Application of an alkaloid compound in the preparation of a drug for preventing and treating diabetic nephropathy

Abstract

The present invention relates to the application field of Chinese medicine compounds, and specifically to the application of an alkaloid compound in the preparation of a drug for preventing and treating diabetic nephropathy. The alkaloid compound can effectively prevent and treat diabetic nephropathy by enhancing the expression of P-GSK-3β / GSK-3β and GSH in cells, inhibiting AR generation and inducing the activation of Nrf2 / HO-1 signaling pathways, thereby exerting an antioxidant effect, and can be used to prepare a drug for preventing and treating diabetic nephropathy.

Description

Technical Field

[0001] The present invention relates to the application field of traditional Chinese medicine compounds, and in particular to the application of an alkaloid compound in the preparation of a medicine for preventing and treating diabetic nephropathy. Background Art

[0002] Diabetic nephropathy (DN) is an abnormal lesion of kidney structure and function caused by diabetic microvascular disease. It is one of the most common microvascular complications of diabetes and an important cause of chronic renal failure and end-stage renal disease. 30%-40% of diabetic patients will develop diabetic nephropathy. Once diabetic nephropathy is formed, most patients will further develop end-stage renal disease. The 5-year survival rate of patients with end-stage renal disease is usually less than 20%.

[0003] The pathogenesis of diabetic nephropathy is complex and involves a variety of different signaling pathways, such as the renin-angiotensin-aldosterone system, oxidative stress, and the polyol pathway. However, there is a relative lack of effective drugs and methods for the treatment of diabetic nephropathy. The current standard treatment strategy for diabetic nephropathy is to strictly control blood sugar and blood pressure and use renin-angiotensin-aldosterone system blockers. However, this treatment method can only slow down the progression rate, but cannot prevent or reverse the disease. Therefore, new drugs targeting the pathological mechanisms of diabetic nephropathy such as oxidative stress and the polyol pathway have become the main focus of developing new therapies.

[0004] Rue (Ruta graveolens L.), commonly known as stinking grass, belongs to the Rutaceae family. It is a small evergreen shrub with a strong and special smell. It is a perennial dicotyledonous herb that is easy to collect and has significant biological activity. It has been used as a medicinal plant since ancient times. It is native to the Mediterranean region and is now distributed all over the world. It is planted in both the north and south of my country, mainly in the southwest and south. The plant contains more than 130 compounds of different categories of natural products, such as alkaloids, coumarins, essential oils, flavonoids and furanoquinolines. Rue extracts are an important area of ​​drug development and have many pharmacological activities. Rue has long been used in traditional medicine to treat a variety of diseases, mainly to relieve pain, eye problems, rheumatism and dermatitis. However, the composition of traditional Chinese medicine is complex, and which components are truly effective is one of the important topics in the field of traditional Chinese medicine research. Summary of the invention

[0005] In view of the problems in the prior art, the present invention provides an application of an alkaloid compound in the preparation of a drug for preventing and treating diabetic nephropathy, wherein the alkaloid compound is: 3-hydroxy-2,4-dimethoxy-10-methyl-9-acridone (structural formula: Figure 1 ).

[0006] The alkaloid compound can enhance the expression of P-GSK-3β / GSK-3β and GSH in cells, inhibit AR generation and induce the activation of Nrf2 / HO-1 signaling pathway.

[0007] The present invention also provides a method for extracting alkaloid compounds:

[0008] (1) 10 kg of the aerial part of Rutaecarpa was crushed, and the powder was ultrasonically extracted twice with distilled water, each time for one hour, to obtain a water extract and a medicinal residue;

[0009] (2) extracting the drug residue once with 80% ethanol to obtain an alcohol extract and drug residue, and discarding the drug residue;

[0010] (3) extracting the alcohol extract with petroleum ether and ethyl acetate in sequence to obtain a petroleum ether extract, an ethyl acetate extract and a water extract;

[0011] (4) Select the ethyl acetate extract, take 80 g of 200-300 mesh silica gel powder, mix the sample, pass it through a silica gel column (100% dichloromethane, dichloromethane: methanol = 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, recover ethanol), collect the fractions, spot plate, combine and concentrate to obtain 18 fractions, among which the 6th fraction precipitates crystals to obtain the alkaloid compound.

[0012] The obtained alkaloid compound is identified as 3-hydroxy-2,4-dimethoxy-10-methyl-9-acridone through structure.

[0013] The structural identification is carried out by comparing the nuclear magnetic resonance and mass spectrometry data of the alkaloid compound with the literature.

[0014] The alkaloid compound provided by the present invention can effectively inhibit the proliferation of human glomerular mesangial cells induced by high glucose. The mechanism of action of the alkaloid compound is to enhance the expression of P-GSK-3β / GSK-3β and GSH in cells, inhibit AR generation and induce the activation of Nrf2 / HO-1 signaling pathway, thereby exerting an antioxidant effect. It provides a good lead compound for the development of new drugs for preventing and / or treating diabetic nephropathy, and has important research value and application prospects for the treatment of diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the mass spectrum of the alkaloid compound described in the present invention.

[0016] Figure 2 It is the hydrogen spectrum of the alkaloid compound described in the present invention.

[0017] Figure 3 It is the carbon spectrum of the alkaloid compound described in the present invention.

[0018] Figure 4 The figure is a structural diagram of the alkaloid compound described in the present invention.

[0019] Figure 5 This is a graph showing the results of the alkaloid compound of the present invention inhibiting the proliferation of human glomerular mesangial cells induced by high glucose.

[0020] Figure 6 This is a graph showing the results of the alkaloid compounds of the present invention acting on high glucose-induced HRMC cells.

[0021] Figure 7 This is a graph showing the regulatory effect of the alkaloid compounds of the present invention on AR, Nrf2, HO-1 and P-GSK-3β / GSK-3β.

[0022] Figure 8 This is a diagram showing the regulatory effect of the alkaloid compounds of the present invention on GSH. DETAILED DESCRIPTION

[0023] The prevention and treatment of diabetic nephropathy described in the present invention refers to the prevention and / or treatment of diabetic nephropathy.

[0024] Example 1: Extraction and structural identification of target alkaloid compounds

[0025] 1. Extraction and separation:

[0026] 10kg of the aerial part of Rutaecarpa is crushed, and the powder is ultrasonically extracted twice with distilled water for one hour each time to obtain a water extract and a drug residue. The drug residue is extracted once with 80% ethanol to obtain an alcohol extract and a drug residue, and the drug residue is discarded. The alcohol extract is extracted with petroleum ether and ethyl acetate in sequence to obtain a petroleum ether extract, an ethyl acetate extract and a water extract. The ethyl acetate extract (about 80g) is selected, 80g of 200-300 mesh silica gel powder is taken and mixed with the sample, and passed through a silica gel column (100% dichloromethane, dichloromethane: methanol = 100: 1, 50: 1, 20: 1, 10: 1, 5: 1, 1: 1, and ethanol is recovered), and the fractions are collected, plated, combined and concentrated to obtain 18 fractions, of which the sixth fraction precipitates crystals to obtain the target compound.

[0027] 2. Structural identification:

[0028] 1. Mass spectrum: HR-ESI-MS: m / z 286.1095[M+H]+, molecular formula is C16H15NO4; mass spectrum is as follows Figure 1 shown.

[0029] 2. NMR: The data tables of hydrogen spectrum and carbon spectrum are shown in Table 1 below. The solvents are MeOH-d4 and DMSO-d6. The hydrogen spectrum is 600MHz and the carbon spectrum is 150MHz. Figure 2 As shown, the carbon spectrum is Figure 3 shown.

[0030] Table 1. Attribution of hydrogen and carbon spectra signals of target compounds (δ in ppm)

[0031]

[0032] By comparing the above NMR and mass spectrometry data with the literature, the compound was analyzed to be the known compound 3-hydroxy-2,4-dimethoxy-10-methyl-9-acridone, with the structural formula as follows Figure 4 shown.

[0033] The literature is: Acridanone alkaloid in Baliospermum montanum-evaluation of its effect against anaphylaxis[J]. Planta Med. 2011 Nov; 77(17): 1947-9. doi: 10.1055 / s-0030-1271198.

[0034] Example 2: Study on the anti-proliferation effect of 3-hydroxy-2,4-dimethoxy-10-methyl-9-acridone on human renal mesangial cells (HRMC) induced by high glucose: that is, the CCK8 method was used to detect the effect of the target compound on the viability of HRMC cells induced by high glucose.

[0035] 1. Cell culture: HRMC-1 cells were cultured in DMEM low-glucose medium containing 10% FBS (fetal bovine serum) + 1% double antibody at 37° C., 5% CO 2 , and a saturated humidity incubator.

[0036] 2. Take the logarithmic growth of 5*10 3 The cells were plated in a 96-well plate. After the cells adhered to the wall, the cells were starved for 24 hours and then treated in the following groups: solvent control group (NG group): after adding complete medium containing 10% fetal bovine serum for 48 hours, the cells were treated with 0.1% DMSO for 24 hours, for a total of 96 hours; high glucose injury group (HG group): after adding 30mmol / L glucose for 48 hours, the cells were treated with 0.1% DMSO for 24 hours, for a total of 96 hours; target compound: after adding 30mmol / L glucose for 48 hours, the cells were treated with 10μmol / L (low-dose group), 20μmol / L (medium-dose group), and 50μmol / L (high-dose group) target compounds for 48 hours, for a total of 96 hours;

[0037] 3. Digest and count the cells in the above groups, with 5*10 3Cells were inoculated at a density of 100 μL per well in a 96-well plate. Each group had 5 replicate wells. After the culture adhered to the wall, the cells were treated as above for a corresponding period of time. 10 μL / well of CCK8 was added to each well. The CCK8 solution was prepared with complete culture medium. The drug-containing culture medium was removed and 100 μL of culture medium containing CCK8 was added to each well. The culture medium was incubated at 37°C and 5% CO. 2 After incubation for 1 hour, the absorbance (OD) value at 450 nm was analyzed using an enzyme reader.

[0038] Cell viability results are as follows Figure 5 As shown, the results demonstrated that low doses of the compound 3-hydroxy-2,4-dimethoxy-10-methyl-9-acridone could effectively inhibit the proliferation of human glomerular mesangial cells induced by high glucose, and medium and high doses of 3-hydroxy-2,4-dimethoxy-10-methyl-9-acridone could also effectively inhibit the proliferation of human glomerular mesangial cells induced by high glucose, but made the cell viability lower than that of the normal group cells.

[0039] Example 3: RT-qPCR combined with CCK8 results detected the effects of different concentrations of target compounds on high glucose-induced HRMC.

[0040] 1. Cell culture: HRMC-1 cells were cultured in DMEM low-glucose medium containing 10% FBS (fetal bovine serum) + 1% double antibody at 37°C and 5% CO 2 , cultured in a saturated humidity incubator.

[0041] 2. Take the logarithmic growth of 5*10 3 The cells were plated in a 96-well plate. After the cells adhered to the wall, the cells were starved for 24 hours and then treated in the following groups: solvent control group (NG group): after adding complete medium containing 10% fetal bovine serum for 48 hours, the cells were treated with 0.1% DMSO for 24 hours, for a total of 96 hours; high glucose injury group (HG group): after adding 30mmol / L glucose for 48 hours, the cells were treated with 0.1% DMSO for 24 hours, for a total of 96 hours; target compound group: after adding 30mmol / L glucose for 48 hours, the cells were treated with 10μmol / L (COM-L), 20μmol / L (COM-M), and 50μmol / L (COM-H) target compounds for 48 hours, for a total of 96 hours;

[0042] 3. RT-qPCR experiment: Total RNA was extracted from cells using Trizol reagent according to the instructions, and total cell mRNA was used as a template for reverse transcription of cDNA. The sequence of the target gene was searched on NCBI, and primers were designed using primer5 software. Primers were synthesized by Beijing Qingke for quantitative PCR amplification. A fluorescent quantitative PCR instrument was used, and the relative content of mRNA in cells was calculated using the 2-ΔΔCt method. The relevant primers for mRNA information are as follows: GAPDH as internal reference: 5'-ACAGCCTCAAGATCATCAGC-3' (forward) and 5'-GGTCATGAGTCCTTCCACGAT-3' (reverse); AR: 5'-CCCAGCGCAACCAATCAGAA (forward), 5'-TAAATAGCCCGTGAGGTCGG-3' (reverse); Nrf2: 5'-CAACTACTCCCAGGTTGCCC-3' (forward), 5'-AGTGACTGAAACGTAGCCGAA-3' (reverse); HO-1: 5'-AAACTTCAGAGGGGGCGAAG-3' (forward), 5'-GACAGCTGCCACATTAGGGT-3' (reverse).

[0043] The experimental results are as follows Figure 6 As shown in the figure, the expression of AR mRNA in HRMC cells increased significantly under high glucose induction, and the AR mRNA level decreased significantly after the action of different concentrations of target compounds; the expression of Nrf2 mRNA and HO-1 mRNA in HRMC cells decreased significantly under high glucose induction, and the Nrf2 mRNA and HO-1 mRNA levels increased significantly after the action of different concentrations of target compounds, exerting an antioxidant effect. Combined with the CCK8 results, a low concentration of target compounds that had no effect on normal cell viability was selected for subsequent research.

[0044] Example 4: Western-blot was used to detect the regulatory effect of the target compound on AR, Nrf2, HO-1 and P-GSK-3β / GSK-3β.

[0045] 1. Cell culture: HRMC-1 cells were cultured in DMEM low-glucose medium containing 10% FBS (fetal bovine serum) + 1% double antibody at 37° C., 5% CO 2 , and a saturated humidity incubator.

[0046] 2. Take the logarithmic growth of 5*10 3The cells were plated in a 96-well plate. After the cells adhered to the wall, the cells were starved for 24 hours and then treated in the following groups: solvent control group (NG group): after adding complete medium containing 10% fetal bovine serum for 48 hours, the cells were treated with 0.1% DMSO for 24 hours, for a total of 96 hours; high glucose injury group (HG group): after adding 30mmol / L glucose for 48 hours, the cells were treated with 0.1% DMSO for 24 hours, for a total of 96 hours; positive control group (EPS group): after adding 30mmol / L glucose for 48 hours, the cells were treated with 10μmol / L epalrestat for 48 hours, for a total of 96 hours; target compound group: after adding 30mmol / L glucose for 48 hours, the cells were treated with 10μmol / L (COM) of the target compound for 48 hours, for a total of 96 hours;

[0047] 3. Western-blot experiment: Extract protein from cells and determine protein concentration according to the instructions of BCA protein quantification kit. Prepare gel and prepare samples, separate by electrophoresis on the electrophoresis instrument, and electrophoresis constant voltage 75V for 130min. Stop electrophoresis when bromophenol blue electrophoresis reaches the bottom of the gel, and then transfer to NC membrane. After transfer, take out the membrane and wash it once in 1*PBST. Prepare 5% skim milk powder with 1*PBST, immerse the membrane, and place it at room temperature for 90min. After that, dilute the primary antibody with 1*PBST in a certain proportion, incubate the membrane with the primary antibody, overnight at 4℃, place at room temperature for 30min the next day, and wash 3 times with 1*PBST at the end of incubation, 10min each time. Dilute the HRP-labeled secondary antibody with 1*PBST, incubate the diluted secondary antibody with the membrane at room temperature for 90min, and wash 3 times with 1*PBST at the end of incubation, 15min each time. Then, incubate the membrane with ECL chemiluminescent solution for 1 minute, absorb the liquid with filter paper, wrap the hybridization membrane with plastic film, and image with gel imaging system. Finally, analyze the exposed film with quantity one professional grayscale analysis software. In the experiment, actin was used as the loading control, and 3 samples were repeated in each group for data statistics.

[0048] The experimental results are as follows Figure 7As shown in the figure, AR levels in HRMC cells induced by high glucose increased significantly, indicating the activation of the polyol pathway, and the expression of Nrf2, HO-1, and P-GSK-3β / GSK-3β decreased significantly, causing oxidative stress. After the action of the compound, it was found that both epalrestat and the target compound could reduce the expression of AR, inhibit the polyol pathway, and thus alleviate the oxidative stress induced by high glucose, and the inhibitory effect of the target compound was more significant than that of the AR inhibitor epalrestat; after the action of the target compound, the expression of P-GSK-3β / GSK-3β could be significantly increased, the phosphorylation modification level of GSK-3β was significantly enhanced, the protein stability of GSK-3β was reduced, and its inhibitory effect on Nrf2 was weakened, thereby significantly enhancing the protein expression levels of Nrf2 and HO-1, exerting an antioxidant effect, and then inhibiting the proliferation of mesangial cells in a high glucose environment and reducing the damage of high glucose to cells.

[0049] Example 5: Biochemical kit to detect the regulatory effect of target compound on GSH.

[0050] 1. Cell culture: HRMC-1 cells were cultured in DMEM low-glucose medium containing 10% FBS (fetal bovine serum) + 1% double antibody at 37° C., 5% CO 2 , and a saturated humidity incubator.

[0051] 2. Take the logarithmic growth of 5*10 3 The cells were plated in a 96-well plate. After the cells adhered to the wall, the cells were starved for 24 hours and then treated in the following groups: solvent control group (NG group): after adding complete medium containing 10% fetal bovine serum for 48 hours, the cells were treated with 0.1% DMSO for 24 hours, for a total of 96 hours; high glucose injury group (HG group): after adding 30mmol / L glucose for 48 hours, the cells were treated with 0.1% DMSO for 24 hours, for a total of 96 hours; positive control group (EPS group): after adding 30mmol / L glucose for 48 hours, the cells were treated with 10μmol / L epalrestat for 48 hours, for a total of 96 hours; target compound group: after adding 30mmol / L glucose for 48 hours, the cells were treated with 10μmol / L (COM) of the target compound for 48 hours, for a total of 96 hours;

[0052] 3. Detection of GSH:

[0053] a) Sample preparation:

[0054] The collected cells were washed with PBS for 1-2 times, and then the precipitated cells were collected by low-speed centrifugation. Then 0.3-0.5 mL of 0.1 MPH = 7.4 isotonic PBS buffer was added to suspend the cells. The cells were broken by ultrasonic or manual grinding. 0.1 mL of the broken cell suspension was taken, 0.1 mL of the precipitant was added, and mixed. The mixture was centrifuged at 3500 rpm for 10 minutes, and the supernatant was taken for testing.

[0055] b) Sample testing:

[0056] Preparation of 1 mmol / L GSH standard solution: The molecular weight of GSH is 307. Before each measurement, add 3.07 mg of GSH standard to 10 mL of GSH solvent application solution, mix well, and store at 2°C to 8°C for 48 hours.

[0057] Preparation of 20μmo1 / L GSH standard solution: Take 0.2mL of 1mmol / L GSH standard solution and add 9.8mL of GSH standard solvent application solution.

[0058] The supernatant obtained after the cells are treated with the precipitant is operated according to the following table:

[0059] Table 2

[0060] Blank hole Standard hole Determination hole Precipitant (μL) 100 20μmo1 / LGSH standard (μL) 100 Supernatant (μL) 100 Buffer (μL) 100 100 100 Color developer (μL) 25 25 25

[0061] Mix well, let it stand for five minutes, and detect the absorbance of each well with an enzyme-labeled instrument at a wavelength of 405 nm.

[0062] Depend on Figure 8 The results shown in the figure show that the target compound and epalrestat effectively enhance the expression of GSH in human glomerular mesangial cells induced by high glucose, thereby protecting the cells from oxidative stress damage.

[0063] In summary, the compound 3-hydroxy-2,4-dimethoxy-10-methyl-9-acridone provided by the present invention can effectively inhibit the proliferation of human glomerular mesangial cells induced by high glucose. The mechanism of action of the compound is to enhance the expression of intracellular P-GSK-3β / GSK-3β and GSH, inhibit AR generation and induce the activation of Nrf2 / HO-1 signaling pathway to exert an antioxidant effect, thereby illustrating the use of the alkaloid compound (3-hydroxy-2,4-dimethoxy-10-methyl-9-acridone) described in the present invention for the preparation of drugs for preventing and / or treating diabetic nephropathy.

[0064] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of an alkaloid compound in the preparation of a drug for preventing and treating diabetic nephropathy, wherein the alkaloid compound is: 3-hydroxy-2,4-dimethoxy-10-methyl-9-acridone.

2. The use according to claim 1, characterized in that: The alkaloid compound can enhance the expression of P-GSK-3β / GSK-3β and GSH in cells, inhibit AR generation and induce the activation of Nrf2 / HO-1 signaling pathway.

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