New application of matricarin

Abstract

The invention discloses an application of matricarin in preparation of uric acid lowering drugs. According to the present invention, the new pharmaceutical activity of the matricarin is found through the research, and the compound has dual effects of uric acid generation inhibition and hyperuricemia induced renal tubular cell injury protection, such that the medicinal field of the matricarin monomeric compound is broadened, and the pharmacological activity of the matricarin monomeric compound is further improved; and a further scientific basis is provided for clinical application of the chicory in treatment of hyperuricemia.

Description

Technical Field

[0001] The invention relates to a new medicinal application of matricolide. Background Art

[0002] Hyperuricemia is a common metabolic disorder, which is typically characterized by imbalanced uric acid levels in the body due to overproduction or impaired excretion, leading to accumulation of uric acid in the body and deposition in joints and kidney tissues, which in turn induces a variety of complications such as gout, hypertension, and diabetes. In recent years, the incidence of hyperuricemia in my country has been rising year by year, and it is showing a trend of younger patients. Gout is one of the main complications of hyperuricemia. Patients not only suffer from long-term pain, but also bear the economic pressure brought by continuous treatment, and the cure of this disease is extremely difficult. Currently, the commonly used uric acid-lowering drugs in clinical practice are mainly divided into two categories. One category acts on the uric acid generation stage, reducing uric acid by inhibiting the activity of key enzymes in the uric acid generation process, including xanthine oxidase inhibitors such as allopurinol and febuxostat; the other category is used in the uric acid excretion stage, by strengthening kidney function and promoting the transport and excretion of uric acid in the body to reduce uric acid, such as benzbromarone. Although these drugs take effect quickly, they are not safe, with obvious side effects and adverse reactions. Long-term use can easily lead to damage to the patient's gastrointestinal and liver and kidney functions. Therefore, finding a treatment method with good uric acid-lowering effect and fewer adverse reactions is an urgent problem to be solved. Summary of the invention

[0003] The purpose of the present invention is to provide a new drug activity of chamaelylactone, which has the dual effects of inhibiting uric acid production and promoting renal excretion of uric acid, which not only broadens the medicinal field of chamaelylactone monomer compounds, but also provides further scientific basis for the clinical application of chicory in treating hyperuricemia.

[0004] The present invention provides application of matricolide in preparing uric acid lowering medicine.

[0005] In some embodiments, when preparing the uric acid-lowering drug, the concentration of matricolide is preferably 0.025 mmol / L to 1 mmol / L.

[0006] Furthermore, when the concentration of matricolide is preferably 0.1 mmol / L to 1 mmol / L, matricolide has a mechanism of inhibiting the activity of xanthine oxidase.

[0007] Furthermore, when the concentration of matricolide is preferably 0.025 mmol / L to 0.1 mmol / L, matricolide has a mechanism of action for protecting HK-2 cells.

[0008] More preferably, when the concentration of matricolide is 0.1 mmol / L, matricolide has the mechanism of inhibiting xanthine oxidase activity and protecting HK-2 cells at the same time.

[0009] Compared with the prior art, the present invention has the following advantages:

[0010] Based on the existing research on chicory crude extract, the present invention further studies monomer compounds and screens out matricolide monomer components with uric acid-lowering activity.

[0011] The present invention has found through research that matricolide has the dual effects of inhibiting uric acid production and protecting HK-2 cells to enhance kidney function, and has potential uric acid-lowering activity. The present invention provides further scientific basis for the clinical application of chicory in treating hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the chemical structural formula of matricolide ketone of the present invention;

[0013] Figure 2 This is the effect curve of uric acid concentration on HK-2 cell viability in Example 3 of the present invention;

[0014] Figure 3 This is the protective effect of matricolide ketone on HK-2 cells in a uric acid environment in Example 3 of the present invention. DETAILED DESCRIPTION

[0015] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0016] Example 1

[0017] Preparation of Matricolide

[0018] 1.1 Experimental Materials

[0019] Chicory roots were collected from the planting nursery of Nanjing Zhongshan Botanical Garden in January 2023 and identified by researcher Ren Bingru of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. The plant specimen (No. 20230101) is stored in the herbarium of Nanjing Zhongshan Botanical Garden. The samples were washed, cut into small pieces and dried in an oven at 50°C for later use.

[0020] 1.2 Instruments and Equipment

[0021] Dion Ultimate 3000 high performance liquid chromatograph (analytical); Shimadzu LC-6AD high performance liquid chromatograph (equipped with SPD-20A UV detector) (preparative); desktop high-speed refrigerated centrifuge (eppendorf, Germany); SpectraMax Plus 384 microplate reader (Molecular Devices, USA); precision single-channel pipette (Thermo Fisher Scientific, USA); precision eight-channel pipette gun (Brand, Germany). YMC-Pack ODS-AQ chromatographic columns (S-5μm, 12nm, 4.6×250mm analytical column; and 20×250mm preparative column), 96-well plate, etc.

[0022] 1.3 Chemical reagents

[0023] Chromatographic grade acetonitrile was purchased from Tiandi Reagent Company, USA; column chromatography materials included D101 macroporous resin (Cangzhou, China), polyamide resin (30-60 mesh, Sinopharm Shanghai Trial), 100-200 mesh silica gel (Qingdao Ocean Chemical Co., Ltd.) and RP-18 reverse filler (Merck, Germany); renal tubular epithelial cells (HK-2 cells) were purchased from the Shanghai Cell Collection of the Chinese Academy of Sciences, DMEM / F12 culture medium and fetal bovine serum were purchased from Hyclone; CCK-8 was purchased from Sigma; matricolide ketone standards were homemade by the research group; uric acid, xanthine oxidase, sodium 1-pentane sulfonate and potassium dihydrogen phosphate and other reagents were purchased from reagent companies.

[0024] 1.4 Extraction and separation methods

[0025] The dried chicory root was extracted with 80% ethanol solution for 3 times, each time for 7 days (it can also be refluxed, the sample is added with 6-10 times the volume of 80% ethanol, and the micro-boiling reflux extraction is performed 2-3 times, each time for 2 hours), the extract was filtered and combined, and the reduced pressure concentration was performed on a rotary evaporator until there was no alcohol, and the chicory crude extract was obtained. The crude extract was suspended with an appropriate amount of water, loaded onto D101 macroporous resin, and adsorbed for several hours. After the solvent was naturally drained, it was first eluted with 4-5 column volumes of pure water to remove the polysaccharide components in the sample. Then the adsorbed sample was eluted with 90-95% ethanol. It was concentrated on a rotary evaporator under reduced pressure until there was no alcohol. The above-obtained sample was vortexed with water again, loaded onto polyamide resin (30-60 mesh), and after the sample was adsorbed, it was eluted with 4-5 column volumes of pure water, and the water elution fraction was collected and concentrated to obtain the total sesquiterpene lactones of chicory.

[0026] The obtained chicory total sesquiterpene lactones are segmented by column chromatography using silica gel column chromatography (100-200 meshes), and gradient eluted with a solvent system of dichloromethane:methanol (v / v=100:1, 50:1, 20:1, 10:1, 5:1, 1:1, 1:2) with 3 column volumes, and are divided into 7 segments Fr.S1-Fr.S7, wherein Fr.S3 is separated by RP-18 reverse column (gradient elution is performed with 40%-90% methanol / water as eluent), wherein the 60% methanol eluate is further prepared by liquid phase preparation to obtain the monomer compound of the present invention, and the compound is compared with the standard by HPLC to determine that the structure is matricarin, such as Figure 1 shown.

[0027] Example 2

[0028] Xanthine oxidase inhibitory activity assay

[0029] 1. Experimental Methods

[0030] After being absorbed by the human body, food is decomposed to produce a large amount of purine. Under the action of xanthine oxidase (XO) and other enzymes in the body, purine is decomposed and converted into uric acid as the final product. The production of uric acid is directly affected by enzyme activity. Inhibiting the activity of xanthine oxidase can reduce the production of uric acid in the body. This experiment detected the inhibitory activity of matricolide ketone obtained in Example 1 on XO by measuring the production rate of uric acid in the enzymatic reaction. The experimental steps are as follows:

[0031] Table 1 Xanthine oxidase reaction system

[0032]

[0033] 1. Matricolide experimental group: Referring to the above table, add 200 μL of xanthine oxidase working solution (0.05U / mL) and 200 μL of matricolide solution (0.5, 0.25, 0.1, 0.05, 0.025mM) to the reaction system in sequence, keep warm in a 37℃ water bath for 10min, then add 300 μL of 37℃ preheated substrate xanthine (1mM), mix well, react at 37℃ for 30min, and add 400 μL of hydrochloric acid solution (1M) to terminate the reaction. Blank control PBS group: 200 μL of phosphate buffer (PBS) was used as a blank control. After the reaction of each group was completed, 200 μL of the reaction solution was taken to detect the uric acid content by HPLC.

[0034] 2.HPLC chromatographic conditions: The chromatographic column was a C18 column (250 mm × 4.6 μm, 5 μm). Mobile phase A was acetonitrile, and mobile phase B was an aqueous solution containing 0.52 mM sodium 1-pentanesulfonate and 0.20 M potassium dihydrogen phosphate (pH = 4.0). The separation condition was isocratic 5% acetonitrile. The detection wavelength was 254 nm, the flow rate was 1 mL / min, and the column temperature was 40°C.

[0035] 3. Uric acid standard curve: Accurately weigh 10 mg of uric acid, dissolve it in 0.02 M NaOH and make up to 10 mL to prepare a 1 mg / mL uric acid standard solution, and dilute it with ultrapure water to 7 concentrations of 0.64, 0.32, 0.16, 0.08, 0.04, 0.02, and 0.01 mg / mL. After HPLC detection, perform linear regression with uric acid concentration as the horizontal axis and peak area as the vertical axis.

[0036] 4. Calculate the uric acid concentration of each reaction group according to the standard curve and convert it into uric acid content. Calculate the xanthine oxidase inhibitory activity using the following formula.

[0037]

[0038] Wherein, A1 is the uric acid content of the blank control PBS group; A2 is the uric acid content of the matricolide experimental group.

[0039] 2. Experimental Conclusion

[0040] As shown in Table 2 below, the inhibitory activity of matricolide on xanthine oxidase.

[0041] Table 2. Inhibitory activity of different concentrations of matricolide on xanthine oxidase

[0042]

[0043] As can be seen from Table 2, chamaelylactone can reduce the production of uric acid by inhibiting the activity of xanthine oxidase, and the inhibitory activity increases significantly with the increase of concentration.

[0044] Example 3

[0045] HK-2 cell protection activity experiment

[0046] 1. Experimental Methods

[0047] 1. Construction of uric acid-induced HK-2 cell apoptosis model

[0048] Dissolve 100 mg of uric acid in 10 mL of serum-free DMEM / F12 medium at 42°C to a concentration of 10 7 ng / mL uric acid solution, filtered through a 0.22μm microporous filter membrane and set aside. Take HK-2 cell fluid in the logarithmic growth phase and prepare 10 5 Cell suspension of 100 μL / mL was inoculated in a 96-well plate, and 100 μL was added to each well. After 24 hours of inoculation, the original culture medium was discarded and replaced with serum-free DMEM / F12 culture medium for 12 hours of synchronization. 7ng / mL uric acid culture medium stock solution was diluted with serum-free DMEM / F12 medium to 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 ng / mL uric acid medium, discard the original medium, replace it with gradient uric acid medium, set 6 replicates for each concentration. After 24 hours of culture, discard the original medium, add fresh medium containing 10% CCK-8 solution, incubate at 37℃ for 2 hours, and measure the absorbance OD 450 , calculate cell viability. According to the results, select appropriate uric acid concentration to study the protective effect of HK-2 cells.

[0049] 2. Cytotoxicity assay of matricolide on HK-2 cells

[0050] Take matricolide ketone, dissolve it in DMSO, and prepare a 10mM drug stock solution. Take HK-2 cell fluid in the logarithmic growth phase and prepare a 10 5 A cell suspension of 100 μL / mL was inoculated in a 96-well plate, and 100 μL was added to each well. After 24 hours of inoculation, the original culture medium was discarded and replaced with serum-free DMEM / F12 culture medium for synchronization for 12 hours. The 10 mM drug stock solution was gradiently diluted with DMSO / F12 complete culture medium to 0.1, 1, 10, and 100 μM drug solutions. The original culture medium was discarded and replaced with gradient drug culture medium. Six replicate wells were set for each concentration. After 24 hours of culture, the original culture medium was discarded, and fresh culture medium containing 10% CCK-8 solution was added. After incubation at 37°C for 2 hours, the absorbance value OD was measured. 450 , calculate cell viability.

[0051] 3. Determination of the protective effect of matricolide on HK-2 cells in uric acid environment

[0052] Take HK-2 cell solution in logarithmic growth phase and prepare 10 5 Cell suspension of 1×10 cells / mL was inoculated in a 96-well plate, and 100 μL was added to each well. After 24 hours of inoculation, the original culture medium was discarded and replaced with serum-free DMEM / F12 culture medium for synchronization for 12 hours. After discarding the original culture medium, 100 μL of 2×10 5 ng / mL of uric acid medium and drug-containing medium, so that the final uric acid concentration of each well medium is 5.8×10 3ng / mL. The concentrations of matricolide in the drug experimental groups were 25, 50, and 100 μM, respectively. After culturing for 24 h, the cells were collected and the SOD activity and MDA content were determined according to the instructions of the kit. The original culture medium was discarded, and fresh culture medium containing 10% CCK-8 solution was added. After incubation at 37°C for 2 h, the absorbance value OD was determined. 450 , calculate cell viability.

[0053] 2. Experimental Conclusion

[0054] like Figure 2 Shown is the HK-2 cell viability at different uric acid concentrations. Figure 2 The results showed that the cell viability of HK-2 cells decreased with the increase of uric acid concentration, showing a dose-dependent manner. Uric acid had a killing effect on HK-2 cells, and its half-maximal inhibitory concentration (IC50) was 2.1×10 4 ng / mL, according to the experimental results, 5.8×10 3 ng / mL concentration of uric acid was used for subsequent model construction.

[0055] Table 3 below shows the effects of different concentrations of matricolide on HK-2 cell viability:

[0056] Table 3. Effects of matricolide on HK-2 cell viability

[0057]

[0058] It can be seen from the cytotoxicity test results in Table 3 that matricolide has no obvious cytotoxicity to HK-2 cells at various doses.

[0059] The protective effect of matricolide on HK-2 cells under uric acid environment is shown in Tables 4-6. In the table, CON is the blank control PBS group, UA is 5.8×10 3 ng / mL concentration of uric acid group, 25μM, 50μM, 100μM are different concentrations of matricolide ketone experimental groups:

[0060] Table 4. Effects of chamaelylactone on the survival rate of HK-2 cells in uric acid environment

[0061]

[0062] Table 5. Effects of chamaelylactone on SOD activity in HK-2 cells in uric acid environment

[0063]

[0064] Table 6. Effects of matricolide on MDA content in HK-2 cells in uric acid environment

[0065]

[0066] Combination Figure 3 It can be seen that matricolide can significantly improve the cell viability of HK-2 cells under uric acid environment. In addition, it can also significantly improve the SOD activity in HK-2 cells under uric acid environment and reduce the content of MDA in a dose-dependent manner (Table 4-6), thereby improving kidney function and promoting uric acid metabolism. This study provides further scientific basis for the uric acid-lowering effect of chicory and its clinical application.

Claims

1. Application of matricolide ketone in the preparation of uric acid-lowering drugs.

2. The use of matricolide ketone in the preparation of uric acid-lowering drugs according to claim 1, characterized in that: The concentration of matricolide in the uric acid-lowering drug is 0.025 mmol / L to 1 mmol / L.

3. Use of matricolide in the preparation of a uric acid-lowering drug capable of inhibiting xanthine oxidase activity, characterized in that: The concentration of matricolide in the uric acid-lowering drug is 0.1 mmol / L to 1 mmol / L.

4. The use of matricolide in the preparation of a uric acid-lowering drug having the effect of protecting HK-2 cells, characterized in that: The concentration of matricolide in the uric acid-lowering drug is 0.025 mmol / L to 0.1 mmol / L.

5. Use of matricolide in the preparation of a uric acid-lowering drug that has the effects of inhibiting xanthine oxidase activity and protecting HK-2 cells, characterized in that: The concentration of matricolide in the uric acid-lowering drug is 0.1 mmol / L.

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