Compounds, methods of making and uses

Abstract

The application discloses a compound, a preparation method and application thereof. The compound has a structure as shown in the following formula (I): wherein R1 and R8 are independently selected from C1-C6 alkyl; R2, R3, R4, R5 and R6 are independently selected from hydrogen or C1-C4 alkyl; and R7 is independently selected from hydrogen or C1-C3 alkyl. The compound can reduce uric acid.

Description

TECHNICAL FIELD

[0001] The present application relates to a compound and a preparation method and use. BACKGROUND

[0002] Hyperuricemia refers to the state that under normal dietary conditions, the body produces too much uric acid and / or excretes too little. Hyperuricemia is a chronic metabolic disease caused by purine metabolism disorder, which is clinically divided into primary and secondary. Generally, a simple state of hyperuricemia does not have subjective symptoms, but if the state lasts for a long time, urate in the blood will crystallize and deposit in joints, subcutaneous tissue, kidneys and other parts, and then a series of clinical manifestations such as ventilation and ventilation complications will appear.

[0003] CN113773361A discloses a compound for treating hyperuricemia, a composition, a preparation method and a medical use thereof. CN111714485A discloses a hyperuricemia pharmaceutical composition and its use. SUMMARY

[0004] Therefore, one object of the present application is to provide a compound. The compound can reduce uric acid. Another object of the present application is to provide a preparation method of the compound. Still another object of the present application is to provide use of the compound in preparing a drug for reducing uric acid level. The present application realizes the above objects through the following technical solutions.

[0005] In one aspect, the present application provides a compound, which has a structure as shown in formula (I):

[0006]

[0007] wherein R1 and R8 are independently selected from C1-C6 alkyl; R2, R3, R4, R5 and R6 are independently selected from hydrogen or C1-C4 alkyl; and R7 is independently selected from hydrogen or C1-C3 alkyl.

[0008] According to the compound of the present application, preferably, R1 and R8 are independently selected from C1-C3 alkyl; R2, R3, R4, R5 and R6 are independently selected from hydrogen or C1-C3 alkyl, and R7 is independently selected from hydrogen or methyl.

[0009] According to the compound of the present application, preferably, R1 and R8 are independently selected from methyl and ethyl; R2, R3, R4, R5 and R6 are independently selected from hydrogen and methyl, and R7 is hydrogen.

[0010] According to the compound of the present application, preferably, the structure of the compound is as shown below:

[0011]

[0012] In another aspect, the present application also provides a preparation method of the compound as described above, comprising the following steps:

[0013] (1) sterilizing a mixture of Astragalus membranaceus powder and water, inoculating the mixture with Paecilomyces japonicus seed liquid after cooling, culturing, drying, and crushing to obtain a fermented mycelium powder;

[0014] (2) extracting the fermented mycelium powder with alcohol, concentrating to obtain a concentrate; adding water to the concentrate to form a sample solution;

[0015] (3) loading the sample solution onto a macroporous resin column, eluting with water, 25-35 vol% ethanol, and 70-80 vol% ethanol in sequence, collecting the eluate eluted with 70-80 vol% ethanol, concentrating to obtain a concentrated residue; redissolving the concentrated residue with 38-42 vol% acetonitrile to obtain a crude product solution;

[0016] (4) separating the crude product solution by semi-preparative liquid chromatography to obtain the compound; wherein the chromatographic column used is a C18 chromatographic column; the mobile phase is acetonitrile-water; the gradient elution conditions are: 0-20 min, 80%-60% water, 20-30 min, 60% water; and the detection wavelength is 254-260 nm.

[0017] According to the preparation method of the present application, preferably, in step (1), the mass ratio of Astragalus membranaceus powder to water is 30 g: 10-16 mL; the sterilization is steam sterilization at 121-123℃ for 20-40 min; the mass-volume ratio of Astragalus membranaceus powder to Paecilomyces japonicus seed liquid is 30 g: 30-33 mL; and the culturing condition is culturing at 23-28℃ in a culture box.

[0018] According to the preparation method of the present application, preferably, in step (1), the Paecilomyces japonicus seed liquid is prepared by the following steps:

[0019] Under a sterile environment, Paecilomyces japonicus is inoculated into potato dextrose agar (PDA) medium, cultured and activated in a 26-28℃ incubator for 5-7 days, and the activated mycelium is picked up with an inoculation loop and placed in a sterilized potato dextrose liquid medium in a 26-28℃, 140-150 r·min -1 shaking bed for 3-5 days to obtain the Paecilomyces japonicus seed liquid.

[0020] According to the preparation method of the present application, preferably, in step (2), the alcohol is selected from methanol or ethanol, and the mass-volume ratio of the fermented mycelium powder to alcohol is 1 g: 20-60 mL; and the extraction is carried out under heating reflux for 1-4 h.

[0021] According to the preparation method of the present application, preferably, in step (3), the macroporous resin column is an AB-8 macroporous resin column; and the macroporous resin in the macroporous resin column is activated by 90-95 vol% of ethanol for 20-30 h.

[0022] In still another aspect, the present application also provides use of the compound as described above in the preparation of a medicament for lowering uric acid level. In particular, use in the preparation of a medicament for lowering uric acid level in hyperuricemia.

[0023] The compound of the present application can lower uric acid level and can be used in the preparation of a medicament for preventing and / or treating hyperuricemia. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 A standard curve of uric acid control.

[0025] Figure 2 A cytotoxicity detection result chart of the compound of the present application (i.e. component I).

[0026] Figure 3 A uric acid-lowering efficacy result chart of the compound of the present application (i.e. component I). DETAILED DESCRIPTION

[0027] The present application is further illustrated by the following specific examples without limiting the scope of the present application.

[0028] <EXPLANATION OF TERMS>

[0029] In the present application, Cm-Cn represents having m-n carbon atoms; for example, C1-C6 alkyl represents alkyl having 1-6 carbon atoms.

[0030] In the present application, "alkyl" represents a group having one attachment point, derived from a straight-chain or branched aliphatic hydrocarbon.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0032] <COMPOUND>

[0033] The compound of the present application has a structure as shown in formula (I):

[0034]

[0035] In the present application, R1is independently selected from C1-C6alkyl, preferably from C1-C3alkyl, more preferably from methyl or ethyl, further preferably methyl. In the present application, C1-C6alkyl can include, but is not limited to, straight chain alkyl or branched alkyl; preferably C1-C3alkyl, more preferably C1-C3straight chain alkyl. Examples of C1-C6alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, i-pentyl, neopentyl, hexyl, and the like.

[0036] R2is independently selected from hydrogen or C1-C4alkyl. R2is preferably selected from hydrogen or C1-C3alkyl, more preferably from hydrogen, methyl or ethyl, further preferably from hydrogen or methyl. In the present application, examples of C1-C4alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl. According to one specific embodiment of the present application, R2is hydrogen.

[0037] R3is independently selected from hydrogen or C1-C4alkyl. R3is preferably selected from hydrogen or C1-C3alkyl, more preferably from hydrogen, methyl or ethyl, further preferably from hydrogen or methyl.

[0038] R4is independently selected from hydrogen or C1-C4alkyl. R4is preferably selected from hydrogen or C1-C3alkyl, more preferably from hydrogen, methyl or ethyl, further preferably from hydrogen or methyl.

[0039] R5is independently selected from hydrogen or C1-C4alkyl. R5is preferably selected from hydrogen or C1-C3alkyl, more preferably from hydrogen, methyl or ethyl, further preferably from hydrogen or methyl.

[0040] R6is independently selected from hydrogen or C1-C4alkyl. R6is preferably selected from hydrogen or C1-C3alkyl, more preferably from hydrogen, methyl or ethyl, further preferably from hydrogen or methyl.

[0041] According to one specific embodiment of the present application, R3, R4, R5and R6are all hydrogen.

[0042] R7is independently selected from hydrogen or C1-C3alkyl, preferably from hydrogen, methyl or ethyl, more preferably from hydrogen or methyl.

[0043] R8is independently selected from C1-C6alkyl, preferably from C1-C3alkyl, more preferably from methyl or ethyl, further preferably methyl.

[0044] According to one specific embodiment of the present application, the structure of the compound is as follows:

[0045]

[0046] Generally, methyl glucose is a non-naturally occurring seven-carbon sugar, which is more stable in chemical properties than glucose, and has been applied in some fields such as chemical materials due to its excellent acid and alkali resistance and oxidation resistance. The methyl groups in these methyl glucoses are usually connected to C3 or C1, while the methyl group in the methyl glucoside obtained in the present application is connected to C4.

[0047] <Preparation method>

[0048] The preparation method of the compound of the present application comprises: (1) preparing a fermentation fungus; (2) obtaining a sample solution; (3) purifying by using a macroporous resin; (4) separating by using a semi-preparative chromatography. Optionally, it also comprises obtaining a paecilomyces japonicas seed liquid. Details are described below.

[0049] Obtaining Paecilomyces japonicus seed liquid

[0050] Under a sterile environment, paecilomyces japonicas is inoculated into a potato dextrose agar (PDA) medium, and is cultured in an incubator at 26-28℃ for 5-7 days. The activated mycelium is picked up by an inoculation loop and is placed in a sterilized potato dextrose liquid medium at 26-28℃, 140-150 r·min -1 in a shaker for 3-5 days to obtain a paecilomyces japonicas seed liquid.

[0051] In some specific embodiments, under a sterile environment, paecilomyces japonicas is inoculated into a potato dextrose agar (PDA) medium, and is cultured in an incubator at 28℃ for 5-7 days. The activated mycelium is picked up by an inoculation loop and is placed in a sterilized potato dextrose liquid medium at 28℃, 145-150 r·min -1 in a shaker for 3-5 days to obtain a paecilomyces japonicas seed liquid, i.e. an activated paecilomyces japonicas seed liquid.

[0052] Preparing fermentation broth

[0053] The mixture of the astragalus membranaceus powder and water is sterilized, and after cooling, the paecilomyces japonicas seed liquid is inoculated, cultured, dried, and crushed to obtain a fermentation fungus powder. This is conducive to obtaining the compound shown in formula (I), especially the compound shown in formula (A).

[0054] In the present application, the astragalus membranaceus powder can be obtained by crushing and sieving astragalus membranaceus decoction pieces. Preferably, it is sieved through a No. 1 sieve.

[0055] The mass-volume ratio of the astragalus membranaceus powder to water is 30 g: 10-16 mL, preferably 30 g: 12-15 mL, and more preferably 30 g: 14-15 mL. The sterilization can be steam sterilization at 121-123℃ for 20-40 min. The steam sterilization can be performed by using equipment known in the art.

[0056] In the present application, the temperature can be lowered to 20-35°C, preferably to the culture temperature for inoculating the Paecilomyces japonica seed liquid. The culture temperature is 23-28°C, preferably 26-28°C, such as 28°C. The culture can be carried out in a culture box known in the art.

[0057] In the present application, the mass-volume ratio of the Astragalus membranaceus powder to the Paecilomyces japonica seed liquid can be 30g:30-33mL, preferably 30g:30-31mL.

[0058] In the present application, the pulverization can employ a pulverizer.

[0059] Obtaining sample loading solution

[0060] The fermented mycelium powder is extracted with alcohol, concentrated to obtain a concentrate; the concentrate is added to water to form a sample loading solution. This is conducive to the next step of macroporous resin purification.

[0061] In the present application, the alcohol is selected from methanol or ethanol, preferably methanol. The mass-volume ratio of the fermented mycelium powder to the alcohol is 1g:20-60mL, preferably 1g:30-60mL, more preferably 1g:40-50mL. The extraction is carried out under heating reflux for 1-4h, preferably 2-4h, more preferably 2-3h.

[0062] The concentrate is reconstituted by adding water (preferably distilled water or deionized water) to obtain a sample loading solution. The mass-volume ratio of the fermented mycelium powder to water is 1g:60-100mL, preferably 1g:80-100mL, more preferably 1g:90-100mL.

[0063] Purification using macroporous resin

[0064] The sample loading solution is loaded onto a macroporous resin column, and eluted with water, 25-35vol% ethanol, and 70-80vol% ethanol in sequence. The eluate eluted with 70-80vol% ethanol is collected, concentrated to obtain a concentrated residue; the concentrated residue is reconstituted with 38-42vol% acetonitrile to obtain a crude product solution.

[0065] The macroporous resin column is preferably an AB-8 macroporous resin column.

[0066] The macroporous resin in the macroporous resin column is activated with 90-95vol% ethanol for 20-30h. In some embodiments, the sample loading flow rate can be 0.9-1.2mL / min, preferably 1.0-1.2mL / min, more preferably 1.0-1.1mL / min.

[0067] The amount of eluent water is 300-600 mL: 1 g, preferably 400-600 mL: 1 g, more preferably 500-600 mL: 1 g, of the volume of water to the mass of the fermented mycelium powder. The amount of eluent 25-35 vol% ethanol is 300-600 mL: 1 g, preferably 400-600 mL: 1 g, more preferably 550-600 mL: 1 g, of the volume of 25-35 vol% ethanol to the mass of the fermented mycelium powder. The amount of eluent 70-80 vol% ethanol is 300-700 mL: 1 g, preferably 400-600 mL: 1 g, more preferably 550-600 mL: 1 g, of the volume of 70-80 vol% ethanol to the mass of the fermented mycelium powder.

[0068] In some preferred embodiments, the sample solution is loaded onto a macroporous resin column, and eluted with water, 30-35 vol% ethanol, and 75-78 vol% ethanol in sequence, and the eluate eluted with 75-78 vol% ethanol is collected.

[0069] In the present application, the concentrated residue is reconstituted with an acetonitrile aqueous solution, which is preferably 38-42 vol% acetonitrile aqueous solution, for example, 40 vol% acetonitrile aqueous solution.

[0070] Separation using preparative chromatography

[0071] The crude product solution is separated by semi-preparative liquid chromatography to obtain the compound. This is advantageous for separating the compound of formula (I), especially the compound of formula (A).

[0072] The semi-preparative liquid chromatography is performed under the following conditions: the column used is a C18 column, preferably a C18 column (20 x 250 mm, 10 μm), for example, a SHIMADZU C18 column (20 x 250 mm, 10 μm). The mobile phase is acetonitrile-water; the gradient elution conditions are 0-20 min, 80%-60% water, 20-30 min, 60% water. The detection wavelength is 254-260 nm, preferably 260 nm. In some embodiments, the flow rate is 13-15 mL / min, preferably 14-15 mL / min. The injection volume is 0.8-1 mL, preferably 0.9-1 mL.

[0073] <Use>

[0074] The present application also provides the use of the compound as described above in the preparation of a medicament for reducing uric acid level.

[0075] The compound of the present application can significantly reduce uric acid level, and can be used in the preparation of a medicament for preventing and treating hyperuricemia. In some embodiments, the compound or a pharmaceutically acceptable salt, hydrate or solvate thereof can be used in the preparation of a medicament for preventing and treating hyperuricemia.

[0076] The raw materials and instruments used in the present application are described below:

[0077] 1. Materials used in the experiment

[0078] Radix Astragali was Mongolian Radix Astragali, purchased from Shanxi Hunyuan Wansheng Radix Astragali Development Co., Ltd. (Batch: SX20191101). Paecilomyces cicadae was from China Forestry Microorganism Collection Center (No. cffc81169). Potato liquid medium (containing chloramphenicol) was purchased from Haibo Biotechnology Co., Ltd. (No. HB0233-8). Potato glucose agar (containing antibiotics) was purchased from Beijing Aobosan Biotechnology Co., Ltd. (No. 20220418). AB-8 macroporous resin was purchased from Cangzhou Baoneng Adsorption Material Technology Co., Ltd.

[0079] DMEM medium was purchased from Beijing Biodot Biotechnology Co., Ltd. (Gibco, C1195500BT). Heat-inactivated special fetal bovine serum was purchased from Beijing Biodot Biotechnology Co., Ltd. (Vivacell, batch number: C04001-500HI). Trypsin was purchased from Beijing Biodot Biotechnology Co., Ltd. (Gibco, batch number: 25200-056). BRL3A cells were purchased from Wuhan Punoxie Biotechnology Co., Ltd. Xanthine (purity ≥98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Batch number: A23GB158367). Probenecid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Batch number: H2117241). Uric acid (purity ≥98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Batch number: A05GB157092).

[0080] 2. Instruments used in the experiment

[0081] The semi-preparative liquid chromatograph was an LC-20AR semi-preparative liquid chromatograph, purchased from Shimadzu International Trade (Shanghai) Co., Ltd. The liquid chromatograph was an LC-20AT high-performance liquid chromatograph, purchased from Shimadzu International Trade (Shanghai) Co., Ltd. The nuclear magnetic resonance data was obtained using a Unity Inova 400MHz nuclear magnetic resonance instrument from Varian Company, USA. The present application used a UHPLC-Q-Exactive-Orbitrap high-resolution mass spectrometer to analyze the compounds.

[0082] Preparation Example 1

[0083] Paecilomyces cicadae seed liquid: Under sterile conditions, Paecilomyces cicadae was inoculated into potato glucose agar (PDA) medium, cultured in a 28°C incubator for 7 days, and a suitable amount of activated mycelium was picked up with an inoculation loop in sterilized potato glucose liquid medium, placed in a 28°C, 150r·min -1The seed liquid of Paecilomyces cicadidae was obtained by culturing for 5 days in a shaker, which was the activated seed liquid of Paecilomyces cicadidae.

[0084] Preparation Example 2

[0085] AB-8 macroporous resin column: AB-8 macroporous resin was activated for 24 h with 95 vol% ethanol to obtain activated AB-8 macroporous resin, which was columned in a wet way, and washed with distilled water until no alcohol smell was left to obtain an AB-8 macroporous resin column.

[0086] Example

[0087] The 30 g of Astragalus membranaceus was crushed through a No. 1 sieve, 15 mL of distilled water was added, and the mixture was sterilized by steam at 121 ℃ for 25 min. Then, 30 mL of the activated seed liquid of Paecilomyces cicadidae prepared in Preparation Example 1 was inoculated when the temperature was lowered to 28 ℃, and the mixture was cultured in an incubator at 28 ℃ until the mycelium filled the bag. Then, the mixture was taken out, dried by blowing air at 60 ℃, crushed, and passed through a No. 4 sieve to obtain a fermented mycelium powder, which was used as needed.

[0088] 1 g of the fermented mycelium powder was added to 50 mL of methanol to extract by reflux for 2 h, and then evaporated on a water bath. Distilled water was added to a volume of 100 mL to obtain a sample solution. The sample solution was columned on the AB-8 macroporous resin column obtained in Preparation Example 2, and the flow rate was adjusted to 1 mL / min. After columnation, 600 mL of distilled water, 600 mL of 30 vol% ethanol, and 600 mL of 75 vol% ethanol were eluted, respectively, and the eluate of 75 vol% ethanol was collected, concentrated at 50 ℃ under reduced pressure, transferred to an evaporating dish, evaporated on a water bath at 70 ℃, and dissolved in 40 vol% acetonitrile water solution by ultrasonic treatment. Then, the solution was filtered through a 0.22 μm microporous filter to obtain a crude product solution.

[0089] The crude product solution was separated by semi-preparative liquid chromatography to obtain the compound, which was referred to as component I. In this process, a SHIMADZU C18 column (20 x 250 mm, 10 μm) was used, the flow rate was 15 mL / min, the injection amount was 1 mL, the mobile phase was acetonitrile A-water B, and the gradient elution conditions were 80% to 60% B for 0-20 min and 60% B for 20-30 min. The detection wavelength was 260 nm.

[0090] The target compound (component I) was subjected to structural identification, and the results were as follows:

[0091]

[0092] 1H NMR (400 MHz, DMSO-D6) δ: 8.32 (s, 1H) (H-2), 7.95 (d, J = 8.8 Hz, 1H) (H-5), 7.29-7.20 (m, 2H) (H-2', 6'), 7.03 (d, J = 8.5 Hz, 1H) (H-5'), 6.92 (dd, J = 8.8, 2.2 Hz, 1H) (H-6), 6.84 (d, J = 2.1 Hz, 1H) (H-8), 4.98 (d, J = 7.8 Hz, 1H) (C1"), 3.80 (s, 3H) (4'-OCH3), 3.45 (s, 3H) (4"-OCH3), 3.02 (t, J = 9.3 Hz, 1H) (H-4").

[0093] 13 C NMR (101 MHz, DMSO-D6) δ: 174.54 (C4), 163.34 (C7), 157.49 (C9), 153.29 (C2), 148.74 (C4'), 145.93 (C3'), 127.25 (C5), 124.50 (C6'), 123.08 (C3), 122.76 (C1'), 116.29 (C10), 115.99 (C2'), 115.52 (C6), 112.23 (C5'), 102.10 (C8), 99.70 (C1"), 79.14 (C4"), 76.61 (C3"), 75.67 (C5"), 73.47 (C2"), 60.30 (C6"), 59.63 (4"-OCH3), 55.76 (4'-OCH3).

[0094] MS: Found (m / z): 459.13. The molecular formula of this compound is C 23 H 23 O 10 .

[0095] Experimental Example

[0096] The above compound was subjected to uric acid-lowering efficacy evaluation, process and results as follows:

[0097] 1. CCK8 determination of cytotoxicity

[0098] 6.9 mg of component I obtained in the example was precisely weighed and dissolved in 150 μL of DMSO to prepare a mother liquor with a concentration of 100 mmol·L -1 , and diluted with DMEM medium to 5 μmol·L -1 , 15 μmol·L -1 , 25 μmol·L -1 , respectively, and filtered to remove bacteria.

[0099] BRL3A cells were seeded in 96-well cell culture plates at a cell density of 3500 cells per well, 100 μL per well, and adhered for 24 h. The drug-containing medium containing Component I was added at concentrations of 5 μmol·L -1 , 15 μmol·L -1 , and 25 μmol·L -1 , respectively, and incubated at 37°C in a 5% CO2 incubator for 48 h. The cell survival rate was determined by the CCK8 method.

[0100] 2. Establishment of hyperuricemia cell model and drug administration

[0101] 57.1 mg of probenecid sample was accurately weighed and dissolved in 1 mL of DMSO to prepare a stock solution at a concentration of 200 mmol·L -1 , and diluted with the medium to 5 μmol·L -1 , 15 μmol·L -1 , and 25 μmol·L -1 , respectively. The solution was filtered to remove bacteria, and the drug administration solution containing Component I of Example 1 was prepared.

[0102] Cell recovery and culture: BRL3A cells stored in a -80°C freezer were quickly thawed at 37°C, centrifuged at 1000 r·min -1 for 3 min to remove DMSO, and then discarded. The supernatant was discarded, and the cells were cultured in a 37°C, 5% CO2 incubator. When the cells reached 90% confluence, they were passaged, and the cell experiments were performed when the cells reached 70% to 75% confluence.

[0103] Cell grouping and establishment of hyperuricemia model: After the cells reached the appropriate density, they were digested with 0.25% trypsin and seeded in a 24-well cell culture plate at a plating density of 1.25 x 10 5 cells / mL. After adhering for 24 h, the cells were grouped and administered. The blank control group was incubated with DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator for 48 h without adding other reagents. The model group was incubated with DMEM medium containing 10% fetal bovine serum and 1 mM xanthine to cause hyperuricemia, and incubated at 37°C in a 5% CO2 incubator for 48 h. The positive control group was incubated in the same manner as the model group, and 15 μmol·L -1 , 25 μmol·L -1 of probenecid was added, and incubated at 37°C in a 5% CO2 incubator for 48 h. The drug administration group was incubated in the same manner as the model group, and 5 μmol·L -1 , 15 μmol·L -1 , 25 μmol·L -1 of the compound Component I obtained in Example 1 was added, and incubated at 37°C in a 5% CO2 incubator for 48 h.

[0104] 3. Uric acid content in cell supernatant

[0105] Standard solution preparation: 0.0051 g of uric acid standard was precisely weighed into a test tube, 250 μL of distilled water was added to prepare a suspension, and a small amount of 0.1 M NaOH solution was added dropwise until it was clear, and then it was transferred to a 50 mL volumetric flask, and distilled water was added to the calibration line to prepare a solution with a concentration of 102 μg·mL -1 The mother liquor was stored in a 4°C refrigerator for standby use. Since uric acid is unstable in solution, a new uric acid standard solution was prepared at regular intervals.

[0106] Liquid phase conditions: After the cell modeling was completed, the supernatant was aspirated and passed through a 0.22 μm microporous filter, and then injected into a LC-20AT Shimadzu high performance liquid chromatograph. The specific liquid phase conditions are as follows.

[0107] Chromatographic column: ZORBAX SB-Aq (4.6 x 150 mm, 3.5 μm); mobile phase A: 7 x 10 -3 mol·L -1 KH2PO4-H3PO4 solution, adjust pH to 3.5; mobile phase B: 7 x 10 -3 mol·L -1 KH2PO4-H3PO4 solution containing 10% acetonitrile, adjust pH to 3.5; column oven: 25°C; injection volume: 10 μL; detection wavelength: 283 nm, elution gradient is shown in Table 1.

[0108] Table 1 Liquid phase elution gradient for uric acid detection

[0109] Time (min) Mobile phase B (%) 0-6 0 6-14 0-70 14-17.4 70

[0110] Standard curve establishment: 12.5 mL of uric acid control mother liquor was precisely measured into a 25 mL volumetric flask, and distilled water was added to the calibration line to obtain a solution with a concentration of 51 μg·mL -1 uric acid control solution, and gradient dilution to concentrations of 40.8 μg·mL -1 , 30.6 μg·mL -1 , 20.4 μg·mL -1 , 10.2 μg·mL -1 , 5.1 μg·mL -1 , 1.02 μg·mL -1 , respectively, were passed through a 0.22 μm microporous filter and injected into a high performance liquid chromatograph. The uric acid chromatographic peak area (Y) and the control product concentration (X) were used to establish a linear regression equation. The regression equation was Y = 44852X - 9828, R 2 = 0.9998. The standard curve is shown in Figure 1 .

[0111] Sample stability study: The supernatant of the model group and a uric acid reference solution with a concentration of 10.2 μg·mL⁻¹ were incubated at room temperature for 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 16 h, and 24 h before being injected into HPLC. The RSD values ​​were calculated to determine the stability of uric acid in the sample and reference solutions. The peak area RSD of the uric acid standard was 0.113%, and the peak area RSD of the model group sample was 0.085%, indicating that both the uric acid standard and the model group sample exhibited good stability at room temperature within 24 h, ensuring the reliability of subsequent sample detection values.

[0112] Instrument precision assessment: Six consecutive injections of uric acid reference solution were performed, and the RSD value was calculated to determine the instrument precision. With six consecutive injections, the RSD value of the reference peak area was 0.085%, indicating good instrument precision and reliable and stable subsequent test results.

[0113] 4. Evaluation of the efficacy of uric acid-lowering drugs

[0114] 4.1 CCK8 assay for cytotoxicity

[0115] The cytotoxicity of component I was detected using the CCK8 assay. The results are shown in [Figure number missing]. Figure 2 The test results show that when the concentration of component I is 5 μg·mL⁻¹, the cytotoxicity is relatively high, and when the concentration is 15 μg·mL⁻¹, the cytotoxicity is relatively high. -1 or 25 μg·mL -1 At that time, the cytotoxicity decreased. Overall, component I was slightly more toxic, with cell viability generally below 0.95, which may cause some damage to cells. Figure 2 In the middle, "I-5μmol·L -1 "This indicates that the dosage concentration of component I is 5 μg·mL" -1 And so on.

[0116] 4.2 Efficacy of uric acid-lowering drugs

[0117] A BRL3A hyperuricemia cell model was established and different concentrations of drugs were administered. The uric acid level in the cell supernatant was detected by HPLC. The results are as follows: Figure 3 As shown. When the dosage concentration of component I obtained in Example 1 is 5 μmol·L⁻¹. -1 15 μmol·L -1 At that time, the dosage concentration was 25 μmol·L. -1 The slightly weaker efficacy at this time suggests that component I may be more suitable for low-concentration administration, at a dosage of 15 μmol·L⁻¹. -1The uric acid-lowering effect reaches the highest. Compared with propyl sulfonamide, the uric acid-lowering effect of propyl sulfonamide increases with the increase of the administration concentration, while the uric acid-lowering effect of the component I increases first and then decreases. Meanwhile, compared with propyl sulfonamide with the same administration concentration, the component I has obvious advantages at low concentration administration, so the component I has good research prospects, but the toxicity needs to be paid attention to.

[0118] Figure 3 Compared with the blank group, "**" represents p<0.01; compared with the model group, "##" represents p<0.01; "###" represents p<0.001; and "####" represents p<0.0001. The ordinate represents the uric acid concentration value.

[0119] The present application is not limited to the above-mentioned embodiments, and any modification, improvement, replacement conceived by those skilled in the art without departing from the essential content of the present application falls within the scope of the present application.

Claims

1. A method of preparing a compound, characterized by, The structure of the compound is shown as follows: The preparation method comprises the following steps: (1) sterilizing a mixture of Astragalus membranaceus powder and water, inoculating Paecilomyces japonicus seed liquid after cooling, culturing, drying, crushing to obtain fermented mycelium powder; (2) extracting the fermented mycelium powder with alcohol, concentrating to obtain a concentrate; adding water to the concentrate to form a sample solution; (3) loading the sample solution on a macroporous resin column, eluting with water, 25-35 vol% ethanol and 70-80 vol% ethanol in sequence, collecting the eluate eluted with 70-80 vol% ethanol, concentrating to obtain a concentrated residue; redissolving the concentrated residue with 38-42 vol% acetonitrile to obtain a crude product solution; (4) separating the crude product solution by semi-preparative liquid chromatography to obtain the compound; wherein the chromatographic column used is a C18 chromatographic column; the mobile phase is acetonitrile-water; the gradient elution conditions are: 0-20 min, 80%-60% water, 20-30 min, 60% water; the detection wavelength is 254-260 nm.

2. The production method according to claim 1, characterized by, In step (1), the mass-volume ratio of Astragalus membranaceus powder to water is 30g:10-16mL; the sterilization is steam sterilization at 121-123℃ for 20-40min; the mass-volume ratio of Astragalus membranaceus powder to Paecilomyces japonicus seed liquid is 30g:30-33mL; the culturing condition is culturing at 23-28℃ in a culture box.

3. The preparation method according to claim 2, characterized in that, In step (1), the Paecilomyces japonicus seed liquid is prepared by the following steps: Under aseptic environment, Paecilomyces cicadidae was inoculated into potato dextrose agar (PDA) medium, and cultured in a 26-28°C incubator for 5-7 days for activation. After activation, the mycelium was picked up with an inoculation loop and inoculated into sterilized potato dextrose liquid medium, and cultured in a 26-28°C, 140-150 r·min -1 shaker for 3-5 days to obtain Paecilomyces cicadidae seed liquid.

4. The method of claim 1, wherein, In step (2), the alcohol is selected from methanol or ethanol, and the mass-volume ratio of fermented mycelium powder to alcohol is 1g:20-60mL; the extraction is under the condition of heating reflux for 1-4h.

5. The process according to any one of claims 1 to 4, characterized in that, In step (3), the macroporous resin column is an AB-8 macroporous resin column; the macroporous resin in the macroporous resin column is obtained by activating 90-95vol% ethanol for 20-30h. In step (3), the macroporous resin column is an AB-8 macroporous resin column; the macroporous resin in the macroporous resin column is obtained by activating 90-95vol% ethanol for 20-30h.

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