal tubular epithelium to the tubular lumen.

[0006] Data from studies of renal cell apoptosis associated with nephrotoxic drugs suggest that apoptosis plays a role in regulating renal growth and remodeling. Apoptosis is also thought to play a key role in uric acid-induced renal injury or chronic gout. Renal biopsies from patients with hyperuricemia and chronic renal failure show typical signs of apoptosis in proximal tubular epithelial cells. The Bcl-2 family is a key regulator of apoptosis. Normally, Bcl-2 forms a heterodimer with Bax, inhibiting the pro-apoptotic effects of Bax.

[0007] In addition to renal cell apoptosis, inflammatory responses in the kidneys of hyperuricemic mice may also contribute to the progression of hyperuricemia-induced nephropathy. The transcription factor NF-κB plays a crucial role in inflammatory responses to various stimuli, and dysregulation of NF-κB may contribute to inflammation-related diseases. The transcription factor NF-κB plays a central role in inflammatory responses by activating proinflammatory genes. Dysregulation or prolonged activation of NF-κB can drive inflammatory diseases. Therefore, balanced NF-κB signaling is crucial for immune homeostasis and the prevention of pathological inflammation.

[0008] Corydalis hendersonii, also known as Nepalese Corydalis, is a plant of the genus Corydalis in the family Papaveraceae. It is primarily found in Tibet, my country, at altitudes between 4,200 and 5,200 meters. Its Tibetan medicinal name is Riguanzima. The Jingzhu Materia Medica records that it is used to clear blood heat, pulse heat, and dry blood stasis, and is used for various inflammatory and febrile conditions. The Complete Collection of Tibetan Prescriptions explicitly mentions Riguanzima in 109 prescriptions, and while Corydalis hendersonii is frequently used in these prescriptions, Tibetan medical literature provides relatively broad descriptions of its functions and indications, lacking specific treatments for its primary diseases. Modern pharmacological research is scarce. Currently, there are few reports on the use of Corydalis hendersonii extracts in preventing and treating hyperuricemia and kidney protection, representing a research gap. Summary of the Invention

[0009] In view of the technical gap in the prior art, the present invention provides a Corydalis dwarfii extract having the activity of preventing and treating hyperuricemia.

[0010] The present invention also provides a method for preparing the Corydalis dwarfii extract.

[0011] Another object of the present invention is to provide the application of the above-mentioned Corydalis dwarfii extract.

[0012] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are as follows:

[0013] The present invention provides a Corydalis dwarf extract having the activity of preventing and treating hyperuricemia. The Corydalis dwarf extract is composed of the following compounds:

[0014] .

[0015] The present invention also provides a method for preparing the above-mentioned total alkaloid extract of Corydalis dwarfi having the activity of preventing and treating hyperuricemia, comprising the following steps:

[0016] (1) Cold-extracting the medicinal material of Corydalis dwarfi with an ethanol solution, filtering the extract and concentrating it under reduced pressure to obtain an extract concentrate;

[0017] (2) The extract was dissolved in a dilute hydrochloric acid aqueous solution to obtain a corydalic acid aqueous solution, and then ethyl acetate was added and ammonia was added dropwise while shaking to extract the solution, thereby obtaining an ethyl acetate extract and an aqueous layer extract. After in vitro XOD enzyme activity-guided tracking, the ethyl acetate extract with the best activity was obtained.

[0018] (3) The ethyl acetate extract was subjected to UPLC-MS / MS targeted alkaloid detection to obtain a total alkaloid extract combination of Corydalis dwarfus, specifically: Berberrubine, 8,14-Dihydroflavinantine*, Tetrahydroprotopapaverine, Zarzissine, reticuline*, Bicuculline*, 1,2-Methylenedioxy-3,10,11-trimethoxynoraporphine, Cheilanthifoline, 13-Hydroxyl-N-methylcanadine, Allocryptopine, Hydrastine, Higenamine*, Norcoclaurine*, 1-(4-hydroxyphenyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-8-ol*, Allocryptopine opine,Salsolidine,(2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-[[(12R)-16-methoxy-11-methyl-3,5-dioxa-11-azapentacyclo[10.7.1.02,6.08,20.0 14,19]icosa-1(20),2(6),7,14,16,18-hexaen-17-yl]oxy]oxane-3,4,5-triol, Norchelerythrine*, Dihydrosanguinarine*, Salsolidine, Zarzissine, Hydrastine, Discretamine-glucose.

[0019] Preferably, in step (1), the ratio of the Corydalis dwarfii medicinal material to the ethanol solution is 1 kg: 5 L; the concentration of the ethanol solution is 95%; the number of cold soaking extractions is 3; each extraction is performed at room temperature of 25°C for 7 days.

[0020] Preferably, in step (2), the concentration of the dilute hydrochloric acid is 5‰, the extract is dissolved in a dilute hydrochloric acid aqueous solution, adjusted to pH 2-3, and filtered to obtain a filtrate; the amount of ethyl acetate added is the same as the volume of the dwarf corydalis acid aqueous solution; the concentration of the ammonia water is 10%, and the ethyl acetate is extracted three times, and the ammonia water is adjusted to pH 9-10 of the dwarf corydalis aqueous solution, and the solution is allowed to stand for 2 h after each extraction.

[0021] Preferably, in step (3), the liquid chromatography detection conditions are as follows: ① chromatographic column: Agilent SB-C18 1.8 µm, 2.1 mm * 100 mm; ② mobile phase: phase A is ultrapure water containing 0.1% formic acid, phase B is acetonitrile containing 0.1% formic acid, gradient elution; ③ flow rate 0.35 mL / min; column temperature 40°C; injection volume 2 µL.

[0022] The conditions of the gradient elution were as follows: the B phase ratio was 5% at 0.00 min, the B phase ratio linearly increased to 95% within 9.00 min and maintained at 95% for 1 min, the B phase ratio decreased to 5% from 10.00 to 11.10 min, and equilibrated at 5% for 14 min.

[0023] Preferably, the mass spectrometry detection conditions are as follows: electrospray ionization (ESI) temperature 550°C; ion spray voltage (IS) 5500 V (positive ion mode) 1-4500 V (negative ion mode); ion source gas I (GSI), gas II (GSII) and curtain gas (CUR) are set to 50, 60 and 25 psi, respectively, and the collision-induced ionization parameter is set to high; QQQ scan uses MRM mode, and the collision gas (nitrogen) is set to medium.

[0024] The present invention also provides the use of the total alkaloid extract of Corydalis dwarfi in preparing a medicine having the activity of preventing and treating hyperuricemia and protecting the kidney.

[0025] The beneficial effects of the present invention are:

[0026] (1) The present invention conducted an in-depth and systematic study on the chemical components of Corydalis dwarfi and its in vitro xanthine oxidase inhibitory activity, in vivo uric acid-lowering activity and kidney-protective activity, providing detailed data support for the clinical application of Corydalis dwarfi.

[0027] (2) The preparation method provided by the present invention can effectively extract the effective active ingredients from Corydalis dwarfi, and the extracted extract has xanthine oxidase inhibitory activity, in vivo uric acid-lowering activity and kidney protection activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1is the inhibitory activity of Corydalis dwarfi against XOD;

[0029] Figure 2 The PPI network of interactions between hyperuricemia-related genes and potential targets of total alkaloids from Corydalis dwarfi.

[0030] Figure 3 A timeline of the animal experiment process;

[0031] Figure 4 This is the analysis chart of uric acid lowering and kidney protection indicators of the total alkaloids;

[0032] Figure 5 The anatomy of the kidneys of mice in each group;

[0033] Figure 6 Pathological sections of kidney tissues of mice in each group (H&E staining, Masson staining);

[0034] Figure 7 The effect of total alkaloids from Corydalis dwarfi on the expression of XOD in the liver and uric acid transporter protein in the kidney tissue of mice;

[0035] Figure 8 The effect of total alkaloids from Corydalis dwarfi on the expression of apoptosis-related proteins in mouse kidney tissue;

[0036] Figure 9 The effect of total alkaloids from Corydalis dwarfi on inflammatory factors and NF-κB signaling pathway in mouse kidney tissue;

[0037] Figure 10 The purpose of cck-8 was to detect the effects of different concentration gradients of total alkaloids from Corydalis dwarf on HK-2 cell viability;

[0038] Figure 11 Detection of HK-2 cell viability for cck-8:

[0039] Figure 12 This is the effect of total alkaloids from Corydalis dwarf on the NF-κB signaling pathway in HK-2 cells. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further explained and illustrated below through specific embodiments.

[0041] Example 1

[0042] (1) 10 kg of dried roots of Corydalis dwarfi were soaked in ethanol at room temperature for 3 times (3 x 50 L), once every 7 days. The extract was recovered under reduced pressure to obtain 1169.4 g of total extract concentrate.

[0043] (2) The total extract was fully dissolved with 5‰ HCl solution and the pH was adjusted to 2. In the acid water portion, the pH was adjusted to 10 with ammonia water, and the extract was extracted three times with equal volumes of ethyl acetate to obtain the ethyl acetate extract and the aqueous layer extract. The ethyl acetate layers were combined and concentrated, and after in vitro XOD enzyme activity-guided tracking, 33 g of total alkaloids in the ethyl acetate layer were finally obtained.

[0044] (3) The ethyl acetate extract was subjected to UPLC-MS / MS targeted alkaloid detection; the specific detection conditions were:

[0045] Liquid chromatography detection conditions are as follows: ① Chromatographic column: Agilent SB-C18 1.8 µ m, 2.1 mm * 100 mm; ② Mobile phase: Phase A is ultrapure water (added with 0.1% formic acid), Phase B is acetonitrile (added with 0.1% formic acid); ③ Elution gradient: Phase B ratio is 5% at 0.00 min, linearly increases to 95% within 9.00 min and maintains at 95% for 1 min, Phase B ratio decreases to 5% from 10.00 to 11.10 min and equilibrates at 5% for 14 min; ④ Flow rate is 0.35 mL / min; Column temperature is 40°C; Injection volume is 2 µ L.

[0046] Mass spectrometry detection conditions were as follows: electrospray ionization (ESI) temperature 550°C; ion spray voltage (IS) 5500 V (positive ion mode) 1-4500 V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and collision-induced ionization parameters were set to high. QQQ scanning was performed in MRM mode, with the collision gas (nitrogen) set to medium. Declustering potential (DP) and collision energy (CE) were further optimized to achieve DP and CE for each MRM transition. A specific set of MRM transitions was monitored in each period, based on the metabolites eluting during that period, as shown in Table 1.

[0047] Table 1

[0048]

[0049] The extract yields obtained from each extraction part are shown in Table 2.

[0050] Table 2

[0051]

[0052] Effect embodiment

[0053] (I) The total extract, total alkaloids, and non-alkaloid extracts were evaluated for their in vitro XOD inhibitory activity. Allopurinol (AP) was used as a positive control. The inhibitory activity of each extract of Corydalis dwarfi against XOD was evaluated, and the inhibition rate and IC were calculated. 50 , as shown in Table 3.

[0054] Table 3

[0055]

[0056] Note: - means not detected.

[0057] like Figures 1-6 As shown, the IC of total alkaloids was calculated based on 50 The value was 0.66 mg / mL, indicating that the total alkaloids were a potential XOD inhibitor.

[0058] (II) Network pharmacology prediction of core uric acid-lowering targets of total alkaloids from Corydalis dwarfi

[0059] The standard SMILES of major alkaloids were retrieved using the PubChem database. The Swiss target prediction database was imported to predict potential drugs. The keyword "hyperuricemia" was searched in the GeneCards, DisGeNET, and DrugBank databases. Duplicate targets were deleted, and the names of all targets were standardized using the UniProt database. The cross-targets of major alkaloids and HUA were uploaded to STRING to obtain the protein-protein interaction (PPI) network. The PPI network of the target protein was then reconstructed in Cytoscape, and the key PPI module was determined using the MCODE algorithm in metscape. The results are shown in Figure 2. Figure 2 As shown in the figure, the core target of total alkaloids of Corydalis dwarfi for lowering uric acid is Bcl-2.

[0060] (3) The total alkaloids of Corydalis dwarfi, the fraction with the best XOD inhibitory activity, were used to verify their in vivo effects in hyperuricemia mice.

[0061] like Figure 3Shown: Kunming mice (male, SPF, 20±2 g) were adapted to the laboratory environment for 1 week. During this period, the mice were fed normally and their body weight was monitored. All mice were randomly divided into two groups, including 6 mice in the normal group and 36 mice in the model group. Except for the mice in the normal group, the mice in other groups were gavaged with 500 mg / kg / d potassium oxonate and 300 mg / kg / d adenine (ground in a mortar and added to 0.5% CMC-Na) every day to induce hyperuricemia. The mice in the normal group were gavaged with 0.5% CMC-Na. All mice received drug gavage once a day. After 2 weeks of drug gavage, 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. After successful establishment (serum uric acid exceeded 300 µ mol / L) for subsequent drug administration.

[0062] The established model mice were randomly divided into six groups (model group, allopurinol-positive group, benzbromarone-positive group, low-dose group, medium-dose group, and high-dose group, with six mice in each group). The model group continued to receive 500 mg / kg / day of potassium oxonate and 300 mg / kg / day of adenine via daily gavage. The normal control group was fed a standard diet and treated with an equal volume of saline mixed with 0.5% CMC-Na via gavage. One hour after the adenine and potassium oxonate co-administration, mice in the low-, medium-, and high-dose groups were gavaged with different concentrations of total alkaloids (TA) from Corydalis dwarfis (50 mg / kg / day, 100 mg / kg / day, and 200 mg / kg / day). Mice in the positive control group were gavaged with allopurinol (0.5% CMC-Na) solution (10 mg / kg / day). The drugs were administered once daily at the same time for two consecutive weeks, and body weights were recorded daily. During the experiment, mice had free access to food and water.

[0063] ① Collection of experimental animal tissue samples:

[0064] 1) Blood sample collection

[0065] After 28 consecutive days of administration, the bedding was changed one day in advance and the mice were fasted for more than 12 h. One hour after administration on the 28th day, the eyeballs of the mice in each group were removed for blood collection. The blood samples were collected in 1.5 mL EP tubes, allowed to stand at 4 °C for 15 min, and then centrifuged at 3000 rpm at 4 °C for 10 min. The supernatant was aspirated and placed in 1.5 mL ordinary EP tubes and kept in a -20 °C refrigerator for later use.

[0066] 2) Liver and kidney tissue collection after autopsy

[0067] After blood collection, mice were sacrificed by cervical dislocation and fixed in the supine position. The skin was then disinfected with 75% alcohol and pre-prepared. Pre-sterilized surgical scissors and forceps were used to open the abdomen layer by layer along the linea alba. The abdominal cavity was cleaned with normal saline, and the liver lobes were carefully excised and placed in 2 mL cryovials at -20°C. Both kidneys were carefully removed from the posterior peritoneal cavity. The renal capsule was carefully removed, and the left kidney was removed and placed in a 2 mL cryovial at -20°C. After liver and left kidney tissue collection, the tissues were transferred to a -80°C freezer until use. The right kidney was fixed in 4% paraformaldehyde in ethanol at a volume approximately 10 times the volume of the tissue and stored at 4°C. Organ tissue was used for subsequent experiments.

[0068] ② Experimental results and analysis:

[0069] like Figure 4 As shown in A, compared with the normal control group (137.96± 35.74 μ mol / L), the serum uric acid (SUA) level of mice in the hyperuricemia model (HUA) group was significantly increased to 235.93 ± 26.3 μ mol / L (p<0.001), indicating that the hyperuricemia model has been successfully established. Compared with the HUA group, the SUA level of mice in the positive control drug allopurinol and benzbromarone groups was significantly reduced to 150.93±29.07 μ mol / L and 181.26 ± 35.64 μ mol / L (p<0.001, p<0.01); while SUA levels were significantly reduced in the low, medium, and high dose groups of Corydalis dwarfi total alkaloids (TA), to 164.67±36.25μmol / L, 155.45±4.41μmol / L, and 143.40±43.33μmol / L, respectively (p<0.001), showing a dose-dependent effect. These data suggest that TA has significant uric acid-lowering activity and is a potential therapeutic agent for hyperuricemia.

[0070] Serum creatinine (SCr) and blood urea nitrogen (BUN) are important indicators for diagnosing renal function. To further evaluate the renal injury repair ability of TA, we measured and analyzed the SCr and BUN levels in hyperuricemic mice given different doses of TA. Figure 4As shown in Figures B and C, compared with the control group, SCr and BUN levels in mice in the HUA group were significantly increased (p < 0.001). Compared with the HUA group, the active drugs allopurinol and benzbromarone reduced SCr levels (p < 0.001) but had no significant effect on BUN levels (p > 0.05). However, compared with the HUA group, different doses of TA (50, 100, and 200 mg / kg) significantly reduced SCr and BUN levels in hyperuricemic mice (p < 0.001), indicating that TA has a certain ability to repair renal damage.

[0071] XOD is the main enzyme driving uric acid synthesis. Hyperuricemia is often associated with upregulation of hepatic XOD activity. We examined whether TA inhibited XOD activity in hyperuricemic mice. Figure 4 As shown in Figures D and E, compared with the control group, serum and liver XOD activities in mice in the HUA group were significantly increased (p < 0.001). Allopurinol (10 mg / kg), a commonly used XOD inhibitor in clinical practice, significantly reduced serum and liver XOD activities in hyperuricemic mice (p < 0.001, p < 0.01). Compared with the HUA group, the low, medium, and high doses of TA (50, 100, and 200 mg / kg) all significantly reduced serum and liver XOD activities in hyperuricemic mice (p < 0.001). These results suggest that TA may exert its uric acid-lowering effect by inhibiting XOD activity and reducing uric acid production.

[0072] ③ Kidney anatomy photos, pathological sections and analysis

[0073] Hyperuricemia is often accompanied by kidney damage, so we observed the effect of TA on the appearance of the kidneys in hyperuricemia mice. Figure 5 As shown: The kidneys of mice in the HUA group were pale and uneven in appearance. However, after TA treatment, the kidney surface became smooth and returned to a reddish-brown color.

[0074] To further investigate the effects of TA on renal pathological changes in hyperuricemia mice, the kidneys of mice in different drug groups were histologically examined. Kidney sections were stained with hematoxylin and eosin (H&E) to observe the morphology, and pathological analysis was performed.

[0075] like Figure 6As shown in Figure (A), the glomeruli of mice in the normal control group were normal in morphology, with clear tubular structures and densely packed tubular epithelial cells. In the hyperuricemia group, numerous renal tubules were dilated, cytoplasmic vacuolation of the tubular epithelial cells was more common, and the tubular structure was unclear. Allopurinol (10 mg / kg) and benzbromarone (10 mg / kg) had no significant effect on the repair of renal damage. Compared with the HUA group, different doses of TA (50, 100, and 200 mg / kg) improved renal damage.

[0076] The above experimental data and histopathological results confirmed that TA has anti-hyperuricemia effect and can effectively improve kidney damage caused by hyperuricemia.

[0077] like Figure 6 As shown in Figure B, kidney tissue sections were stained with Masson staining to observe collagen structure. Masson staining revealed significant tubular dilatation and interstitial fibrosis in the kidneys of hyperuricemia mice. Allopurinol-treated mice showed significant interstitial fibrosis and numerous vacuoles. Compared with the HUA group, TA at different doses (50, 100, and 200 mg / kg) improved renal damage. Kidney tissue in the TA group showed uniform staining, normal glomerular morphology, clear tubular structure, and densely packed tubular epithelial cells, indicating that TA has a protective effect against hyperuricemia-induced renal damage.

[0078] ④ Expression of XOD in liver and uric acid transporter protein in kidney tissue of mice in each group

[0079] like Figure 7 As shown, compared with the normal control group, HUA group showed significantly upregulated hepatic XOD protein expression (p < 0.001), while TA significantly reduced hepatic XOD protein expression in hyperuricemic mice (p < 0.1, p < 0.01, p < 0.001) in a dose-dependent manner. The potency of 100 mg / kg of total alkaloids was comparable to that of 10 mg / kg of allopurinol. These data suggest that downregulating XOD protein expression and thus reducing uric acid production is one of the mechanisms of TA's anti-hyperuricemic action.

[0080] Compared with the normal group, the expression of URAT1 and GLUT9 proteins in the kidneys of mice in the model group was significantly increased (p<0.01). Compared with the model group, the expression of URAT1 and GLUT9 proteins in the kidneys of mice in the TA-treated group was significantly decreased (p<0.01, p<0.001). This suggests that TA can downregulate the expression of URAT1 and GLUT9 proteins in the kidneys, thereby reducing renal uric acid reabsorption and exerting a uric acid-lowering effect.

[0081] Compared with the normal group, the expression of ABCG2 protein in the kidneys of mice in the model group was significantly decreased (p<0.01). Compared with the model group, the expression of ABCG2 protein in the kidneys of mice in the TA-treated groups was significantly increased (p<0.01, p<0.001). This indicates that TA can upregulate ABCG2 protein expression and promote uric acid excretion.

[0082] ⑤ Expression of anti-apoptotic proteins in the kidneys of mice in each group

[0083] like Figure 8 As shown, compared with the normal group, Bax protein expression in the renal tissue of mice in the model group was increased (p < 0.01), while Bcl-2 protein expression was decreased (p < 0.01). Compared with the model group, there was no significant difference in Bax and Bcl-2 protein expression in the positive drug allopurinol and benzbromarone groups. However, Bax protein expression in the renal tissue of mice in the TA group was significantly decreased (p < 0.1, p < 0.01), while Bcl-2 protein expression was significantly increased (p < 0.1, p < 0.01) in a dose-dependent manner. These results suggest that total alkaloids from Corydalis dwarfi ameliorates hyperuricemia-induced nephropathy by inhibiting cell apoptosis.

[0084] ⑥ Expression of inflammatory factors and related signaling pathways in the kidneys of mice in each group

[0085] like Figure 9 As shown, compared with the normal group, TNF-α expression was upregulated in the HUA group (p<0.01). TA significantly inhibited the expression of the proinflammatory cytokine TNF-α in a dose-dependent manner (p<0.01, p<0.001). Compared with the normal group, IL-6 expression was significantly upregulated in the HUA group (p<0.001). TA significantly inhibited the expression of the proinflammatory cytokine IL-6 in a dose-dependent manner (p<0.1, p<0.01, p<0.001). Compared with the normal group, IL-1β protein expression was significantly upregulated in the model group (p<0.001). Compared with the model group, TA significantly downregulated IL-1β protein expression in both groups in a dose-dependent manner (p<0.001). These results suggest that the renal protective effect of TA includes alleviating the inflammatory response in HUA mice.

[0086] Compared with the normal group, the model group p -IκBα / IκBα levels increased (p<0.01). Compared with the model group, allopurinol and benzbromarone in the positive control group showed no significant changes. p -IκBα / IκBα levels were decreased (p<0.1, p<0.01). Compared with the normal group, the HUA group p-p65 / p-65 increased significantly compared with the model group. There was no significant change in allopurinol and benzbromarone in the positive control group. Compared with the model group, the TA group p -p65 / p-65 were significantly downregulated (p<0.1, p<0.01). Therefore, TA may alleviate renal inflammation caused by hyperuricemia by inhibiting NF-κB signaling and suppressing the secretion of inflammatory factors.

[0087] (IV) The total alkaloids of Corydalis dwarfi, the fraction with the best XOD inhibitory activity, were used to verify the in vitro effect on HK-2 cells.

[0088] ① Cell culture

[0089] 1) Cell Passaging: When the cell density in the culture dish reaches approximately 80%-90%, the cells are passaged. Move the culture dish to a clean bench, discard the original culture medium, and wash twice with 2 mL of PBS. Discard the PBS, add 2 mL of trypsin to digest the cells. Discard the trypsin, and add 2 mL of the appropriate culture medium to terminate the digestion. Repeatedly pipette the bottom of the dish until a single-cell suspension is formed. Transfer 1 mL of the cell suspension to a new 10 cm culture dish, add 7-8 mL of the appropriate culture medium, and gently shake the dish back and forth to mix. Place the dish in a 37°C, 5% CO2 incubator.

[0090] 2) Cell Cryopreservation: Cryopreserve cells that are in good growth condition. Move the culture dish to a clean bench, discard the original culture medium, and rinse once with 2 mL of PBS. Discard the PBS and add 2 mL of trypsin for digestion. Discard the trypsin and add 2 mL of prepared freezing solution (200 µL DMSO + 1800 µL fetal bovine serum) and mix thoroughly by inhalation. Transfer the tube to a cryovial, seal with parafilm, label, and incubate at 4°C for 30 min, then at -20°C for 30 min. Finally, store in a -80°C freezer.

[0091] 3) Cell Thawing: Remove the frozen renal tubular epithelial cells from the -80°C freezer and immediately place them in a 37°C constant-temperature water bath. Shake rapidly until the freezing solution is completely thawed. Wipe the outer surface of the container dry and spray with 75% ethanol. Quickly transfer the container to a clean bench, remove the sealing film, and disinfect the container with an alcohol burner. Blow the freezing solution evenly through the air and transfer it to a 10 cm culture dish. Add 7 mL of culture medium containing 10% fetal bovine serum and 1% double-antibody. Gently shake the culture medium back and forth to evenly distribute the solution. Place the dish in a 37°C, 5% CO2 incubator. Once the cells have fully adhered, replace the culture medium with fresh medium.

[0092] ②CCK-8 assay for cell viability

[0093] The cells in the logarithmic growth phase were digested with trypsin, culture medium was added and blown into a single cell suspension, and counted using a counting plate. The control group and the experimental group were both counted at 1*105 Cells were seeded / well in a 96-well plate. 100 µL of culture medium was added to each well, and the 96-well plate was incubated in a 37°C, 5% CO2 incubator for 24 h. After the cells had fully adhered, the 96-well plate was removed and placed in a clean bench. The original culture medium was discarded. 100 µL of normal culture medium was added to the control group, and 100 µL of uric acid and culture medium containing different concentrations of TA were added to the experimental groups, respectively. Three replicates were set up, and the 96-well plate was incubated in a 37°C, 5% CO2 incubator for 48 h. After 48 h, the original culture medium of the experimental and control groups was discarded, and 100 µL of culture medium containing 10% CCK-8 solution was added to each well. The blank group was treated with 100 µL of culture medium containing 10% CCK-8 solution without cells. The plates were incubated in a 37°C, 5% CO2 incubator for 2 h. The OD value of each well was measured at a wavelength of 450 nm using a microplate reader. The cell growth inhibition rate was calculated as follows:

[0094] Inhibition rate (%) = (OD e -OD0) / (OD c -OD0)×100%

[0095] Here, OD e Represents the OD value of each experimental group, OD c represents the OD value of the control group, and OD0 represents the OD value of the blank group.

[0096] ③Experimental results and analysis

[0097] In order to detect the maximum safe concentration of TA that can play a protective role on HK-2 cells, the cell activity of each experimental group was studied by CCK-8 method. Figure 10 As shown, compared with the normal control group, as the TA concentration increased, cell viability gradually decreased, demonstrating that increasing TA concentrations have a deleterious effect on HK-2 cells. At a TA concentration of 40 µg / mL, cell viability exceeded 85%, indicating that this concentration is the maximum safe TA concentration.

[0098] ④Expression of anti-apoptotic proteins in HK-2 cells

[0099] As shown in Figure 11, compared with the normal group, the model group showed increased Bax protein expression (p < 0.01) and decreased Bcl-2 protein expression (p < 0.01). There were no significant differences in Bax and Bcl-2 protein expression in the positive drug allopurinol and benzbromarone groups compared with the model group. However, the TA group showed a significant decrease in Bax protein expression (p < 0.1, p < 0.01), while a significant increase in Bcl-2 protein expression (p < 0.01). These results suggest that total alkaloids from Corydalis dwarfi improve hyperuricemia-induced nephropathy by inhibiting cell apoptosis.

[0100] ⑤Expression of inflammatory factors and related signaling pathway proteins in HK-2 cells

[0101] As shown in Figure 12, compared with the normal group, the model group p -IκBα / IκBα levels increased (p<0.001). Compared with the model group, there was no significant change in the positive control group after allopurinol and benzbromarone administration. p -IκBα / IκBα levels were decreased (p<0.01). Compared with the normal group, the HUA group p -p65 / p-65 increased significantly (p<0.01), compared with the model group, there was no significant change in the positive control group after allopurinol and benzbromarone administration, compared with the model group, TA group p -p65 / p-65 were significantly downregulated in a dose-dependent manner (p<0.1, p<0.01). Therefore, TA may alleviate renal inflammation caused by hyperuricemia by inhibiting NF-κB signaling and suppressing the secretion of inflammatory factors.

Claims

1. A method for preparing a total alkaloid extract of Corydalis dwarfi having activity in preventing and treating hyperuricemia, characterized in that: The following steps are involved: (1) Cold-extracting the medicinal material of Corydalis dwarfi with an ethanol solution, filtering the extract and concentrating it under reduced pressure to obtain an extract concentrate; (2) The extract was dissolved in a dilute hydrochloric acid aqueous solution to obtain a corydalic acid aqueous solution, and then ethyl acetate was added and ammonia was added dropwise while shaking to extract the solution, thereby obtaining an ethyl acetate extract and an aqueous layer extract. After in vitro XOD enzyme activity-guided tracking, the ethyl acetate extract was obtained; (3) Targeted alkaloid detection of the ethyl acetate extract by UPLC-MS / MS; The total alkaloid extract of Corydalis dwarfi consists of the following compounds: 。 2. The preparation method according to claim 1, characterized in that In step (1), the ratio of the dwarf Corydalis medicinal material to the ethanol solution is 1 kg: 5 L; the concentration of the ethanol solution is 95%; the number of cold soaking extractions is 3 times; each extraction is performed at room temperature of 25°C for 7 days.

3. The preparation method according to claim 1, characterized in that In step (2), the concentration of the dilute hydrochloric acid is 5‰, the extract is dissolved in a dilute hydrochloric acid aqueous solution, the pH is adjusted to 2-3, and the filtrate is filtered; the amount of ethyl acetate added is the same as the volume of the dwarf corydalis acid aqueous solution; the concentration of the ammonia water is 10%, and the ethyl acetate is extracted three times, and the ammonia water is adjusted to a pH of 9-10 of the dwarf corydalis aqueous solution, and the solution is allowed to stand for 2 hours after each extraction.

4. The preparation method according to any one of claims 1 to 3, characterized in that In step (3), the liquid chromatography detection conditions are as follows: ① Chromatographic column: Agilent SB-C18 1.8 µ m, 2.1 mm * 100 mm; ② Mobile phase: Phase A is ultrapure water containing 0.1% formic acid, Phase B is acetonitrile containing 0.1% formic acid, gradient elution; ③ Flow rate is 0.35 mL / min; Column temperature is 40°C; Injection volume is 2 µL.

5. The preparation method according to claim 4, characterized in that The gradient elution conditions were as follows: the B phase ratio was 5% at 0.00 min, the B phase ratio linearly increased to 95% within 9.00 min and maintained at 95% for 1 min, the B phase ratio decreased to 5% from 10.00 to 11.10 min, and equilibrated at 5% for 14 min.

6. The preparation method according to claim 4, characterized in that The mass spectrometry detection conditions were as follows: electrospray ionization source temperature of 550°C; ion spray voltage: 5500 V, positive ion mode; 1-4500 V, negative ion mode; ion source gas I, gas II, and curtain gas were set to 50, 60, and 25 psi, respectively, and the collision-induced ionization parameter was set to high; QQQ scanning used the MRM mode, and the collision gas nitrogen was set to medium.

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