Isoprenyl xanthone compound, preparation method and application thereof

Abstract

The application relates to a kind of isoprenyl xanthone compounds and preparation method and application thereof, relate to the medical technical field, comprising the following steps: 1) after cutting, cold extraction of the whole herb of Eupatorium fortunei, filtrate is collected and concentrated under reduced pressure to obtain extract;2) the extract obtained in step 1) is chromatographed by silica gel column, gradient elution is carried out, and 3 fractions Fr.A-Fr.C are obtained;3) fraction Fr.A in step 2) is chromatographed by MCI column, gradient elution is carried out, and 6 fractions Fr.A1-Fr.A6 are obtained;4) fraction Fr.A3 in step 3) is chromatographed by silica gel column, gradient elution is carried out, and 11 fractions Fr.A3.1-Fr.A3.11 are obtained;5) compound is prepared and separated by HPLC high performance liquid chromatography method in fraction Fr.A3.10 in step 4), for preparing the drug for resisting Alzheimer disease.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an isopentenyl xanthonesone compound, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide, primarily characterized by cognitive impairment and progressive memory loss. World Health Organization data shows that the estimated number of people with dementia in 2019 was 55 million, and this number is projected to rise to 139 million by 2050 (Philip S, Bart De S, Miia K, Henne H, Gael C, Charlotte ET, Jeffrey C, Van Der Flier W M. Lancet, 2021, 397(10284):1577-1590.). With in-depth research into the pathogenesis of AD, neuroinflammation is considered a significant contributing factor. Neuroinflammation specifically refers to the immune response activated in the central nervous system (CNS). Typically, neuroinflammation occurs under the stimulation of CNS damage, infection, toxins, or the influence of autoimmunity. AD-related inflammatory components include microglia and astrocytes in the brain, cytokines, and chemokines. Aβ amylase activates neuroimmune cells, inducing the release of chemokines and cytokines, disrupting the neuronal environment, damaging neurons, and promoting oxidative stress or apoptosis, thus leading to neuroinflammation (Twarowski B, Herbet M. Inflammatory Processes in Alzheimer's Disease-Pathomechanism, Diagnosis and Treatment: A Review. Int J Mol Sci. 2023 Mar30;24(7):6518.). This process is mainly mediated by microglia. Abnormal microglia activation releases various pro-inflammatory factors and mediators, including nitric oxide (NO) and reactive oxygen species (ROS). Nitric oxide synthase (iNOS) is the rate-limiting enzyme for NO synthesis, and NO can promote the release of the inflammatory cytokine COX-2, which is a key enzyme that exacerbates the inflammatory response. Therefore, compounds that inhibit NO production may be potential major candidate compounds for treating neuroinflammatory diseases. It is of great significance to find highly effective and low-toxicity anti-Alzheimer's drugs from natural products to inhibit neuroinflammation. Summary of the Invention

[0003] The purpose of this invention is to solve the above-mentioned problems in the prior art and to provide an isopentenyl xanthonesone compound, its preparation method and application.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] An isopentenyl xanthonesone compound has the following structural formula:

[0006] .

[0007] The method for preparing an isopentenyl xanthonesone compound includes the following steps:

[0008] 1) After chopping the whole plant of *Gynostemma pentaphyllum*, extract it by cold soaking in methanol-water solution, collect the filtrate and concentrate it under reduced pressure to obtain the extract;

[0009] 2) The extract obtained in step 1) was subjected to silica gel column chromatography with gradient elution of the mobile phase to obtain three fractions Fr.A to Fr.C;

[0010] 3) The fraction Fr. A from step 2) was subjected to MCI column chromatography and eluted with a methanol and water gradient to obtain 6 fractions Fr. A1 to Fr. A6;

[0011] 4) The fraction Fr. A3 from step 3) was subjected to silica gel column chromatography, eluted with a gradient of petroleum ether and ethyl acetate, to obtain 11 fractions Fr. A3.1 to Fr. A3.11;

[0012] 5) The target compound was obtained by separating the fraction Fr. A3.10 from step 4) using HPLC.

[0013] In step 1), the cold maceration extraction is performed 2 to 4 times, each time for 12 to 24 hours.

[0014] In step 2), the mobile phase is dichloromethane, ethyl acetate, and methanol; the volume ratio of dichloromethane, ethyl acetate, and methanol can be 1:0:0, 0:1:0, or 0:0:1, respectively.

[0015] In step 3), the volume ratio of methanol to water can be 8:2, 17:3, 9:1, 19:1, or 10:0.

[0016] In step 4), the volume ratio of petroleum ether to ethyl acetate can be 1:0, 100:1, 50:1, 25:1, or 10:1, respectively.

[0017] The application of the aforementioned isopentenyl xanthonesone compound is for the preparation of drugs targeting iNOS to combat Alzheimer's disease.

[0018] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0019] The isopentenyl xanthonesone compounds of this invention are obtained from the whole herb *Lysimachia christinae*, and the extraction and separation methods are simple, with readily available raw materials. These compounds can target iNOS to inhibit NO production in mouse microglia (BV-2) cell line after stimulation, and hold promise for development as anti-Alzheimer's disease drugs. Attached Figure Description

[0020] Figure 1 The results of the Griess kit experiment on the inhibition of NO production in LPS-induced BV-2 cells by 10 μM compound 1 (drug treatment for 24 h) show that the bars in the graph represent, from left to right, the blank group treated with 1‰ DMSO, the model group treated with 1 μg / mL LPS, the positive control group treated with 1 μM TPCA and 1 μg / mL LPS, and the experimental group treated with 10 μM compound 1 and 1 μg / mL LPS.

[0021] Figure 2 The figure shows the results of different concentrations (2.5, 5, 10 μM) of compound 1 inhibiting the expression of iNOS and COX-2 proteins in BV-2 cells; among them, Figure 2 Figure A shows the Western blotting results of compound 1 downregulating the expression of iNOS and COX-2 proteins in a concentration-dependent manner, with α-tubulin as the reference. Figure 2 In Figure B, the band intensity ratio of iNOS / α-tubulin was analyzed using ImageJ. The bar chart from left to right represents the blank group treated with 1‰ DMSO, the model group treated with 1 μg / mL LPS, and the experimental group treated with 2.5 μM, 5 μM, and 10 μM compound 1, respectively. Figure 2 In the middle C, the intensity ratio of COX-2 / α-tubulin bands was analyzed using ImageJ. The bar chart from left to right represents the blank group treated with 1‰ DMSO, the model group treated with 1 μg / mL LPS, and the experimental group treated with 2.5 μM, 5 μM, and 10 μM compound 1, respectively. Detailed Implementation

[0022] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0023] Example 1

[0024] This invention relates to the preparation and structural identification of isopentenyl xanthonesone compounds.

[0025] 1. Preparation of Compound 1:

[0026] Step 1: Chop the whole plant of *Gynostemma pentaphyllum* (7.5 kg) and extract it by cold soaking in methanol-water solution 2–4 times, 12–24 h each time. Recover the soaking solution and concentrate under reduced pressure to obtain 800.0 g of crude methanol extract.

[0027] Step 2: 760g of the crude methanol extract from Step 1 was subjected to silica gel column chromatography with a gradient elution system using dichloromethane, ethyl acetate, and methanol as mobile phases (1:0:0, 0:1:0, 0:0:1) to obtain three fractions (Fr.A~Fr.C).

[0028] Step 3: The fraction Fr. A (69.5 g) from Step 2 was subjected to MCI column chromatography with methanol and water as the mobile phase (8:2, 17:3, 9:1, 19:1, 10:0) as the system gradient elution to obtain 6 fractions (Fr. A1 to Fr. A6).

[0029] Step 4: The fraction Fr. A3 (20.92 g) from Step 3 was subjected to silica gel column chromatography with petroleum ether and ethyl acetate as mobile phases (1:0, 100:1, 50:1, 25:1, 10:1) as mobile phases to obtain 11 fractions (Fr. A3.1 to Fr. A3.11).

[0030] Step 5: The fraction Fr. A3.10 (81.2 mg) from Step 4 was subjected to high performance liquid chromatography (HPLC) (ODS Rp-18, 5 μm, 250 × 10 mm, 75% ACN / H2O, 5 mL / min) to obtain compound 1 (3.4 mg).

[0031] The structural formula of compound 1 is:

[0032] .

[0033] 2. Structural identification of compound 1:

[0034] Compound 1, a yellow powdery solid;

[0035]

[0036] Example 2

[0037] Anti-Alzheimer's disease effect of compound 1 of the present invention

[0038] 1. Materials and Instruments

[0039] 1.1 Experimental cells: Mouse microglia BV-2

[0040] 1.2 Experimental Apparatus

[0041] This includes an inverted microscope (Olympus Leica, Japan), a cell culture incubator (Thermo Fisher Scientific, USA), a Millipore ultrapure water system (Millipore, USA), a high-speed refrigerated centrifuge (BeckMan Coulter, USA), micropipettes (Eppendorf, USA), a protein vertical electrophoresis system (Bio-Rad, USA), a wet transfer tank (Bio-Rad, USA), disposable culture dishes (Orange Scientific, Belgium), 48-well culture plates (Orange Scientific, Belgium), 96-well culture plates (Orange Scientific, Belgium), a vertical automatic electric heating pressure steam sterilizer (Shanghai Shenan Medical Instrument Factory), an electric thermostatic water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.), an ELISA reader (Shanghai Jinghong Experimental Equipment Co., Ltd.), a WH-3 vortex mixer (Shanghai Huxi Analytical Instrument Co., Ltd.), and a clean bench (Suzhou Antai Air Technology Co., Ltd.).

[0042] 1.3 Main Reagents

[0043] DMEM (High glucose) Hyclone

[0044] Fetal bovine serum Pricella

[0045] Hyclone, a pancreatic enzyme digestive solution

[0046] Opti-MEM Hyclone

[0047] BCA Kit Thermo Scientific

[0048] 1.4 Solution Preparation

[0049] ① RIPA cell lysis buffer:

[0050] 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 5 μg / mL Aprotenin, 5 μg / mL Leupeptasin.

[0051] ②PBS buffer solution:

[0052] 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, adjust pH to 7.4 with hydrochloric acid.

[0053] ③ Reagents for protein electrophoresis:

[0054] 1) 1.0 M Tris-HCl buffer

[0055] Take 12.12 g of Tris, add hydrochloric acid to adjust the pH to 6.8, add deionized water, bring the volume to 100 mL, adjust the pH to 6.8 again, sterilize at high temperature, and store at room temperature.

[0056] 2) 1.5 M Tris-HCl buffer

[0057] Take 18.2 g of Tris, add hydrochloric acid to adjust the pH to 8.8, add deionized water, bring the volume to 100 mL, adjust the pH to 8.8 again, sterilize at high temperature, and store at room temperature.

[0058] 3) Electrophoresis buffer

[0059] Take 3.03 g of Tris-HCl, 18.77 g of glycine, and 1 g of SDS and dissolve them in 1000 mL of distilled water.

[0060] 4) 2×SDS loading buffer

[0061] 6 mL dd H₂O, 5 mL 0.5 M Tris (pH 6.8), 4 mL 20% SDS, bromophenol blue.

[0062] 4 mg, 50% glycerol 4 mL, β-mercaptoethanol 1 mL

[0063] 5) 1×TBST

[0064] 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween-20

[0065] 6) Sealing liquid

[0066] 5% (w / v) skim milk powder dissolved in 1×TBST.

[0067] 7) Electro-hydraulic transfer

[0068] 3.03 g of Tris and 14.4 g of glycine were dissolved in 900 mL of distilled water, and finally 100 mL of methanol solution was added and mixed well.

[0069] 8) 10% SDS

[0070] Dissolve 10 g SDS in distilled water to 100 mL, in a 50 °C water bath, and store at room temperature.

[0071] 9) 10% Ammonium Persulfate (AP)

[0072] Dissolve 0.1 g of ammonium persulfate in 1.0 mL of ultrapure water and store at 4 °C.

[0073] 2. Compound 1 inhibits LPS-induced NO production.

[0074] NO is extremely unstable in solution. Under acidic conditions, NO can undergo a diazo reaction with the diazonium salt sulfanilamide to produce a pale yellow diazo compound. This product then couples with naphthylvinyldiamine to produce a purplish-red product. The amount of product generated is linearly related to the NO concentration, and a maximum absorption peak is observed at 540 nm. Therefore, the NO content in the sample can be calculated by constructing a standard curve.

[0075] 2.1 Experimental Methods

[0076] Experimental cells: BV-2 (mouse microglia)

[0077] ② Experimental procedure:

[0078] 1) Take an appropriate amount of DMEM culture medium (containing 10% fetal bovine serum) and accurately dilute the NO standard NaNO2 to a final concentration of 0, 1, 2, 5, 10, 20, 40, 60, and 100 μM. Add the standard dilution buffer to each well of a 96-well microplate at a volume of 50 μL, setting up 3 replicates. Then add 50 μL each of Griess Reagent I and Griess Reagent II sequentially, shake, and store in the dark for 10 min. Measure the absorbance at 540 nm using a microplate reader. Construct a standard curve with concentration on the x-axis and OD value on the y-axis.

[0079] 2) Collect BV-2 cells in a stable state, prepare a cell suspension, and filter it at 4 × 10⁻⁶ cells per well. 4 The cells were seeded at a density of 70% to 80% into 96-well cell culture plates and cultured in a constant temperature cell culture incubator at 37 ℃ with 5% CO2 for 12 to 24 h until the cells adhered to the plate and the cell density reached 70% to 80%.

[0080] 3) After microscopic observation, carefully aspirate the supernatant from the 96-well plate. Prepare LPS to a concentration of 1 μg / mL using DMEM medium, and prepare compound 1 to a concentration of 10 μM using diluted LPS medium. Add 100 μL to each well of the 96-well plate, setting up four replicates. Incubate at 37°C with 5% CO2 for 24 h. The experiment also included a blank control group (containing DMEM medium without LPS), an LPS model group, and a TPCA group.

[0081] 4) Transfer 50 μL of supernatant to a 96-well microplate, add 50 μL each of Griess Reagent I and Griess Reagent II, shake, and store in the dark for 10 min. Measure the absorbance at 540 nm using a microplate reader. Calculate the NO content using the standard curve to perform initial screening for the compound's anti-inflammatory activity.

[0082] 2.2 Experimental Results

[0083] The inhibitory effect of compound 1, isolated from *Gynostemma pentaphyllum*, on NO production in LPS-stimulated model cells (BV-2 cells) was preliminarily investigated using the Griess method. See also... Figure 1 The results showed that 1 μg / mL LPS could significantly induce NO production, and compound 1 had certain NO production inhibitory activity at a concentration of 10 μM.

[0084] 3. Compound 1 downregulates LPS-induced iNOS protein expression in BV-2 cells.

[0085] Western blotting separates samples based on differences in molecular weight and charge using polyacrylamide gel electrophoresis (PAGE). The samples are then transferred to a solid-phase support where proteins are adsorbed non-covalently, stabilizing the types and biological activities of the separated peptides. The proteins or peptides on the solid-phase support then act as antigens, triggering an immunoreaction with the corresponding primary antibody. The specific primary antibody then reacts with an enzyme-coupled secondary antibody. Finally, under the action of the enzyme, the substrate undergoes color development or chemiluminescence imaging, thereby detecting the separated specific target protein.

[0086] 3.1 Experimental Methods

[0087] Experimental cells: BV-2 (mouse microglia)

[0088] ② Experimental procedure:

[0089] 1) Collect BV-2 cells in a stable state and prepare a cell suspension. Then, filter the suspension at 3 × 10⁻⁶ cells per well. 5The cells were seeded at a density of 70% to 80% into 6-well cell culture plates and cultured in a constant temperature cell culture incubator at 37°C with 5% CO2 for 12 to 24 hours until the cells adhered to the plate and the cell density reached 70% to 80%.

[0090] 2) After microscopic observation, the supernatant in the culture dish was aspirated. Compound 1 was prepared to 10 μM in DMEM medium containing 10% fetal bovine serum, and 2 mL was added to each well of a 6-well plate. The plate was incubated at 37 ℃ with 5% CO2 for 2 h. LPS was then added to a final concentration of 1 μg / mL. The plate was incubated at 37 ℃ with 5% CO2 for 24 h. A blank control group (containing DMEM medium without LPS) and an LPS model group were also included in the experiment.

[0091] 3) After 24 h, discard the old culture medium, wash the sample once with pre-cooled PBS buffer, and carefully aspirate the PBS buffer. Add an appropriate volume of RIPA lysis buffer (prepared with protease inhibitors and phosphatase inhibitors) to fully cover the cells, and lyse on ice for 2 min. Scrape adherent cells off the surface using a pipette tip and transfer them to a labeled 1.5 mL EP tube. Lyse on ice for 15 min, vortexing once every 5 min, repeating 3 times. Then centrifuge at 4 ℃, 12000 rpm for 15 min, and transfer the supernatant to a new EP tube.

[0092] 4) Prepare BCA working solution according to the ratio of reagent A to reagent B = 50:1. Add cell lysis supernatant and BCA working solution (1:199) to each well of a 96-well microplate with a total volume of 200 μL, setting up 2 replicates. Mix the sample solution thoroughly by shaking for 30 s, incubate at 37℃ for 30 min, and measure the absorbance at 540 nm using a microplate reader. Calculate the protein concentration by substituting the values ​​into the standard curve.

[0093] 5) Based on the calculated protein concentration, add an appropriate amount of lysis buffer to ensure that each protein sample has the same concentration and volume. Then add an appropriate amount of loading buffer (containing 5% β-mercaptoethanol), boil the samples at 100 ℃ for 5 min, and centrifuge at 1000 rpm / min for 30 s at room temperature.

[0094] 6) SDS-PAGE electrophoresis: Place the prepared gel plate in the electrophoresis tank, add an appropriate amount of electrophoresis buffer, and load the sample (10 μL per well). Add an appropriate amount of protein marker to both sides of the sample. Set the initial voltage to 80 V. After about 30 minutes, introduce the separating gel. The protein markers will begin to separate. Change the voltage to 120 V. Adjust the electrophoresis time according to the experimental requirements.

[0095] 7) Electrotransfer: While electrophoresis is in progress, pre-cool the electrotransfer buffer and activate the PVDF membrane with methanol. After electrophoresis, remove the gel plate, accurately install the membrane transfer device, add the pre-cooled electrotransfer buffer, set the voltage to 90 V and the upper limit of the current to 399 mA, and place it in an ice box for membrane transfer for 90 min.

[0096] 8) Sealing: After the transfer is completed, transfer the PVDF membrane to the sealing box, add 5% skim milk, and seal by shaking at low speed on a shaker at room temperature for more than 1 hour.

[0097] 9) Primary antibody incubation: Cut the membrane according to the molecular weight of the target protein. Wash the cut PVDF membrane with TBST and transfer it to an incubation chamber. Prepare primary antibodies (anti-β-tublin; anti-iNOS; anti-COX-2; anti-p38; anti-p-p38) using TBST with 1% skim milk or 5% BSA solution. Add the prepared primary antibody solution to the incubation chamber and incubate overnight at 4 °C.

[0098] 10) Incubation of secondary antibody: Prepare secondary antibody (anti-mouse; anti-RAbbit) using 1% skim milk prepared with TBST. Recover the primary antibody, add an appropriate amount of TBST to wash the PVDF membrane 3 times, 10 min each time. Then add the secondary antibody and incubate at room temperature for 1 h. Add TBST again to wash for 10 min, and repeat 4 times.

[0099] 11) Development: Mix solutions A and B of the ECL luminescent substrate solution in equal proportions, and take an appropriate amount to drop evenly onto the PVDF film placed in the developing clamp for a light-protected reaction. Press the film in a dark room, and then expose the protein bands sequentially with the developer and fixer. Quantify the protein band intensity using ImageJ.

[0100] 3.2 Experimental Results

[0101] See Figure 2 At 10 μM, with p < 0.01 as the criterion for significant activity, LPS significantly activated the expression of related proteins in inflammatory cells. Compound 1 significantly inhibited the expression levels of iNOS and COX-2 proteins in model cells, and this effect was concentration-dependent.

Claims

1. A method for preparing an isopentenyl xanthonesone compound, characterized in that, Includes the following steps: 1) After chopping the whole plant of *Gynostemma pentaphyllum*, extract it by cold soaking in methanol-water solution, collect the filtrate and concentrate it under reduced pressure to obtain the extract; 2) The extract obtained in step 1) is subjected to silica gel column chromatography with gradient elution of the mobile phase to obtain three fractions Fr.A to Fr.C; the mobile phase is dichloromethane, ethyl acetate and methanol; the volume ratio of dichloromethane, ethyl acetate and methanol can be 1:0:0, 0:1:0, 0:0:1 respectively; 3) Perform MCI column chromatography on the fraction Fr. A from step 2), elute with a methanol and water gradient to obtain 6 fractions Fr. A1 to Fr. A6; the volume ratio of methanol to water can be 8:2, 17:3, 9:1, 19:1, 10:0, respectively. 4) The fraction Fr. A3 from step 3) was subjected to silica gel column chromatography and eluted with a gradient of petroleum ether and ethyl acetate to obtain 11 fractions Fr. A3.1 to Fr. A3.11; the volume ratio of petroleum ether to ethyl acetate could be 1:0, 100:1, 50:1, 25:1, or 10:1, respectively. 5) The target compound was obtained by separating the fraction Fr. A3.10 from step 4) using HPLC. The target compound is an isopentenyl xanthonone compound with the following structural formula: 。 2. The method for preparing an isopentenyl xanthonesone compound as described in claim 1, characterized in that: In step 1), the cold maceration extraction is performed 2 to 4 times, each time for 12 to 24 hours.

3. An isoprenylxanthone compound, characterized by: Obtained by the preparation method according to any one of claims 1 to 2.

4. The use of a compound according to claim 3, characterized in that: Used to prepare drugs for Alzheimer's disease.

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