Isopentenyl xanthone compound as well as preparation method and application thereof

Abstract

The invention discloses an isopentenyl xanthone compound as well as a preparation method and application thereof, and relates to the technical field of medicines, and the preparation method comprises the following steps: 1) chopping the whole herb of hypericum ascyron, performing cold soaking extraction, collecting filtrate, and performing vacuum concentration to obtain an extract; 2) subjecting the extract obtained in the step 1) to silica gel column chromatography and mobile phase gradient elution to obtain three fractions Fr.A-Fr.C; the preparation method comprises the following steps: 1) preparing a fraction Fr.A, 2) preparing a fraction Fr.A, 3) carrying out MCI column chromatography and gradient elution on the fraction Fr.A in the step 2) to obtain six fractions Fr.A1 to Fr.A6, 4) carrying out silica gel column chromatography and gradient elution on the fraction Fr.A3 in the step 3) to obtain 11 fractions Fr.A3.1 to Fr.A3.11, and 5) carrying out HPLC high performance liquid chromatography on the fraction Fr.A3.10 in the step 4) to prepare and separate the compound which is used for preparing the anti-Alzheimer disease medicine.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an isopentenyl xanthone compound and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease in the world, with the main clinical manifestations of cognitive impairment and progressive memory loss. According to data from the World Health Organization, the number of people suffering from dementia in 2019 was estimated to be 55 million, and it is expected to increase to 139 million in 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 the in-depth study of the pathogenesis of AD, neuroinflammation is considered to be an important pathogenic factor of AD. Neuroinflammation specifically refers to the immune response activated in the central nervous system (CNS). Usually, neuroinflammation occurs under the stimulation of CNS damage, infection, toxins or under the action of autoimmunity. AD-related inflammatory components include brain microglia and astrocytes, cytokines and chemokines. Aβ amyloid activates neural immune cells, induces the release of chemokines and cytokines, destroys the environment around neurons, damages neurons, promotes oxidative stress or apoptosis, and thus leads to the occurrence of 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 will release a variety of proinflammatory factors and mediators, including nitric oxide (NO) and reactive oxygen species (ROS). Nitric oxide synthase (iNOS) is the rate-limiting enzyme for NO synthesis. At the same time, NO can promote the release of inflammatory cytokine COX-2, which is a key enzyme for inflammatory cells to aggravate inflammatory response. Therefore, compounds that inhibit NO production may be potential candidate compounds for the treatment of neuroinflammatory diseases. It is of great significance to find highly effective and low-toxic anti-Alzheimer's disease drugs that inhibit neuroinflammation from natural products. Summary of the invention

[0003] The purpose of the present invention is to solve the above problems in the prior art and to provide an isopentenyl xanthone compound and a preparation method and application thereof.

[0004] In order to achieve the above object, the present invention adopts the following technical scheme:

[0005] An isopentenyl xanthone compound, the structural formula of which is as follows:

[0006] .

[0007] The method for preparing an isopentenyl xanthone compound comprises the following steps:

[0008] 1) Chop the whole herb of the Herba Lycopodii into pieces and extract it with a methanol-water solution, collect the filtrate and concentrate it under reduced pressure to obtain an extract;

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

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

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

[0012] 5) The fraction Fr. A3.10 in step 4) was separated by HPLC to obtain the target compound.

[0013] In step 1), the cold soaking 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 ratios of dichloromethane, ethyl acetate and methanol are 1:0:0, 0:1:0 and 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, 10:0 respectively.

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

[0017] The application of the isopentenyl xanthone compound is used to prepare a drug for fighting Alzheimer's disease with iNOS as the target.

[0018] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0019] The isopentenyl xanthone compounds of the present invention are obtained from the herb of Glehnia littoralis, and the extraction and separation method is simple, and the raw materials are easily available. The compounds can target iNOS to inhibit the production of NO after the mouse microglial cell line (BV-2) is stimulated, and are expected to be developed into anti-Alzheimer's disease drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the Griess kit experiment in which 10 μM compound 1 inhibited the production of NO in BV-2 cells induced by LPS (drug action for 24 hours); the bar graphs represent from left to right the blank group treated with 1‰ DMSO, the model group treated with 1 μg / mL LPS, the positive drug 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 results of the inhibition of iNOS and COX-2 protein expression in BV-2 cells by compound 1 at different concentrations (2.5, 5, 10 μΜ) are shown in FIG. Figure 2 A in the middle is the Western Blotting result diagram showing that compound 1 down-regulated the expression of iNOS and COX-2 proteins in a concentration-dependent manner, with α-tubulin as the reference object; Figure 2 Middle B is the iNOS / α-tubulin band intensity ratio analyzed by ImageJ, and its bar graphs from left to right represent the blank group treated with 1‰ DMSO, the model group treated with 1 μg / mL LPS, and the experimental groups treated with 2.5 μΜ, 5 μΜ, and 10 μΜ compound 1; Figure 2 Middle C is the COX-2 / α-tubulin band intensity ratio analyzed by ImageJ. The bar graphs from left to right represent the blank group treated with 1‰ DMSO, the model group treated with 1 μg / mL LPS, and the experimental groups treated with 2.5 μΜ, 5 μΜ, and 10 μΜ compound 1. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0023] Example 1

[0024] The invention relates to the preparation and structural identification of isopentenyl xanthone compounds.

[0025] 1. Preparation of compound 1:

[0026] Step 1: Chop the whole herb of the material, Achyranthes bidentata (7.5 kg), and then use a methanol-water solution for cold extraction 2 to 4 times, each time for 12 to 24 hours. Recover the soaking liquid and concentrate it under reduced pressure to obtain 800.0 g of crude methanol extract.

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

[0028] Step 3: The fraction Fr. A (69.5 g) in step 2 was chromatographed on an MCI column using methanol and water as the mobile phase (8:2, 17:3, 9:1, 19:1, 10:0) for systematic gradient elution to obtain 6 fractions (Fr. A1 to Fr. A6).

[0029] Step 4: The fraction Fr. A3 (20.92 g) in step 3 was subjected to silica gel column chromatography with petroleum ether and ethyl acetate as the mobile phase (1:0, 100:1, 50:1, 25:1, 10:1) for systematic gradient elution 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 purified by HPLC (ODS Rp-18, 5 μm, 250×10 mm, 75% ACN / H 2 O, 5 mL / min) to give compound 1 (3.4 mg).

[0031] The structural formula of the compound 1 is:

[0032] .

[0033] 2. Structural identification of compound 1:

[0034] Compound 1, 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 microglial cells BV-2

[0040] 1.2 Experimental instruments

[0041] Including inverted microscope - Olympus Leica, Japan, cell culture incubator - Thermo, USA, Millipore ultrapure water instrument - Millipore, USA, high-speed refrigerated centrifuge - Beckman Coulter, USA, micropipette - Eppendorf, USA, protein vertical electrophoresis instrument - Bio-Rad, USA. Wet transfer tank - Bio-Rad, USA, disposable culture dish - Orange Scientific, Belgium, 48-well culture plate - Orange Scientific, Belgium, 96-well culture plate - Orange Scientific, Belgium, vertical automatic electric pressure steam sterilizer - Shanghai Shen'an Medical Equipment Factory, electric constant temperature water bath - Shanghai Jinghong Laboratory Equipment Co., Ltd., microplate reader - Shanghai Jinghong Laboratory Equipment Co., Ltd., WH-3 vortex instrument - Shanghai Huxi Analytical Instrument Co., Ltd., clean bench - Suzhou Antai Air Technology Co., Ltd.

[0042] 1.3 Main Reagents

[0043] DMEM (High glucose) Hyclone

[0044] Fetal Bovine Serum Pricella

[0045] Pancreatic enzyme digestion solution Hydrone

[0046] Opti-MEM Hyclone

[0047] BCA Kit Thermo Scientific

[0048] 1.4. Solution preparation

[0049] ① RIPA cell lysis buffer:

[0050] 50 mMTris-HCl (pH 7.4), 150 mMNaCl, 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 mMNaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, adjust pH to 7.4 with hydrochloric acid.

[0053] ③ Protein electrophoresis related reagents:

[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, make up 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, make up 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] dd 2 O 6 mL, 0.5 M Tris pH 6.8 5 mL, 20% SDS 4 mL, bromophenol blue

[0062] 4 mg, 50% glycerol 4 mL, β-mercaptoethanol (2-mercaptoethanol) 1 mL

[0063] 5) 1× TBST

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

[0065] 6) Blocking solution

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

[0067] 7) Electroporation solution

[0068] Dissolve 3.03 g of Tris and 14.4 g of glycine in 900 mL of distilled water, and finally add 100 mL of methanol solution and mix well.

[0069] 8) 10% SDS

[0070] 10 g SDS, add distilled water to 100 mL, dissolve in a 50 ℃ water bath, and store at room temperature.

[0071] 9) 10% ammonium persulfate (AP)

[0072] 0.1 g ammonium persulfate was dissolved in 1.0 mL ultrapure water and stored at 4 °C.

[0073] 2. Compound 1 inhibits the production of NO induced by LPS

[0074] NO is extremely unstable in solution. Under acidic conditions, NO can react with diazonium salt sulfonamide to form a light yellow diazo compound. This product will react with naphthyl vinyl diamine to form a purple-red product. The amount of the product is linearly related to the concentration of NO, and there is a maximum absorption peak at a wavelength of 540 nm. Therefore, the content of NO in the sample can be calculated by making a standard curve.

[0075] 2.1 Experimental Methods

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

[0077] ② Experimental operation:

[0078] 1) Take an appropriate amount of DMEM medium (containing 10% fetal bovine serum) and accurately dilute the NO standard NaNO 2 , so that the final concentration of the standard is 0, 1, 2, 5, 10, 20, 40, 60, 100 μM. Add 50 μL of the standard diluent to each well of the 96-well ELISA plate, and set up 3 replicate wells. Then add 50 μL of Griess Reagent Ⅰ and Griess Reagent Ⅱ respectively, shake and store in the dark for 10 min, use an ELISA reader to measure the absorbance at a wavelength of 540 nm, and establish a standard curve with concentration as the horizontal axis and OD value as the vertical axis.

[0079] 2) Collect the BV-2 cells in a stable state and prepare a cell suspension. 4 The cells were inoculated into a 96-well cell culture plate at a density of 1.50 μg / mL and placed in a constant temperature cell culture incubator at 37 °C with 5% CO 2 The cells were cultured for 12-24 h until the cells adhered to the wall and the cell density reached 70%-80%.

[0080] 3) After microscopic observation, carefully aspirate the supernatant from the 96-well plate. Use DMEM medium to prepare LPS to 1 μg / mL, and use the diluted LPS medium to prepare compound 1 to 10 μM. Add 100 μL to each well of the 96-well plate and set up 4 replicates. Incubate at 37°C with 5% CO 2 The cells were cultured for 24 h under the conditions of 4% paraformaldehyde and 1% paraformaldehyde. A blank group (DMEM medium without LPS), an LPS model group, and a TPCA group were also set up.

[0081] 4) Take 50 μL of the supernatant and transfer it to a 96-well ELISA plate. Add 50 μL of Griess Reagent Ⅰ and 50 μL of Griess Reagent Ⅱ respectively. After shaking, store in the dark for 10 min. Use an ELISA reader to measure the absorbance at a wavelength of 540 nm. Substitute the standard curve to calculate the NO content and conduct a preliminary screening of the anti-inflammatory activity of the compound.

[0082] 2.2 Experimental Results

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

[0084] 3. Compound 1 down-regulates the expression of iNOS protein induced by LPS in BV-2 cells

[0085] Western blotting separates samples by polyacrylamide gel electrophoresis (PAGE) based on the molecular weight and charge differences of the samples, and then transfers them to a solid phase carrier that adsorbs proteins in a non-covalent bond form and can stabilize the type of electrophoretically separated polypeptides and their biological activities. Subsequently, the protein or polypeptide on the solid phase carrier is used as an antigen to react with the corresponding primary antibody. The specific primary antibody then reacts with the secondary antibody coupled to the enzyme. Finally, under the action of the enzyme, the substrate is colored or chemiluminescently developed to detect the separated specific target protein.

[0086] 3.1 Experimental Methods

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

[0088] ② Experimental operation:

[0089] 1) Collect BV-2 cells in a stable state to prepare a cell suspension and plate them at 3×10 5The cells were inoculated into a 6-well cell culture plate at a density of 1.50 μg / mL and placed in a constant temperature cell culture incubator at 37°C with 5% CO 2 The cells were cultured for 12-24 h until the cells adhered to the wall and the cell density reached 70%-80%.

[0090] 2) After microscopic observation, the supernatant was discarded. Compound 1 was prepared into 10 μM in DMEM medium containing 10% fetal bovine serum and added to a 6-well plate at a volume of 2 mL per well. Incubate the plate at 37 °C and 5% CO 2 The cells were cultured for 2 h under the conditions of 5% CO. LPS was then added to a final concentration of 1 μg / mL. 2 The cells were cultured for 24 h under the condition of 4% paraformaldehyde. A blank group (DMEM medium without LPS) and an LPS model group were also set up.

[0091] 3) After 24 hours, discard the old culture medium, wash the sample once with pre-cooled PBS buffer, and carefully dry 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 minutes. Use a pipette tip to scrape off the adherent cells and transfer them to a labeled 1.5 mL EP tube. Lyse on ice for 15 minutes, vortex and oscillate once every 5 minutes, and repeat 3 times. Then centrifuge at 4°C, 12,000 rmp / min for 15 minutes, and transfer the supernatant to a new EP tube.

[0092] 4) Prepare BCA working solution at a ratio of 50:1 for reagent A solution: B solution, add cell lysis supernatant and BCA working solution (1:199) to a 96-well ELISA plate with a total volume of 200 μL per well, and set up 2 replicate wells. Mix the sample solution evenly on a shaker for 30 s, incubate at 37°C for 30 min, and measure the absorbance at 540 nm on an ELISA reader. Substitute the standard curve to calculate the protein concentration.

[0093] 5) According to the calculated protein concentration, add an appropriate amount of lysis buffer to make the protein concentration and volume of each sample equal. Then add an appropriate amount of loading buffer (containing 5% β-mercaptoethanol), cook the sample at 100°C for 5 min, and centrifuge at room temperature at 1000 rpm / min for 30 s.

[0094] 6) SDS-PAGE electrophoresis: Place the prepared gel plate in the electrophoresis tank, add an appropriate amount of electrophoresis solution, load the sample (10 μL per well), and add an appropriate amount of protein marker on both sides of the sample. Set the initial voltage to 80 V. After about 30 minutes, the separation gel is entered and the protein marker begins to separate. Change the voltage to 120 V and arrange the electrophoresis time according to the experimental requirements.

[0095] 7) Electrotransfer: Pre-cool the electrotransfer solution and methanol-activated PVDF membrane while electrophoresis is in progress. After electrophoresis, remove the gel plate, accurately install the membrane transfer device, add the pre-cooled electrotransfer solution, set the voltage to 90 V, the current limit to 399 mA, and place the membrane in an ice box for 90 minutes.

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

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

[0098] 10) Incubate with secondary antibody: Use TBST to prepare 1% skim milk to prepare secondary antibody (anti-Mouse; anti-RAbbit), recover the primary antibody, add appropriate amount of TBST to wash the PVDF membrane 3 times, 10 min each time. Then add secondary antibody, incubate at room temperature for 1 h, add TBST again to wash for 10 min, repeat 4 times.

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

[0100] 3.2 Experimental Results

[0101] See also Figure 2 When compound 1 was 10 μM, **p < 0.01 was used as the basis for judging significant activity. LPS could significantly activate 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. An isopentenyl xanthone compound, characterized in that: The structural formula is as follows: 。 2. The method for preparing an isopentenyl xanthone compound according to claim 1, characterized in that: The following steps are involved: 1) Chop the whole herb of the Herba Lycopodii into pieces and extract it with a methanol-water solution, collect the filtrate and concentrate it under reduced pressure to obtain an extract; 2) The extract obtained in step 1) is subjected to silica gel column chromatography with gradient elution of mobile phase to obtain three fractions Fr.A to Fr.C; 3) The fraction Fr. A in step 2) was subjected to MCI column chromatography and gradient eluted with methanol and water to obtain 6 fractions Fr. A1 to Fr. A6; 4) The fraction Fr. A3 in step 3) was subjected to silica gel column chromatography, and gradient elution was performed with petroleum ether and ethyl acetate to obtain 11 fractions Fr. A3.1 to Fr. A3.11; 5) The fraction Fr. A3.10 in step 4) was separated by HPLC to obtain the target compound.

3. The method for preparing an isopentenyl xanthone compound according to claim 2, characterized in that: In step 1), the cold soaking extraction is performed 2 to 4 times, each time for 12 to 24 hours.

4. The method for preparing an isopentenyl xanthone compound according to claim 2, characterized in that: In step 2), the mobile phase is dichloromethane, ethyl acetate and methanol; the volume ratios of dichloromethane, ethyl acetate and methanol are 1:0:0, 0:1:0 and 0:0:1 respectively.

5. The method for preparing an isopentenyl xanthone compound according to claim 2, characterized in that: In step 3), the volume ratio of methanol to water may be 8:2, 17:3, 9:1, 19:1, 10:0, respectively.

6. The method for preparing an isopentenyl xanthone compound according to claim 2, characterized in that: In step 4), the volume ratio of petroleum ether to ethyl acetate can be 1:0, 100:1, 50:1, 25:1, 10:1, respectively.

7. The use of an isopentenyl xanthone compound according to claim 1, characterized in that: Used for preparing drugs against Alzheimer's disease.

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