N-Acetyl dopamine derivatives, their preparation methods and applications, and anti-oxidative stress drugs

Abstract

The present invention provides N-acetyl dopamine derivatives, a preparation method and application thereof, and an antioxidant stress drug, belonging to the technical field of drugs. The present invention provides an N-acetyl dopamine derivative having the structure shown in Formula I. The N-acetyl dopamine derivative provided by the present invention is a new compound and has an antioxidant effect, and can be used for preparing an antioxidant stress drug. Moreover, it is extracted and separated from the body of Vespa velutina nigrithorax, which has great significance for the research on the active ingredients of Vespa velutina nigrithorax. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to N-acetyl dopamine derivatives, their preparation methods and applications, and antioxidant stress drugs. Background Art

[0002] Vespa velutina auraria Smith is an insect of the order Hymenoptera, suborder Apocrita, family Vespidae. It is mainly distributed in Yunnan, Guizhou, Sichuan and other places, and is a relatively common wasp in cultivation, having both edible and medicinal values. The properties of wasps are sweet, cool, and slightly poisonous, and are mainly used to treat abdominal distension and pain, vomiting, light body and qi tonifying, dispelling wind, detoxifying, and treating freckles and facial sores. The toxin components of wasps mainly consist of amines, polypeptides, enzymes and other proteins. The wasp bodies often contain alkaloids, phenols, proteins, polypeptides, amino acids, reducing sugars and other substances.

[0003] Since the components contained in insect bodies are extremely complex, how to more deeply and detailedly analyze the specific chemical components and their related pharmacological effects of Vespa velutina auraria Smith is of great significance for the development and utilization of Vespa velutina auraria Smith. Summary of the Invention

[0004] The purpose of the present invention is to provide an N-acetyl dopamine derivative, its preparation method and application, and an antioxidant stress drug. The N-acetyl dopamine derivative provided by the present invention has an antioxidant effect, and is extracted and separated from the body of Vespa velutina auraria Smith, which is of great significance for the research of the active ingredients of Vespa velutina auraria Smith.

[0005] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides an N-acetyl dopamine derivative having the structure shown in Formula I:

[0007]

[0008] The present invention provides a preparation method of the N-acetyl dopamine derivative according to the above technical solution, comprising the following steps:

[0009] Extract the body of Vespa velutina auraria Smith with an ethanol aqueous solution, concentrate the obtained extract to obtain an extraction paste;

[0010] Mix the extraction paste with water, sequentially extract the obtained suspension mother liquor with petroleum ether, chloroform and ethyl acetate, and concentrate the obtained ethyl acetate extract to obtain an ethyl acetate fraction;

[0011] Under gradient elution conditions, the ethyl acetate fraction was subjected to first silica gel column chromatography, and the eluents used were petroleum ether-chloroform eluent and chloroform-methanol eluent in sequence; when gradient elution was carried out with the petroleum ether-chloroform eluent, the volume ratios of petroleum ether to chloroform were 1:1, 3:7, and 0:1 in sequence, and when gradient elution was carried out with the chloroform-methanol eluent, the volume ratios of chloroform to methanol were 25:1, 10:1, and 5:1 in sequence. The eluate with a volume ratio of chloroform to methanol of 5:1 was collected and concentrated to obtain fraction C5;

[0012] Under gradient elution conditions, fraction C5 was subjected to second silica gel column chromatography, and the eluent used was chloroform-methanol eluent. The volume ratios of chloroform to methanol in the chloroform-methanol eluent were 10:1, 5:1, 7:3, and 6:4 in sequence. The eluate with a volume ratio of chloroform to methanol of 6:4 was collected and concentrated to obtain fraction C5-11;

[0013] Fraction C5-11 was purified by Sephadex LH-20 gel column to obtain a purified fraction;

[0014] The purified fraction was subjected to third silica gel column chromatography to obtain an N-acetyl dopamine derivative having the structure shown in Formula I.

[0015] Preferably, the volume fraction of the ethanol aqueous solution is 75-95%.

[0016] Preferably, the number of extractions is 3-5 times; the temperature of each extraction is independently 10-30 °C, the time of each extraction is independently 24-72 h, and the material-liquid ratio of each extraction is independently 1:1.5-5.

[0017] Preferably, the particle size of the first silica gel used in the first silica gel column chromatography is 200-300 mesh, and the mass of the first silica gel is 10-20 times the mass of the ethyl acetate fraction;

[0018] The particle size of the second silica gel used in the second silica gel column chromatography is 300-400 mesh, and the mass of the second silica gel is 20-30 times the mass of fraction C5.

[0019] Preferably, the eluent used in the Sephadex LH-20 gel column purification is methanol.

[0020] Preferably, the eluent used in the third silica gel column chromatography is chloroform-methanol eluent, and the volume ratio of chloroform to methanol in the chloroform-methanol eluent is 5:3.

[0021] Preferably, the particle size of the third silica gel used in the third silica gel column chromatography is 300-400 mesh, and the mass of the third silica gel is 20-30 times the mass of the purified fraction.

[0022] The present invention provides the use of the N-acetyl dopamine derivative described in the above technical solution or the N-acetyl dopamine derivative prepared by the preparation method described in the above technical solution in the preparation of an antioxidant stress drug.

[0023] The present invention provides an antioxidant stress drug, which comprises an active ingredient and a pharmaceutically acceptable excipient, and the active ingredient is the N-acetyl dopamine derivative described in the above technical solution or the N-acetyl dopamine derivative prepared by the preparation method described in the above technical solution.

[0024] The present invention provides an N-acetyl dopamine derivative. The N-acetyl dopamine derivative provided by the present invention (denoted as Compound I) is a new compound and has an antioxidant effect. It can be used in the preparation of an antioxidant stress drug, and it is extracted and separated from the body of Vespa velutina nigrithorax, which is of great significance for the research of the active ingredients of Vespa velutina nigrithorax.

[0025] The present invention provides a preparation method of the Compound I. The present invention uses the body of Vespa velutina nigrithorax as a raw material to extract and separate Compound I. The raw material is relatively easy to obtain, and the extraction and separation steps are simple to operate. Description of the Drawings

[0026] Figure 1 For the main 1 H- 1 HCOSY (red line) and HMBC (blue arrow) correlation diagram;

[0027] Figure 2 Graph showing the effects of different concentrations and different action times of H2O2 solution on cell viability;

[0028] Figure 3 Graph showing the results of detecting the ROS level in PC12 cells by DCFH-DA fluorescence labeling method. Detailed Embodiments

[0029] The present invention provides an N-acetyl dopamine derivative having the structure shown in Formula I:

[0030]

[0031] The present invention provides a preparation method of the N-acetyl dopamine derivative described in the above technical solution, which comprises the following steps:

[0032] Extract the body of Vespa velutina nigrithorax with an aqueous ethanol solution, concentrate the obtained extract to obtain an extraction paste;

[0033] Mix the extraction paste with water, sequentially extract the obtained suspension mother liquor with petroleum ether, chloroform and ethyl acetate, and concentrate the obtained ethyl acetate extract to obtain an ethyl acetate fraction;

[0034] Under gradient elution conditions, the ethyl acetate fraction was subjected to the first silica gel column chromatography separation, and the eluents used were petroleum ether-chloroform eluent and chloroform-methanol eluent in sequence; when using the petroleum ether-chloroform eluent for gradient elution, the volume ratios of petroleum ether to chloroform were 1:1, 3:7, and 0:1 in sequence, and when using the chloroform-methanol eluent for gradient elution, the volume ratios of chloroform to methanol were 25:1, 10:1, and 5:1 in sequence. The eluent with a chloroform to methanol volume ratio of 5:1 was collected and concentrated to obtain fraction C5;

[0035] Under gradient elution conditions, the fraction C5 was subjected to the second silica gel column chromatography separation, and the eluent used was chloroform-methanol eluent. The volume ratios of chloroform to methanol in the chloroform-methanol eluent were 10:1, 5:1, 7:3, and 6:4 in sequence. The eluent with a chloroform to methanol volume ratio of 6:4 was collected and concentrated to obtain fraction C5-11;

[0036] The fraction C5-11 was purified by Sephadex LH-20 gel column to obtain a purified fraction;

[0037] The purified fraction was subjected to the third silica gel column chromatography separation to obtain an N-acetyl dopamine derivative having the structure shown in Formula I.

[0038] In the present invention, the body of Vespa velutina nigrithorax was extracted with an ethanol aqueous solution, and the obtained extract was concentrated to obtain an extraction extract. Before extracting the body of Vespa velutina nigrithorax, the present invention preferably pulverized the body of Vespa velutina nigrithorax; the present invention preferably pulverized the body of Vespa velutina nigrithorax to 20 mesh; in the examples of the present invention, it was specifically pulverized by a pulverizer. In the present invention, the volume fraction of the ethanol aqueous solution is preferably 75-95%, more preferably 90-95%. In the present invention, the extraction method is preferably soaking extraction; the number of extractions is preferably 3-5 times, more preferably 4 times; the temperature of each extraction is independently preferably 10-30°C, more preferably room temperature (25°C); the time of each extraction is independently preferably 24-72 h, more preferably 48-72 h. Specifically, the time of the first extraction is preferably 72 h, and the time of each subsequent extraction is preferably 48 h; the solid-liquid ratio of each extraction is independently preferably 1:1.5-5, more preferably 1:3, and the solid-liquid ratio is based on kg:L, that is, 1 kg:1.5-5 L. In the present invention, after each extraction, the obtained material liquid is preferably filtered successively through a 400-mesh filter cloth and a Buchner funnel by suction filtration, and the residue continues for the next extraction; after the last extraction, the filtrates obtained by suction filtration are combined to obtain an extract. The present invention preferably concentrates the extract to be ethanol-free to obtain an extraction extract; the concentration method is preferably vacuum concentration.

[0039] After obtaining the extraction extract, the present invention mixes the extraction extract with water, and successively extracts the obtained suspension mother liquor with petroleum ether, chloroform and ethyl acetate. The obtained ethyl acetate extract is concentrated to obtain an ethyl acetate fraction. In the present invention, the water is preferably pure water; based on the mass of the Vespa velutina nigrithorax body, the dosage ratio of the Vespa velutina nigrithorax body to water is preferably 32 kg: 0.5 - 1.5 L, more preferably 32 kg: 0.8 L. The present invention preferably mixes the extraction extract with water and heats it, stirs until a uniform suspension mother liquor is obtained, and then cools it to room temperature for subsequent extraction; the heating temperature is preferably 50 - 85°C, more preferably 80°C. The present invention successively uses petroleum ether, chloroform and ethyl acetate as extraction agents to extract the suspension mother liquor. Each time of extraction, the volume ratio of the material to be extracted to the extraction agent is preferably independently 1: 0.5 - 3, more preferably 1: 1; the extraction times of each extraction agent are preferably independently 3 - 5 times, more preferably 4 times. In the present invention, after the last extraction with ethyl acetate, the extraction liquids obtained from each ethyl acetate extraction are combined to obtain an ethyl acetate extract; the ethyl acetate extract is concentrated to obtain an ethyl acetate fraction; the concentration method is preferably vacuum concentration, and the degree of concentration can be determined according to the routine selection of those skilled in the art, and the present invention has no special limitation on this.

[0040] After obtaining the ethyl acetate fraction, the present invention performs first silica gel column chromatography separation on the ethyl acetate fraction under gradient elution conditions. In the present invention, the particle size of the first silica gel used in the first silica gel column chromatography separation is preferably 200 - 300 mesh, and the mass of the first silica gel is preferably 10 - 20 times the mass of the ethyl acetate fraction, more preferably 15 times. In the present invention, the eluents used in the first silica gel column chromatography separation are successively petroleum ether - chloroform eluent and chloroform - methanol eluent; when performing gradient elution with the petroleum ether - chloroform eluent, the volume ratio of petroleum ether to chloroform is successively 1: 1, 3: 7, 0: 1, and the volume of the eluent used for each gradient is preferably 2.3 - 2.7 L, more preferably 2.5 L (based on the mass of the Vespa velutina nigrithorax body being 32 kg) to remove low - polarity impurities; when performing gradient elution with the chloroform - methanol eluent, the volume ratio of chloroform to methanol is successively 25: 1, 10: 1, 5: 1, and the volume of the eluent used for each gradient is preferably 1.8 - 2.2 L, more preferably 2 L (based on the mass of the Vespa velutina nigrithorax body being 32 kg); the eluate with a chloroform - methanol volume ratio of 5: 1 is collected and concentrated to obtain a C5 component. In the examples of the present invention, specifically, the eluate with a chloroform - methanol volume ratio of 5: 1 is collected, and combined with TLC identification, the eluates with the same results are combined and concentrated to obtain a C5 component. In the present invention, the concentration method is preferably vacuum concentration, and the degree of concentration can be determined according to the routine selection of those skilled in the art, and the present invention has no special limitation on this.

[0041] After obtaining the C5 fraction, the present invention performs a second silica gel column chromatography separation on the C5 fraction under gradient elution conditions. In the present invention, the particle size of the second silica gel used in the second silica gel column chromatography separation is preferably 300 to 400 mesh, and the mass of the second silica gel is preferably 20 to 30 times the mass of the C5 fraction, more preferably 25 times. In the present invention, the eluent used in the second silica gel column chromatography separation is a chloroform-methanol eluent, and the volume ratio of chloroform to methanol in the chloroform-methanol eluent is successively 10:1, 5:1, 7:3, 6:4. The volume of the eluent used for each gradient is preferably 0.8 to 1.2 L, more preferably 1 L (based on the mass of the Vespa bicolor body being 32 kg). The eluate with a chloroform-methanol volume ratio of 6:4 is collected and concentrated to obtain the C5-11 fraction. In the examples of the present invention, specifically, the eluate with a chloroform-methanol volume ratio of 6:4 is collected, and combined with TLC identification, the eluates with the same results are combined and concentrated to obtain the C5-11 fraction. In the present invention, the concentration method is preferably vacuum concentration, and the degree of concentration can be determined by the conventional selection of those skilled in the art. The present invention has no special limitation on this.

[0042] After obtaining the C5-11 fraction, the present invention purifies the C5-11 fraction by Sephadex LH-20 gel column to obtain a purified fraction. In the present invention, the eluent used in the Sephadex LH-20 gel column purification is preferably methanol, and the volume of the methanol used is preferably 500 to 700 mL, more preferably 600 mL (based on the mass of the Vespa bicolor body being 32 kg). When eluting with the methanol, starting from when the eluate begins to flow out, the present invention preferably collects the eluate after 300 mL and concentrates it to obtain a purified fraction. In the examples of the present invention, after eluting with methanol, specifically, combined with TLC identification, the eluates corresponding to the main spot components are combined and concentrated to obtain a purified fraction. In the present invention, the concentration method is preferably vacuum concentration, and the degree of concentration can be determined by the conventional selection of those skilled in the art. The present invention has no special limitation on this.

[0043] After obtaining the purified component, the present invention performs a third silica gel column chromatography separation on the purified component to obtain an N-acetyl dopamine derivative having the structure shown in Formula I. In the present invention, the particle size of the third silica gel used in the third silica gel column chromatography separation is preferably 300-400 mesh, and the mass of the third silica gel is preferably 20-30 times, more preferably 25 times, the mass of the purified component; the eluent used in the third silica gel column chromatography separation is preferably a chloroform-methanol eluent, and the volume ratio of chloroform to methanol in the chloroform-methanol eluent is preferably 5:3, and the volume of the chloroform-methanol eluent used is preferably 450-550 mL, more preferably 500 mL (based on the mass of the wasp larvae being 32 kg). When eluting with the chloroform-methanol eluent, starting from when the eluate begins to flow out, the present invention preferably collects the eluate after 250 mL for concentration to obtain an N-acetyl dopamine derivative having the structure shown in Formula I, which is a yellow powder. In the examples of the present invention, after eluting with the chloroform-methanol eluent, specifically in combination with TLC detection, the eluates corresponding to the single spot components are combined and concentrated to obtain an N-acetyl dopamine derivative having the structure shown in Formula I. In the present invention, the concentration method is preferably vacuum concentration, and the concentration is based on completely removing the eluent.

[0044] The present invention provides the use of the N-acetyl dopamine derivative described in the above technical solution or the N-acetyl dopamine derivative prepared by the preparation method described in the above technical solution in the preparation of an antioxidant stress drug. The N-acetyl dopamine derivative provided by the present invention can significantly reduce the level of ROS in cells, and the ability of the N-acetyl dopamine derivative to reduce ROS is comparable to that of ascorbic acid, indicating that it can reduce the oxidative stress response in cells and has a certain antioxidant ability, and can be used in the preparation of antioxidant stress drugs.

[0045] The present invention provides an antioxidant stress drug, comprising an active ingredient and a pharmaceutically acceptable excipient, and the active ingredient is the N-acetyl dopamine derivative described in the above technical solution or the N-acetyl dopamine derivative prepared by the preparation method described in the above technical solution. In the present invention, the content of the active ingredient in the antioxidant stress drug is preferably 10-99 wt%, more preferably 30-90 wt%. The present invention has no special limitation on the specific type of the pharmaceutically acceptable excipient, and pharmaceutically acceptable excipients well-known to those skilled in the art can be used. The present invention has no special limitation on the dosage form, administration method and dosage of the antioxidant stress drug, and appropriate dosage form, administration method and dosage can be selected according to actual needs.

[0046] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative work belong to the scope of protection of the present invention.

[0047] Example 1

[0048] (1) Take 32 kg of Vespa bicolor bodies, crush them to 20 mesh through a pulverizer, use an ethanol aqueous solution with a volume fraction of 95% as the extractant, soak and extract at room temperature (25 °C) for 72 h according to a solid-liquid ratio of 1 kg: 3 L, and sequentially filter the obtained soaking liquid through a 400-mesh filter cloth and a Buchner funnel; the residue is continuously soaked and extracted 3 times by the same method as above, and the soaking and extraction time for each time is 48 h. All the extracts are combined and concentrated under reduced pressure until there is no ethanol, obtaining an extraction extract, which is stored at 4 °C for standby.

[0049] (2) Mix the extraction extract with 0.8 L of pure water and heat it in a water bath at 80 °C, stir until a uniform suspension mother liquor is obtained; cool the suspension mother liquor to room temperature, use petroleum ether as the extractant to extract the cooled suspension mother liquor 3 times, and the volume ratio of the extractant to the suspension mother liquor is 1:1 each time to remove low-polarity impurities; then use chloroform as the extractant and continue to extract the obtained mother liquor 3 times with the same operation; then use ethyl acetate as the extractant and continue to extract the obtained mother liquor 5 times with the same operation. The obtained ethyl acetate extracts are combined and concentrated under reduced pressure to obtain the ethyl acetate part (92 g) of the Vespa bicolor extract.

[0050] (3) Perform silica gel column (the silica gel used has a particle size of 200 - 300 mesh) chromatography separation on the ethyl acetate part, use petroleum ether-chloroform as the eluent, perform gradient elution according to the volume ratio of petroleum ether to chloroform of 1:1, 3:7, 0:1, and the volume of the eluent used for each gradient is 2.5 L to remove low-polarity impurities; continue to use chloroform-methanol as the eluent, elute the silica gel column after petroleum ether-chloroform elution according to the volume ratio of chloroform to methanol of 25:1, 10:1, 5:1, the volume of the eluent used for each gradient is 2 L, collect the eluent with a volume ratio of chloroform to methanol of 5:1, and combine the eluents with the same results through TLC identification, and concentrate under reduced pressure to obtain the C5 component;

[0051] (4) Subject the C5 component to silica gel column chromatography (the silica gel used has a particle size of 300 - 400 mesh), using chloroform - methanol as the eluent, and perform gradient elution according to the volume ratios of chloroform to methanol of 10:1, 5:1, 7:3, and 6:4. The volume of the eluent used for each gradient is 1 L. Collect the eluent with a volume ratio of chloroform to methanol of 6:4, and combine the eluents with the same results through TLC identification. After concentration under reduced pressure, obtain the C5 - 11 component;

[0052] (5) Purify the C5 - 11 component by Sephadex LH - 20 gel column, using methanol as the eluent with a volume of 600 mL. Starting from when the eluent begins to flow out, collect the eluent after 300 mL. Combine the main spot components through TLC detection, and obtain a concentrated sample after concentration under reduced pressure. Subject the concentrated sample to silica gel column chromatography (the silica gel used has a particle size of 300 - 400 mesh), elute with an eluent with a volume ratio of chloroform to methanol of 5:3, with a volume of 500 mL. Starting from when the eluent begins to flow out, collect the eluent after 250 mL. Combine the single - spot components through TLC detection, and obtain 9.7 mg of the target compound I (yellow powder) after concentration under reduced pressure.

[0053] Perform structure identification on the compound I as follows:

[0054] The HR - ESI - MS of the compound I m / z: 346.0943 (calcd. 346.0927, [M + H] + ), and its molecular formula is C 17 H 15 NO7, with an unsaturation degree of 11. The 1 1H NMR (Table 1) and HSQC spectra 7.59 (1H, dd, J = 8.4, 2.2 Hz, H - 7), 7.53 (1H, d, J = 2.0 Hz, H - 5), 7.01 (1H, d, J = 8.4 Hz, H - 8), 6.97 (1H, d, J = 2.1 Hz, H - 2'), 6.84 (2H, overlap, H - 5', 6') show the presence of two 1,3,4 - trisubstituted aromatic rings. 1 The 1H NMR and HSQC spectra show δ H 5.86 (1H, d, J = 7.1 Hz, H - 3), 4.93 (1H, d, J = 7.0 Hz, H - 2) have two oxygen - containing methine signals, and one methyl δ H 1.85 (3H, s, H - 2”'). 13 The 13C NMR and HSQC spectra show that compound I has 17 carbon signals, including two carbonyl signals δ C 170.3, 167.1, and 12 aromatic carbon signals δC 148.2, 146.7, 145.9, 143.2, 128.3, 125.1, 124.5, 120.6, 119.4, 117.7, 116.0, 115.6, two oxygenated methylene carbon signals δ C 78.2, 77.7 and a methyl carbon signal δ C 22.9. Through the HMBC correlation signals ( Figure 1 ) H-2 / C-8a and the molecular weight speculation, it may be an acetyl dopamine derivative. δ H 7.59 (1H, dd, J = 8.4, 2.2 Hz, H-7) correlated with C-8a indicates that there is a side chain attached to C-6, and H-7 / C-9, H-5 / C-9 (δ C 167.1) indicates that the carboxyl group at C-9 is connected to the benzene ring. The HMBC correlations of H-3 / C-1" (δ C 170.3), H-2" / C-1" show that there is an acetamido group at C-3. The correlations of H-2 / C-1', H-2 / C-2', H-2 / C-6' and H-3 / C-1' suggest that C-2 is connected to a 1,3,4-trisubstituted benzene ring. The vicinal coupling constant J = 7.0, 7.1 Hz of H-2 / H-3 in compound I indicates the existence of a trans H-2 / H-3 relationship. In summary, the structure of compound I is determined as Figure 1 shown and named trans-2-(3',4'-dihydroxyphenyl)-3-acetylamino-6-carboxy-1,4-benzodioxane. After database retrieval, compound I has not been reported and is a new compound.

[0055] Table 1 1 H (400 MHz) and 13 C (100 MHz) NMR data of compound I

[0056]

[0057]

[0058] Test Example 1

[0059] In this test example, the antioxidant stress activity of compound I was detected. Specifically, the level of ROS in PC12 cells (rat adrenal medulla pheochromocytoma differentiated cell line) was detected, and ascorbic acid was used as the positive control drug to study the cellular antioxidant stress ability of compound I in the present invention.

[0060] DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) is a non-polar chemical substance that can freely pass through the cell membrane. After entering the cell, it is removed of two ethyl groups by intracellular esterase to form polar DCFH, which remains inside the cell. When cells produce ROS, it will react with DCFH, oxidizing DCFH to DCF by ROS. By measuring the fluorescence intensity of DCF in cells with a multifunctional microplate reader, the in-situ real-time detection of intracellular ROS based on single cells can be carried out, and thus the degree of change of free radicals in cells can be directly reflected according to the fluorescence intensity.

[0061] In the following experiments, SPSS was used for the statistics and analysis of data, and GraphPad Prism, version 5.0 software, was used for graphing. When comparing between groups, one-way ANOVA in parametric tests was used.

[0062] The reagents, equipment and specific detection steps required for this test example are as follows:

[0063] 1. Cell type and source: Highly differentiated PC12 cells, purchased from Wuhan Punosai Life Science Co., Ltd.

[0064] 2. Main instrument equipment: Carbon dioxide constant temperature incubator (Thermoscientific), vertical air-pressurized steam sterilizer (Shanghai Boxun), multifunctional microplate reader (SpectraMax M2).

[0065] 3. Main reagents: DMEM high-glucose medium (Wuhan Punosai), trypsin (Wuhan Punosai), fetal bovine serum (GIBCO), thiazolyl blue (Shanghai Beyotime), reactive oxygen species detection kit (Beijing Solarbio).

[0066] 4. MTT was used to detect the effects of different concentrations and different action times of H2O2 solution on the survival rate of PC12 cells:

[0067] (1) Take 5.1 μL of 30 wt% hydrogen peroxide solution and add it to 1 mL of deionized water to prepare a stock solution of 50000 μM. Then, H2O2 solutions with concentrations of 100 μM, 200 μM, 400 μM, 600 μM, and 800 μM were prepared with high-glucose medium respectively.

[0068] (2) Take PC12 cells in the logarithmic growth phase, digest them with trypsin and make a cell suspension of 1×10 5 cells / mL. Set up a blank group, a negative control group, and an H2O2 group in a 96-well plate. In the H2O2 group and the negative control group, 100 μL of cell suspension was inoculated into each well, and only cell-free culture medium of equal volume was added to the blank group. Incubate in a 37°C, 5% CO2 incubator for 24 h.

[0069] (3) The H2O2 group was added with the prepared H2O2 solution, and the negative control group and the blank group were added with an equal volume of complete medium, with 5 replicate wells in each group.

[0070] (4) After incubating for 1 h, 2 h, 3 h, and 4 h respectively in an incubator at 37 °C and 5% CO2, the supernatant was discarded, and 20 μL of MTT with a concentration of 5 mg / mL was added to each well. Then, it was continuously incubated in an incubator at 37 °C and 5% CO2 for 4 h, and 50 μL of DMSO was added to each well to dissolve the purple formazan crystals.

[0071] (5) Shake well, measure the optical density value (OD value) of each well at a wavelength of 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate.

[0072] Figure 2 It is a graph showing the effects of different concentrations and different action times of the H2O2 solution on the cell survival rate. As can be seen from Figure 2 it that when the concentration of the H2O2 solution is 400 μM, the cell survival rate reaches 55.2%, and the effect of incubating for 4 h is better. Therefore, 400 μM is selected as the optimal working concentration, and 4 h is selected as the optimal working time.

[0073] 5. Determination of reactive oxygen species (ROS):

[0074] (1) PC12 cells in the logarithmic growth phase were taken, digested with trypsin, and made into a cell suspension of 3×10 5 cells / mL. A blank control group, a model group, an ascorbic acid group, and a compound I group were set up in a 6-well plate. 2 mL of the cell suspension was inoculated into each well and incubated in an incubator at 37 °C and 5% CO2 for 24 h.

[0075] (2) After 24 h, ascorbic acid solution (final concentration of 14 μg / mL) was added to the ascorbic acid group, and compound I solution (final concentration of 14 μg / mL) was added to the compound I group. The other groups were added with an equal volume of PBS and incubated in an incubator at 37 °C and 5% CO2 for 24 h.

[0076] (3) The model group, the ascorbic acid group, and the compound I group were stimulated with H2O2 solution (final concentration of 400 μM), and the blank control group was added with an equal volume of complete medium and incubated in an incubator at 37 °C and 5% CO2 for 4 h.

[0077] (4) Cell sample treatment: The incubated cells were taken out of the incubator, the medium was discarded, 1.5 mL of DCFH-DA with a concentration of 10 μM was added, and it was placed in the incubator for dark incubation for 30 min; after the incubation was completed, DCFH-DA was discarded, and the cells were washed twice with PBS to fully remove DCFH-DA, and then the cells were resuspended with 800 μL of serum-free medium.

[0078] Take 100 μL of each group of cells into a 96-well plate, and detect the fluorescence intensity of each cell on a multifunctional microplate reader with an excitation light of 488 nm and an emission light of 525 nm.

[0079] (6) Count each group of cells and calculate the fluorescence intensity of each group at the same number of cells (5×10 4 cells).

[0080] Figure 3 It is a result graph for detecting the ROS level in PC12 cells using the DCFH-DA fluorescence labeling method. As can be seen from Figure 3 it, compared with the blank control group, the level of ROS in the model group increased significantly. Both ascorbic acid and Compound I can significantly reduce the level of ROS in cells, and the ability of Compound I to reduce ROS is equivalent to that of ascorbic acid, indicating that Compound I can reduce the oxidative stress response in cells and has certain antioxidant ability.

[0081] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An N-acetyl dopamine derivative having the structure shown in Formula I:

2. A method for preparing the N-acetyl dopamine derivative according to Claim 1, comprising the following steps: Extracting the body of Vespa velutina with an aqueous ethanol solution, concentrating the obtained extract to obtain an extraction paste; Mixing the extraction paste with water, sequentially extracting the obtained suspension mother liquor with petroleum ether, chloroform and ethyl acetate, concentrating the obtained ethyl acetate extract to obtain an ethyl acetate fraction; Performing first silica gel column chromatography separation on the ethyl acetate fraction under gradient elution conditions, and the eluents used are a petroleum ether-chloroform eluent and a chloroform-methanol eluent in sequence; when performing gradient elution with the petroleum ether-chloroform eluent, the volume ratios of petroleum ether to chloroform are 1:1, 3:7, 0:1 in sequence, and when performing gradient elution with the chloroform-methanol eluent, the volume ratios of chloroform to methanol are 25:1, 10:1, 5:1 in sequence. Collect the eluate with a volume ratio of chloroform to methanol of 5:1 and concentrate it to obtain a C5 fraction; Performing second silica gel column chromatography separation on the C5 fraction under gradient elution conditions, and the eluent used is a chloroform-methanol eluent. The volume ratios of chloroform to methanol in the chloroform-methanol eluent are 10:1, 5:1, 7:3, 6:4 in sequence. Collect the eluate with a volume ratio of chloroform to methanol of 6:4 and concentrate it to obtain a C5-11 fraction; Purifying the C5-11 fraction by Sephadex LH-20 gel column to obtain a purified fraction; Performing third silica gel column chromatography separation on the purified fraction to obtain an N-acetyl dopamine derivative having the structure shown in Formula I; the eluent used for the third silica gel column chromatography separation is a chloroform-methanol eluent, and the volume ratio of chloroform to methanol in the chloroform-methanol eluent is 5:

3.

3. The preparation method according to claim 2, characterized in that, The volume fraction of the aqueous ethanol solution is 75-95%.

4. The preparation method according to claim 2 or 3, characterized in that, The number of extractions is 3-5 times; the temperature of each extraction is independently 10-30 °C, the time of each extraction is independently 24-72 h, and the material-liquid ratio of each extraction is independently 1:1.5-5.

5. The preparation method according to claim 2, characterized in that, The particle size of the first silica gel used for the first silica gel column chromatography separation is 200-300 mesh, and the mass of the first silica gel is 10-20 times the mass of the ethyl acetate fraction; The particle size of the second silica gel used for the second silica gel column chromatography separation is 300-400 mesh, and the mass of the second silica gel is 20-30 times the mass of the C5 fraction.

6. The preparation method according to claim 2, wherein The eluent used for the Sephadex LH-20 gel column purification is methanol.

7. The preparation method according to claim 2, characterized in that, The particle size of the third silica gel used for the third silica gel column chromatography separation is 300-400 mesh, and the mass of the third silica gel is 20-30 times the mass of the purified fraction.

8. Use of the N-acetyl dopamine derivative according to Claim 1 or the N-acetyl dopamine derivative prepared by the preparation method according to any one of Claims 2-7 in the preparation of an antioxidant stress drug.

9. An antioxidant stress drug, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is the N-acetyl dopamine derivative as claimed in claim 1 or the N-acetyl dopamine derivative prepared by the preparation method as claimed in any one of claims 2 to 7.

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