Corydalis pygmaea total alkaloid extract for preventing and treating hyperuricemia as well as preparation method and application of corydalis pygmaea total alkaloid extract

Abstract

The invention belongs to the technical field of extraction of active compounds, and particularly relates to a corydalis pygmaea total alkaloid extract with hyperuricemia prevention and treatment activity and a preparation method and application of the corydalis pygmaea total alkaloid extract. The method comprises the following steps: performing cold soaking extraction on a corydalis pygmaea medicinal material by using an ethanol solution, filtering an extracting solution, and performing vacuum concentration to obtain an extract; and dissolving the extract in a diluted hydrochloric acid aqueous solution, oscillating with ethyl acetate while dropwise adding stronger ammonia water for extraction, and then post-processing the extracted part to obtain the corydalis pygmaea total alkaloid extract. According to the application, deep and systematic research is carried out on the secondary metabolite of the corydalis pygmaea total alkaloids and the in-vitro xanthine oxidase inhibition activity, the in-vivo uric acid reduction activity and the kidney protection activity of the secondary metabolite, and detailed data support is provided for clinical application of the Tibetan medicine corydalis pygmaea. According to the preparation method provided by the invention, effective active ingredients in corydalis pygmaea can be effectively extracted, and the extracted extract has xanthine oxidase inhibitory activity, in-vivo uric acid reduction activity and kidney protection 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 for preventing and treating hyperuricemia, and a preparation method and application thereof. Background Art

[0002] Hyperuricemia is a chronic metabolic disease caused by excessive production or reduced excretion of uric acid. The kidney is an important regulatory organ for the uric acid cycle. About 70% of the uric acid in the human body is excreted in the urine through the kidney. When uric acid flows into the glomerulus through the blood circulation, more than 98% of the uric acid is filtered through the glomerulus, most of which is reabsorbed by the proximal renal tubules and then secreted by the distal convoluted tubules and excreted in the urine. Hyperuricemia patients are in a state of supersaturated blood uric acid for a long time, and the excretion burden of the kidneys is increased. About 90% of hyperuricemia patients have renal excretion disorders, causing kidney damage. At present, hyperuricemia in my country has become the second largest chronic metabolic disease after diabetes, and the incidence of hyperuricemia nephropathy has also increased year by year.

[0003] The drug treatment strategy for hyperuricemia is to control blood uric acid first. At present, 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 concentration of uric acid in the body. However, there are currently few available drugs for this disease, and the toxic and 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 decisive role in the production of uric acid. Allopurinol reduces uric acid by inhibiting XOD activity, thereby reducing uric acid concentration in the body. This is the principle of allopurinol in reducing uric acid, so XOD inhibitors are an important direction for the development of hyperuricemia drugs.

[0005] The uric acid transport system includes reabsorptive and secretory proteins that work together to 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 tubular lumen back to the blood, thereby regulating reabsorption through the renal apical membrane. At the same time, the excretion of uric acid is mainly promoted by the secretory protein ABCG2, which actively transports uric acid from the tubular epithelial cells to the tubular lumen.

[0006] Data from studies on renal cell apoptosis associated with nephrotoxic drugs indicate that apoptosis plays a role in regulating kidney growth and remodeling. Apoptosis is also thought to play an important role in uric acid-induced renal damage or chronic gout. Renal biopsies of patients with hyperuricemia and chronic renal failure show typical signs of apoptosis of proximal tubular epithelial cells. The Bcl-2 family is an important regulatory factor involved in apoptosis. Under normal circumstances, Bcl-2 forms a heterodimer with Bax, inhibiting the pro-apoptotic effect of Bax.

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

[0008] Corydalis hendersonii is a plant of the genus Corydalis in the family Papaveraceae, also known as Nepalese Corydalis. It is mainly distributed in the area of ​​4200-5200m above sea level in Tibet, my country. The Tibetan name of Corydalis hendersonii is Riguanzima. According to Jingzhu Bencao, it is mainly used to clear blood heat, pulse heat, dry bad blood, and is used for a variety of inflammations and fevers. There are 109 prescriptions of Riguanzima clearly recorded in the Tibetan Medicine Prescriptions Collection. Although Corydalis hendersonii is frequently used in them, the Tibetan medical literature records its functions and indications in a relatively broad manner, lacking its dominant diseases, and there are very few modern pharmacological studies. At present, there are almost no reports on the research of Corydalis hendersonii extract in the prevention and treatment of hyperuricemia and kidney protection, which is 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 activity in preventing and treating hyperuricemia.

[0010] The 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 Corydalis dwarfii extract.

[0012] The technical solution adopted by the present invention to achieve the above-mentioned purpose is as follows: 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: .

[0013] The present invention also provides a method for preparing the above-mentioned Corydalis dwarfi total alkaloid extract having the activity of preventing and treating hyperuricemia, comprising the following steps: (1) 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) dissolving the extract in a dilute hydrochloric acid aqueous solution to obtain a corydalis acid aqueous solution, then adding ethyl acetate and dropping ammonia water to extract while shaking to obtain an ethyl acetate extract and an aqueous layer extract, and tracking the XOD enzyme activity in vitro to obtain the ethyl acetate extract with the best activity; (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.

[0014] 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 times; each extraction is performed at room temperature of 25°C for 7 days.

[0015] Preferably, 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%, ethyl acetate is extracted three times, the ammonia water is adjusted to a pH of 9-10 of the dwarf corydalis aqueous solution, and the extraction is allowed to stand for 2 h after each extraction.

[0016] Preferably, in step (3), the liquid chromatography detection conditions are: 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.

[0017] The conditions of the above gradient elution are as follows: the proportion of phase B is 5% at 0.00 min, the proportion of phase B increases linearly to 95% within 9.00 min and is maintained at 95% for 1 min, the proportion of phase B decreases to 5% between 10.00 and 11.10 min, and is balanced at 5% for 14 min.

[0018] Preferably, the mass spectrometry detection conditions are: 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.

[0019] The present invention also provides the use of the above-mentioned total alkaloid extract of Corydalis dwarfi in preparing a medicine having the activity of preventing and treating hyperuricemia and protecting kidneys.

[0020] The beneficial effects of the present invention are: (1) The present invention has conducted in-depth and systematic research 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.

[0021] (2) The preparation method provided by the present invention can effectively extract the effective active ingredients in 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

[0022] Figure 1 is the inhibitory activity of Corydalis dwarfi on XOD; Figure 2 The PPI network of interactions between hyperuricemia-related genes and potential targets of total alkaloids from Corydalis dwarfi.

[0023] Figure 3 A timeline of the animal experiment process; Figure 4 This is the analysis chart of uric acid lowering and kidney protection indexes of the total alkaloids; Figure 5 The kidney anatomy of mice in each group; Figure 6 Pathological sections of kidney tissues of mice in each group (H&E staining, Masson staining); Figure 7 The effect of total alkaloids of Corydalis dwarfi on the expression of XOD in the liver and uric acid transporter protein in the kidney tissue of mice; Figure 8 The effect of total alkaloids of Corydalis dwarfi on the expression of apoptosis-related proteins in mouse kidney tissue; Fig. 9 The effect of total alkaloids of Corydalis dwarfi on inflammatory factors and NF-κB signaling pathway in mouse kidney tissue; Fig.10 The purpose of cck-8 was to detect the effects of different concentration gradients of total alkaloids of Corydalis dwarf on HK-2 cell viability; Fig.11 Detection of HK-2 cell viability for cck-8: Fig.12 This is the effect of total alkaloids from Corydalis dwarfi on the NF-κB signaling pathway in HK-2 cells. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0025] Example 1 (1) 10 kg of dried roots of Corydalis dwarfi were soaked in ethanol at room temperature for 3 times (3x50L), once every 7 days, and the extract was recovered under reduced pressure to obtain 1169.4 g of total extract; (2) The total extract was fully dissolved with 5‰ HCl solution and the pH was adjusted to 2. In the acid water part, the pH was adjusted to 10 with ammonia water, and the extract was performed three times with an equal volume of ethyl acetate to obtain the ethyl acetate extract part and the water layer extract part respectively. The ethyl acetate layers were combined and concentrated, and the total alkaloids of 33g were finally obtained in the ethyl acetate layer after in vitro XOD enzyme activity-guided tracking. (3) The ethyl acetate extract was subjected to UPLC-MS / MS targeted alkaloid detection; the specific detection conditions were: 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 (with 0.1% formic acid added), Phase B is acetonitrile (with 0.1% formic acid added); ③ Elution gradient: 0.00min B phase ratio is 5%, within 9.00min the B phase ratio increases linearly to 95% and is maintained at 95% for 1 min, 10.00-11.10min the B phase ratio decreases to 5%, and is balanced at 5% for 14 min; ④ Flow rate 0.35 mL / min; Column temperature 40℃; Injection volume 2 µ L.

[0026] The mass spectrometry detection conditions were as follows: electrospray ionization (ESI) temperature 550℃; 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. The QQQ scan used the MRM mode, and the collision gas (nitrogen) was set to medium. The DP and CE of each MRM ion pair were completed by further optimization of the declustering potential (DP) and collision energy (CE). According to the metabolites eluted in each period, a specific set of MRM ion pairs was monitored in each period, as shown in Table 1.

[0027] Table 1 The extract yields obtained from each extraction part are shown in Table 2.

[0028] Table 2 Effect Example (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 50 , as shown in Table 3.

[0029] Table 3 Note: - means not detected.

[0030] like Figure 1-Figure 6 As shown, the IC of total alkaloids was calculated based on 50The value was 0.66 mg / mL, indicating that the total alkaloids were a potential XOD inhibitor.

[0031] (II) Network pharmacology prediction of core uric acid-lowering targets of total alkaloids of Corydalis dwarfi The standard SMILES of major alkaloids were retrieved using PubChem database. The Swiss target prediction database was imported to predict potential drugs. The keyword "hyperuricemia" was searched in GeneCards, DisGeNET, and DrugBank databases. Duplicate targets were removed, 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 modules were determined using the MCODE algorithm in metscape. The results are shown in Figure 2. Figure 2 As shown, the core target of total alkaloids of Corydalis dwarfi for lowering uric acid is Bcl-2.

[0032] (III) The total alkaloids of Corydalis dwarfi, the part with the best XOD inhibitory activity, were used to verify the in vivo effect in hyperuricemia mice.

[0033] like Figure 3 As shown: Kunming mice (male, SPF, 20±2 g) were adapted to the laboratory environment for 1 week, during which time 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.

[0034] The model group mice were randomly divided into 6 groups (model group, allopurinol positive group, benzbromarone positive group, low-dose administration group, medium-dose administration group, and high-dose administration group, 6 mice in each group). The model group continued to be gavaged with 500 mg / kg / d potassium oxonate and 300 mg / kg / d of adenine daily, and the normal control group was fed with ordinary feed and gavaged with 0.5% CMC-Na prepared with an equal amount of normal saline. One hour after the model was established by the combined administration of adenine and potassium oxonate, the low, medium, and high-dose groups were gavaged with different concentrations of total alkaloids of Corydalis dwarfus (TA) solution (50 mg / kg / d, 100 mg / kg / d, and 200 mg / kg / d), and the mice in the positive control group were gavaged with allopurinol (0.5% CMC-Na) solution (10 mg / kg / d). The drugs were administered once a day at the same time for 2 consecutive weeks, and the body weight was recorded once a day. During the experiment, the mice were free to eat and drink water.

[0035] ① Collection of experimental animal tissue samples: 1) Blood sample collection 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 each group of mice 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 stored in a -20 °C refrigerator for later use.

[0036] 2) Collection of liver and kidney tissues after autopsy After blood was taken from the experimental mice, they were killed by cervical dislocation, fixed in supine position, and disinfected with 75% alcohol to prepare the skin. The surgical scissors and forceps that had been sterilized in advance were taken out, and the mouse's abdomen was opened layer by layer along the white line of the abdomen. The abdominal cavity was cleaned with normal saline, and the liver lobe was carefully cut off and placed in a 2 mL cryopreservation tube and stored in a -20°C refrigerator; the left and right kidneys were carefully separated and removed from the posterior position of the abdominal cavity, and then the upper capsule of the kidney was carefully removed, the left kidney was taken, and placed in a 2 mL cryopreservation tube and stored in a -20°C refrigerator. After the liver tissue and left kidney tissue were collected, they were transferred to a -80°C refrigerator for storage until use. The right kidney was fixed in a 4% paraformaldehyde ethanol solution that accounted for about 10 times the volume of the tissue and stored in a 4°C refrigerator. The organ tissue was used for subsequent experiments.

[0037] ② Experimental results and analysis: 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 μmol / 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 the SUA levels of the three low, medium and high dose groups of Corydalis dwarfi total alkaloids (TA) were significantly reduced to 164.67±36.25μmol / L, 155.45±4.41μmol / L, and 143.40±43.33μmol / L (p<0.001), showing a certain dose dependence. The above data show that TA has significant uric acid-lowering activity and is a potential drug for the treatment of hyperuricemia.

[0038] Serum creatinine (SCr) and 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 of hyperuricemic mice given different doses of TA. As shown in Figures B and C, compared with the Control group, the SCr and BUN levels of mice in the HUA group were significantly increased (p<0.001). Compared with the HUA group, the positive drugs allopurinol and benzbromarone reduced the SCr level (p<0.001) but had no significant effect on the BUN level (p>0.05). However, compared with the HUA group, different doses of TA (50, 100, 200 mg / kg) significantly reduced the SCr and BUN levels of hyperuricemic mice (p<0.001), indicating that TA has a certain ability to repair renal injury.

[0039] XOD is the main enzyme driving uric acid synthesis. Hyperuricemia is usually associated with upregulation of liver XOD activity. We tested whether TA inhibited XOD activity in hyperuricemia mice. As shown in Figures D and E, compared with the Control group, the serum and liver XOD activities of mice in the HUA group were significantly increased (p<0.001). As a commonly used XOD inhibitor in clinical practice, allopurinol (10 mg / kg) can significantly reduce the serum and liver XOD activities of hyperuricemia mice (p<0.001, p<0.01). Compared with the HUA group, the low, medium, and high dose groups of TA (50, 100, 200 mg / kg) all significantly reduced the serum and liver XOD activities of hyperuricemia mice (p<0.001). The results showed that TA may reduce uric acid production by inhibiting XOD activity, thereby exerting its uric acid-lowering effect.

[0040] ③ Renal anatomical photos, pathological sections and analysis The development of hyperuricemia is often accompanied by kidney damage, so we observed the effect of TA on the appearance of the kidneys of hyperuricemia mice. Figure 5 As shown: The kidneys of mice in the HUA group were pale and uneven in appearance, but after TA treatment, the kidney surface became smooth and restored to reddish brown.

[0041] To further study the effect of TA on renal pathological changes in hyperuricemia mice, the kidneys of mice in different drug groups were histologically examined, and kidney sections were stained with hematoxylin and eosin (H&E) to observe the morphology, and pathological analysis was performed.

[0042] like Figure 6 As shown in A, the glomerular morphology of mice in the normal control group was normal, the tubular structure was clear, and the tubular epithelial cells were closely arranged. In the mice in the hyperuricemia group, a large number of renal tubules were dilated, cytoplasmic vacuolation of renal tubular epithelial cells was more common, and the renal tubular structure was unclear. Allopurinol (10 mg / kg) and benzbromarone (10 mg / kg) had no significant effect on the repair of renal injury. Compared with the HUA group, different doses of TA (50, 100, 200 mg / kg) improved renal injury.

[0043] The above experimental data and histopathological results confirmed that TA has anti-hyperuricemia effect and can effectively improve kidney damage caused by hyperuricemia.

[0044] like Figure 6 As shown in B, Masson staining was used to observe the collagen structure of kidney tissue sections. The results of Masson staining showed that obvious tubular dilatation was observed in the kidneys of hyperuricemia mice, and obvious renal fibrosis was observed in the interstitial part; the renal interstitial fibrosis of mice in the allopurinol group was obvious, and a large number of tissue vacuoles were produced. Compared with the HUA group, different doses of TA (50, 100, and 200 mg / kg) improved renal damage. The renal tissue of mice in the TA group was uniformly stained, the glomerular morphology and structure were normal, the renal tubular structure was clear, and the renal tubular epithelial cells were closely arranged, indicating that the TA group had a certain protective effect on renal damage caused by hyperuricemia.

[0045] ④ Expression of XOD in liver and uric acid transporter protein in kidney tissue of mice in each group like Figure 7 As shown in the results, compared with the normal group, the expression of XOD protein in the liver of the HUA group was significantly upregulated (p<0.001), while TA significantly reduced the expression of XOD protein in the liver of hyperuricemia mice (p<0.1, p<0.01, p<0.001), and it was dose-dependent. The potency of 100mg / kg total alkaloids was equivalent to that of 10mg / kg allopurinol. The data showed that downregulating XOD protein expression to reduce the production of uric acid is one of the mechanisms of TA against hyperuricemia.

[0046] Compared with the normal group, the expression of URAT1 and GLUT9 proteins in the kidneys of the mice in the model group was significantly upregulated (p<0.01). Compared with the model group, the expression of URAT1 and GLUT9 proteins in the kidneys of the mice in the TA administration group was significantly decreased (p<0.01, p<0.001). This indicates that TA can downregulate the protein expression levels of URAT1 and GLUT9 in the kidneys, thereby reducing the reabsorption of uric acid by the kidneys and playing a role in lowering uric acid. Compared with the normal group, the expression of ABCG2 protein in the kidney of the mice in the model group was significantly decreased (p<0.01). Compared with the model group, the expression of ABCG2 protein in the kidney of the mice in the TA administration group was significantly increased (p<0.01, p<0.001). This shows that TA can upregulate the expression of ABCG2 protein and promote the excretion of uric acid.

[0047] ⑤ Expression of anti-apoptotic proteins in the kidneys of mice in each group like Figure 8 As shown in the figure, compared with the normal group, the expression of Bax protein in the renal tissue of the mice in the model group was increased (p<0.01), and the expression of Bcl-2 protein was decreased (p<0.01). Compared with the model group, there was no significant difference in the expression of Bax and Bcl-2 proteins in the positive drug allopurinol and benzbromarone mice groups, while the expression of Bax protein in the renal tissue of the mice in the TA administration group was significantly decreased (p<0.1, p<0.01), and the expression of Bcl-2 protein was significantly increased (p<0.1, p<0.01) in a dose-dependent manner. The above results indicate that the total alkaloids of Corydalis dwarfi improve nephropathy caused by hyperuricemia by inhibiting cell apoptosis.

[0048] ⑥ Expression of inflammatory factors and related signaling pathways in the kidneys of mice in each group like Fig. 9 As shown in the results, compared with the normal group, the expression of TNF-α in the HUA group was upregulated (p<0.01). Compared with the HUA group, TA significantly inhibited the expression of the proinflammatory cytokine TNF-α (p<0.01, p<0.001) in a dose-dependent manner. Compared with the normal group, the expression of IL-6 in the HUA group was significantly upregulated (p<0.001). Compared with the HUA group, TA significantly inhibited the expression of the proinflammatory cytokine IL-6 (p<0.1, p<0.01, p<0.001) in a dose-dependent manner. Compared with the normal group, the expression of IL-1β protein in the model group mice was significantly upregulated (p<0.001). Compared with the model group, the TA group significantly downregulated the expression of IL-1β protein in a dose-dependent manner (p<0.001). The results showed that the renal protective effect of TA includes reducing the inflammatory response in HUA mice.

[0049] 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, while the TA group p -IκBα / IκBα level 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, allopurinol and benzbromarone in the positive control group showed no significant changes. 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.

[0050] (IV) The total alkaloids of Corydalis dwarfi, the part with the best XOD inhibitory activity, were used to verify the in vitro effect of HK-2 cells.

[0051] ① Cell culture 1) Cell passaging: When the cell density in the culture dish reaches about 80%-90%, the cells are passaged. Move the culture dish into the clean bench, discard the original culture medium, wash twice with 2mL PBS, discard the PBS, add 2mL trypsin to digest, discard the trypsin, add 2mL of the corresponding culture medium to terminate the digestion, and repeatedly blow the bottom of the dish until a single cell suspension is formed. Take 1mL of cell suspension to a new 10 cm culture dish, add 7-8mL of the corresponding culture medium, shake gently back and forth, and place the culture dish in a 37℃, 5% CO2 incubator.

[0052] 2) Cryopreservation of cells: Cryopreservation of cells in good growth condition, move the culture dish into the clean bench, discard the original culture medium, wash once with 2 mL PBS, discard the PBS, add 2 mL trypsin for digestion, discard the trypsin, add 2 mL of prepared cryopreservation solution (200 µL DMSO + 1800 µL fetal bovine serum) and blow evenly. Transfer to a cryopreservation tube, seal with sealing film, mark, place at 4℃ for 30 min, then at -20℃ for 30 min, and finally store in a -80℃ refrigerator.

[0053] 3) Cell recovery: Take the frozen renal tubular epithelial cells out of the -80℃ refrigerator and immediately place them in a 37℃ constant temperature water bath. Shake quickly until the cryopreservation solution is completely melted. Wipe the outer wall and spray 75% ethanol. Move quickly into the clean bench, discard the sealing film, and disinfect with an alcohol lamp. Blow the cryopreservation solution evenly and move 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 and place the culture dish in a 37℃, 5% CO2 incubator. After the cells are completely attached to the wall, the cells need to be replaced with new culture medium.

[0054] ②CCK-8 detection of cell viability The cells in the logarithmic growth phase were digested with trypsin, and the culture medium was added and blown into a single cell suspension. The cells were counted using a counting plate. The control group and the experimental group were counted at 1*10 5 Cells / well were inoculated in a 96-well plate. 100 μL of culture medium was added to each well, and the 96-well plate was placed in a 37°C, 5% CO2 incubator for incubation for 24 h. After the cells were completely attached to the wall, the 96-well plate was removed and moved to the 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 group, and 3 replicates were set up. The 96-well plate was then placed in a 37°C, 5% CO2 incubator for incubation for 48 h. After 48 h, the original culture medium of the experimental and control groups was discarded, 100 μL of culture medium containing 10% CCK-8 solution was added to each well, and the blank group was 100 μL of culture medium containing 10% CCK-8 solution without cells. The cells were incubated in a 37°C, 5% CO2 cell incubator for 2 h, and then the OD value of each well was detected at a wavelength of 450 nm using an enzyme marker. The cell growth inhibition rate was calculated according to the following formula: Inhibition rate (%) = (OD e -OD0) / (OD c -OD0)×100% Here, OD e Represents the OD value of each experimental group, OD c OD represents the OD value of the control group, and OD0 represents the OD value of the blank group.

[0055] ③ Experimental results and analysis In order to detect the maximum safe concentration of TA that can protect HK-2 cells from kidney damage, the cell activity of each experimental group was studied by CCK-8 method. Fig.10 It can be seen that compared with the normal control group, with the increase of TA concentration, the cell viability gradually decreased, which proves that the gradually increasing TA concentration has a harmful stimulation on HK-2 cells. When the TA concentration is 40 µg / mL, the cell survival rate reaches more than 85%, indicating that this concentration of TA is the maximum safe concentration.

[0056] ④Expression of anti-apoptotic proteins in HK-2 cells As shown in Figure 11, compared with the normal group, the expression of Bax protein in the model group increased (p<0.01), and the expression of Bcl-2 protein decreased (p<0.01). Compared with the model group, there was no significant difference in the expression of Bax and Bcl-2 proteins in the positive drug allopurinol and benzbromarone groups, while the expression of Bax protein in the TA administration group was significantly reduced (p<0.1, p<0.01), and the expression of Bcl-2 protein was significantly increased (p<0.01). The above results show that the total alkaloids of Corydalis dwarfi improve nephropathy caused by hyperuricemia by inhibiting cell apoptosis.

[0057] ⑤Expression of inflammatory factors and related signaling pathway proteins in HK-2 cells As shown in Figure 12, compared with the normal group, the model group p -IκBα / IκBα level increased (p<0.001). Compared with the model group, there was no significant change in the positive control group after allopurinol and benzbromarone administration, while in the TA group p -IκBα / IκBα level was decreased (p<0.01). Compared with the normal group, the HUA group p -p65 / p-65 was significantly increased (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, the TA group p -p65 / p-65 was 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 total alkaloid extract of Corydalis dwarfi having activity in preventing and treating hyperuricemia, characterized in that: The total alkaloid extract of Corydalis dwarfi consists of the following compounds: 。 2. A method for preparing the total alkaloid extract of Corydalis dwarfensis having activity for preventing and treating hyperuricemia as claimed in claim 1, characterized in that: The following steps are involved: (1) 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) dissolving the extract in a dilute hydrochloric acid aqueous solution to obtain a corydalis acid aqueous solution, then adding ethyl acetate and dropping ammonia water to extract while shaking to obtain an ethyl acetate extract and an aqueous layer extract, and tracking the XOD enzyme activity in vitro to obtain an ethyl acetate extract; (3) The ethyl acetate extract was subjected to UPLC-MS / MS targeted alkaloid detection.

3. The preparation method according to claim 2, characterized in that: 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 times; each extraction is performed at room temperature of 25°C for 7 days.

4. The preparation method according to claim 2 or 3, 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%, ethyl acetate is extracted three times, the ammonia water is adjusted to a pH of 9-10 of the dwarf corydalis aqueous solution, and the extraction is allowed to stand for 2 h after each extraction.

5. The preparation method according to any one of claims 2 to 4, 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 0.35 mL / min; Column temperature 40℃; Injection volume 2 µL.

6. The preparation method according to claim 5, characterized in that: The conditions of the gradient elution are as follows: the B phase ratio is 5% at 0.00 min, the B phase ratio increases linearly to 95% within 9.00 min and is maintained at 95% for 1 min, the B phase ratio decreases to 5% from 10.00 to 11.10 min, and is balanced at 5% for 14 min.

7. The preparation method according to claim 1, 5 or 6, characterized in that: The 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 the collision-induced ionization parameter was set to high; QQQ scanning used the MRM mode, and the collision gas (nitrogen) was set to medium.

8. Use of the total alkaloid extract of Corydalis dwarfi as claimed in claim 1 in the preparation of a medicament having the activity of preventing and treating hyperuricemia and protecting kidneys.

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