CN111875513B - Resveratrol A ring N (CH) 3 ) 2 Base derivatives, preparation method and application thereof - Google Patents
Resveratrol A ring N (CH) 3 ) 2 Base derivatives, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides resveratrol A ring N (CH) 3 ) 2 A derivative, a preparation method and application thereof, belonging to the technical field of medicine. The invention provides resveratrol A ring N (CH) with a structure shown in a formula I 3 ) 2 The derivative, as a sodium hydrogen exchanger-1 (NHE 1) receptor antagonist, acts on an NHE1 receptor in a targeting way, and simultaneously inhibits 2 signal conduction pathways of a phosphatidylinositol 3-kinase/protein serine threonine kinase (PI 3K/AKT) signal pathway and a Janus kinase/signal transduction and transcription activator (JAK/STAT), thereby playing a role in treating alcoholic fatty liver.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to resveratrol A-ring N (CH) 3 ) 2 A derivative, a preparation method and application thereof.
Background
Alcoholic fatty liver is a liver disease caused by long-term drinking of a large amount of alcohol, and is one of the types of alcoholic liver diseases. Alcoholic fatty liver is a common pathological change of the liver, not an independent disease. When liver cells are damaged, alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) overflow causes the ALT and AST contents in serum to be increased, so the degree of damage of the liver cells of the alcoholic fatty liver patients and the repair condition of the liver cells can be reflected by measuring the ALT and AST activities in the serum.
At present, there is no unified standard for treating alcoholic fatty liver clinically, and the commonly used auxiliary intervention means include insulin sensitizer, lipid regulating drug, antioxidant and the like. Common drugs for treating alcoholic fatty liver include: (1) western medicines such as methionine choline, lecithin, silymarin, inosine, coenzyme A, carnitine orotate, etc. mainly have the functions of protecting liver cells and increasing fat transport; (2) antioxidants such as resveratrol, reduced glutathione, taurine, vitamin E, and mainly have effects of inhibiting cholesterol, triglyceride oxidation and lipid accumulation; (3) the Chinese medicinal materials such as Curcuma rhizome, radix Polygoni Multiflori Preparata, and fructus crataegi have effects of reducing blood lipid and preventing cholesterol deposition in liver.
In the prior art, resveratrol has a good antioxidation effect. However, resveratrol is not ideal in its therapeutic effect on alcoholic fatty liver.
Disclosure of Invention
In view of the above, the present invention aims to provide a resveratrol A ring N (CH) 3 ) 2 A derivative, a preparation method and application thereof. The invention provides resveratrol A ring N (CH) 3 ) 2 The derivative has therapeutic effect on alcoholic fatty liver.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides resveratrol A-ring N (CH) 3 ) 2 A group derivative having the structure shown in formula I:
the invention also providesResveratrol A ring N (CH) in the technical scheme 3 ) 2 A process for the preparation of a derivative comprising the steps of:
mixing diethyl phosphite, an organic solvent, naH and 4-methoxybenzyl bromide to carry out debromination reaction to obtain a compound with a structure shown in a formula 1;
mixing 4-dimethylaminosalicylaldehyde, an organic solvent, chloromethyl methyl ether and diisopropylethylamine for substitution reaction to obtain a compound with a structure shown in a formula 2;
mixing a compound with a structure shown in a formula 1, a compound with a structure shown in a formula 2, an organic solvent and NaH for condensation reaction to obtain a compound with a structure shown in a formula 3;
mixing the compound with the structure shown in the formula 3, tert-butyl alcohol and pyridinium p-toluenesulfonate, and refluxing to obtain resveratrol A ring N (CH) with the structure shown in the formula I 3 ) 2 A derivative of phenyl;
preferably, the time of the debromination reaction is 1 to 3 hours.
Preferably, the time of the substitution reaction is 10 to 30min.
Preferably, the condensation reaction is carried out at a temperature of 0-100 ℃ for 1-3 h.
Preferably, the reflux temperature is 90-100 ℃ and the reflux time is 1-3 h.
Preferably, the molar ratio of the compound having the structure shown in formula 3 to the pyridinium p-toluenesulfonate is 1.
The invention also provides resveratrol A ring N (CH) in the technical scheme 3 ) 2 The resveratrol A ring N (CH) prepared by the preparation method of the base derivative or the technical scheme 3 ) 2 The application of the derivative in preparing medicines for treating alcoholic fatty liver is provided.
Preferably, the drug for treating alcoholic fatty liver comprises an effective dose of a structure shown as a formula IResveratrol A Ring N (CH) 3 ) 2 The derivatives, the stereoisomers, the pharmaceutically acceptable salts and pharmaceutically acceptable carriers, auxiliary materials, excipients and diluents.
Preferably, the dosage form of the drug for treating alcoholic fatty liver disease comprises pharmaceutically acceptable dosage forms of tablets, injections, capsules, granules, pills, powders, oral liquids, sustained release preparations, controlled release preparations or nano preparations.
The invention provides a resveratrol A ring N (CH) with a structure shown in a formula I 3 ) 2 The derivative, as a sodium hydrogen exchanger-1 (NHE 1) receptor antagonist, acts on an NHE1 receptor in a targeting way, and simultaneously inhibits 2 signal conduction pathways of a phosphatidylinositol 3-kinase/protein serine threonine kinase (PI 3K/AKT) signal pathway and a Janus kinase/signal transduction and transcription activator (JAK/STAT), thereby playing a role in treating alcoholic fatty liver.
The invention also provides resveratrol A ring N (CH) in the technical scheme 3 ) 2 The preparation method of the derivative has the advantages of mild conditions, low raw material cost, easy operation and high yield.
Drawings
FIG. 1 shows that resveratrol A ring N (CH) prepared by the invention 3 ) 2 The signal path diagram of the mechanism of treating the alcoholic fatty liver by the derivative;
FIG. 2 shows that ring A N (CH) of resveratrol prepared by the present invention 3 ) 2 Reaction schemes for the derivatives;
FIG. 3 shows the effect of different experimental groups on the quality of the initial body;
FIG. 4 is a graph of the effect of different experimental groups on end body mass;
FIG. 5 is the effect of different experimental groups on hepatic index;
FIG. 6 is a graph of the effect of different experimental groups on serum TG content;
FIG. 7 is a graph of the effect of different experimental groups on serum TC content;
FIG. 8 is a graph of the effect of different experimental groups on serum HDL-C levels;
FIG. 9 is a graph of the effect of different experimental groups on serum ALT levels;
FIG. 10 is a graph of the effect of different experimental groups on serum AST levels;
FIG. 11 shows the effect of different experimental groups on serum SOD level;
FIG. 12 is a graph of the effect of different experimental groups on serum MDA content;
FIG. 13 shows the effect of different experimental groups on the subregional structure of the liver (400X);
FIG. 14 is the effect of different experimental groups on immunohistochemistry for hepatic NHE-1 expression (400X);
FIG. 15 is a graph of the effect of different experimental groups on hepatic NHE-1 expression of WesternBlot (400X);
FIG. 16 is a statistical plot of the effect of different experimental groups on hepatic NHE-1 expression of WesternBlot;
FIG. 17 shows the effect of different experimental groups on hepatic PI3K and STAT immunofluorescence (400 ×).
Detailed Description
The invention provides resveratrol A-ring N (CH) 3 ) 2 A group derivative having the structure shown in formula I:
in the invention, the resveratrol A ring N (CH) with the structure shown in formula I 3 ) 2 The chemical name of the derivative is 2-dihydroxy-4' -methoxy-4-dimethylamino-1, 2-diphenylethylene ((E) -5- (dimethylamino) -2- (4-methoxystyryl) phenol), and the molecular formula is C 17 H 19 NO 2 And a molecular weight of 269.
FIG. 1 shows resveratrol A ring N (CH) 3 ) 2 Resveratrol A-Ring N (CH) as a signaling pathway map for the treatment of fatty liver 3 ) 2 The derivative acts on NHE1 receptor as the targeting of a sodium-hydrogen exchanger-1 (NHE 1) receptor antagonist, and simultaneously inhibits 2 signals of a phosphatidylinositol 3-kinase/protein serine threonine kinase (PI 3K/AKT) signal path and a Janus kinase/signal transduction and transcription activator (JAK/STAT)The conduction pathway plays a role in treating alcoholic fatty liver.
The invention also provides resveratrol A ring N (CH) in the technical scheme 3 ) 2 A process for the preparation of a derivative comprising the steps of:
mixing diethyl phosphite, an organic solvent, naH and 4-methoxybenzyl bromide to carry out debromination reaction to obtain a compound with a structure shown in a formula 1;
mixing 4-dimethylamino salicylaldehyde, an organic solvent, chloromethyl methyl ether and diisopropylethylamine for a substitution reaction to obtain a compound with a structure shown in a formula 2;
mixing a compound with a structure shown in a formula 1, a compound with a structure shown in a formula 2, an organic solvent and NaH for a condensation reaction to obtain a compound with a structure shown in a formula 3;
mixing the compound with the structure shown in the formula 3, tert-butyl alcohol and p-toluenesulfonate, and refluxing to obtain resveratrol A ring N (CH) with the structure shown in the formula I 3 ) 2 A derivative of phenyl;
in the present invention, the raw materials used in the present invention are all commercially available products unless otherwise specified.
In the present invention, the reaction principle of the preparation method is shown in fig. 2.
The invention mixes diethyl phosphite, organic solvent, naH and 4-methoxybenzyl bromide to carry out debromination reaction, and obtains a compound with a structure shown in a formula 1. In the present invention, the organic solvent is preferably DMF.
In the present invention, the amount ratio of diethyl phosphite, organic solvent, naH and 4-methoxybenzyl bromide is preferably 10mmol:8mL of: 15mmol:15mmol of the total amount of the solution.
In the invention, the time of the debromination reaction is preferably 1 to 3 hours, and the temperature of the debromination reaction is preferably room temperature, and no additional heating or cooling is required.
According to the invention, preferably, diethyl phosphite is dissolved in DMF, naH is added under the condition of ice water bath, and 4-methoxybenzyl bromide is added after half an hour.
After the debromination reaction is finished, a saturated ammonium chloride solution is preferably added to quench the reaction, then dichloromethane is used for extraction, and after reduced pressure distillation and column chromatography purification, the compound with the structure shown in formula 1 is obtained. The present invention is not particularly limited to the specific operations of the extraction and vacuum distillation of dichloromethane, and may be performed in a manner well known to those skilled in the art. In the present invention, the eluent used for the column chromatography purification is preferably a petroleum ether-ethyl acetate mixture, and the volume ratio of petroleum ether to ethyl acetate in the mixture is preferably 1.
The method comprises the step of mixing 4-dimethylamino salicylaldehyde, an organic solvent, chloromethyl methyl ether and diisopropylethylamine for substitution reaction to obtain a compound with a structure shown in a formula 2. In the present invention, the organic solvent is preferably dichloromethane.
In the invention, the dosage ratio of the 4-dimethylamino salicylaldehyde, the organic solvent, the chloromethyl methyl ether and the diisopropylethylamine is preferably 10mmol:40mL of: 15mmol:15mmol.
In the present invention, the time for the substitution reaction is preferably 10 to 30min.
In the invention, 4-dimethylamino salicylaldehyde is preferably dissolved in dichloromethane, and chloromethyl methyl ether and diisopropylethylamine are added under the condition of ice-water bath.
After the substitution reaction is finished, preferably, saturated ammonium chloride solution is added to quench the reaction, then dichloromethane is used for extraction, and column chromatography purification is carried out after reduced pressure distillation to obtain the compound with the structure shown in the formula 2. The present invention is not particularly limited to the specific operations of the extraction and vacuum distillation of dichloromethane, and may be performed in a manner well known to those skilled in the art. In the present invention, the eluent used for the column chromatography purification is preferably a petroleum ether-ethyl acetate mixture, and the volume ratio of petroleum ether to ethyl acetate in the mixture is preferably 5.
After obtaining the compounds with the structures shown in the formulas 1 and 2, the invention mixes the compound with the structure shown in the formula 1, the compound with the structure shown in the formula 2, an organic solvent and NaH for condensation reaction to obtain the compound with the structure shown in the formula 3. In the present invention, the organic solvent is preferably DMF.
In the present invention, the compound having the structure represented by formula 1, the compound having the structure represented by formula 2, the organic solvent and NaH are preferably used in a ratio of 3mmol:4.5mmol:4mL of: 4.5mmol.
In the present invention, the condensation reaction is preferably carried out at a temperature of 0 to 100 ℃ for a time of 1 to 3 hours. In the present invention, the condensation reaction is preferably carried out in an oil bath pan.
According to the invention, the compound with the structure shown in the formula 1 is preferably dissolved in DMF, naH is added under the condition of ice-water bath, and then the compound with the structure shown in the formula 2 is added.
After the condensation reaction is finished, the condensation product is preferably naturally cooled to room temperature, then a saturated ammonium chloride solution is added for quenching reaction, then dichloromethane is used for extraction, and column chromatography purification is carried out after reduced pressure distillation, so as to obtain the compound with the structure shown in the formula 3. The present invention is not particularly limited to the specific operations of the extraction and vacuum distillation of dichloromethane, and may be performed in a manner well known to those skilled in the art. In the present invention, the eluent used for the column chromatography purification is preferably a petroleum ether-ethyl acetate mixture, and the volume ratio of petroleum ether to ethyl acetate in the mixture is preferably 10.
After the compound with the structure shown in the formula 3 is obtained, the compound with the structure shown in the formula 3, tert-butyl alcohol and pyridinium p-toluenesulfonate are mixed and refluxed to obtain resveratrol A ring N (CH) with the structure shown in the formula I 3 ) 2 A derivative of (I) or (II).
In the present invention, the reflux temperature is preferably 90 to 100 ℃, more preferably 95 ℃, and the time is preferably 1 to 3 hours. In the present invention, the reflux is preferably carried out in an oil bath pan.
In the present invention, the molar ratio of the compound having the structure represented by formula 3 to pyridinium p-toluenesulfonate is preferably 1.
In the present invention, the amount ratio of the compound having the structure represented by formula 3 to t-butanol is preferably 1 mmol:4mL.
In the invention, the compound with the structure shown in the formula 3 is preferably dissolved in tert-butyl alcohol, and then p-toluenesulfonic acid pyridinium salt is added.
After the reflux is finished, the reflux product is preferably naturally cooled to room temperature, water is added for quenching reaction, then dichloromethane is used for extraction, and after reduced pressure distillation and column chromatography purification, the compound with the structure shown in the formula 3 is obtained. The present invention is not particularly limited to the specific operations of the extraction and distillation under reduced pressure of dichloromethane, and the methods known to those skilled in the art can be adopted. In the present invention, the eluent used for the column chromatography purification is preferably a petroleum ether-ethyl acetate mixture, and the volume ratio of petroleum ether to ethyl acetate in the mixture is preferably 3.
The invention also provides resveratrol A ring N (CH) in the technical scheme 3 ) 2 The resveratrol A ring N (CH) prepared by the preparation method of the base derivative or the technical scheme 3 ) 2 The application of the derivative in preparing medicines for treating alcoholic fatty liver is provided.
In the invention, the medicine for treating alcoholic fatty liver preferably comprises resveratrol A ring N (CH) with a structure shown in formula I, wherein the effective dose of the medicine is more than or equal to 10mg/kg 3 ) 2 The derivatives, the stereoisomers, the pharmaceutically acceptable salts and pharmaceutically acceptable carriers, auxiliary materials, excipients and diluents. In the present invention, the effective dose is preferably not less than 10mg/kg.
In the invention, the dosage form of the drug for treating alcoholic fatty liver preferably comprises a pharmaceutically acceptable dosage form of tablets, injections, capsules, granules, pills, powder, oral liquid, sustained release preparations, controlled release preparations or nano preparations.
To further illustrate the present invention, the following examples are given to provide resveratrol A ring N (CH) 3 ) 2 The derivatives, their preparation and use, and the microbial fuel cells on the sea floor are described in detail, but they should not be construed as limiting the scope of the inventionAnd (4) limiting.
Example 1
1. Medicine preparation: resveratrol (Nanjing spring and autumn bioengineering, inc.); p-methoxybenzyl bromide, 2-hydroxy-5-dimethylaminobenzaldehyde, chloromethyl methyl ether, tetrahydrofuran, sodium hydride, methylene chloride, diethyl phosphite, N-dimethylformamide, pyridinium p-toluenesulfonate, (ann naiji chemical limited); compound methionine choline tablet (Tonghua Dongbao pharmaceutical industry Co., ltd., china); NHE1 primary antibody (BM 5689), PI3K primary antibody (BM 4344), STAT primary antibody (M00337) (Strobile bioengineering, gmbH, wuhan).
Resveratrol A Ring N (CH) 3 ) 2 The synthesis method of the derivative comprises the following steps:
(1) Diethyl phosphite (10mmol, 1.38g) was dissolved in DMF (8 mL), naH (15mmol, 0.36g) was added under ice-water bath conditions, and after half an hour, 4-methoxybenzyl bromide (15 mmol, 3.02g) was added to the reaction mixture, and the reaction was carried out for 1 hour. After the reaction is completed, adding a saturated ammonium chloride solution to quench the reaction, extracting with dichloromethane, distilling under reduced pressure, and purifying by column chromatography (petroleum ether: ethyl acetate = 1) to obtain a compound having a structure shown in formula 1 with a yield of 90%;
(2) 4-Dimethylaminosalicylaldehyde (10mmol, 1.65g) was dissolved in methylene chloride (40 mL), and chloromethyl methyl ether (15mmol, 1.21g) and diisopropylethylamine (15 mmol, 1.94 g) were added under ice-water bath conditions to react for 2 hours. Adding a saturated ammonium chloride solution to quench the reaction, extracting with dichloromethane, distilling under reduced pressure, and purifying by column chromatography (petroleum ether: ethyl acetate =5: 1) to obtain a compound having a structure represented by formula 2, wherein the conversion rate is 98%;
(3) The compound (3 mmol, 0.774g) having the structure shown in the formula 1 is dissolved in DMF (4 mL), naH (4.5mmol, 0.11g) is added under the condition of ice water bath, after reaction for half an hour, the compound (4.5mmol, 0.94g) having the structure shown in the formula 2 is added, and the mixture is placed in an oil bath at 100 ℃ for reaction for 1 hour. After the reaction is completed, cooling to room temperature, adding a saturated ammonium chloride solution to quench the reaction, extracting with dichloromethane, and performing column chromatography purification after reduced pressure distillation (petroleum ether: ethyl acetate =10 = 1) to obtain a compound having a structure represented by formula 3 with a yield of 95%;
(4) Having a structure represented by formula 3The compound (1mmol, 0.313g) was dissolved in t-butanol (4 mL), and pyridinium p-toluenesulfonate (2mmol, 0.502g) was added and heated under reflux in an oil bath at 95 ℃ for 1 hour. After the reaction is completed, cooling to room temperature, adding water to quench the reaction, extracting with dichloromethane, distilling under reduced pressure, and purifying by column chromatography (petroleum ether: ethyl acetate =3 3 ) 2 Derivative, yield 84%;
for the prepared resveratrol A ring N (CH) 3 ) 2 Carrying out structural characterization on the derivative, white solid; 1 H NMR(400MHz,DMSO-d 6 )δ:2.88(s,6H),3.75(s,3H),6.23-6.28(m,2H), 6.88-6.92(m,3H),7.15(d,J＝16.4Hz,1H),7.36(d,J＝8.4Hz,1H),7.40(d,J＝8.4Hz,2H),9.41(brs,1H). 13 C NMR(150MHz,DMSO-d 6 )δ:40.6,55.5,105.2, 114.6,122.3,123.4,127.2,127.4,127.9,130.6,131.7,151.0,156.1,158.5.
2. animals: sprague-Dawley (SD) rats, 6 weeks old, male, 180-220 g, clean grade, were offered by the university of Hunan university.
3. Grouping experiments: the rats are adapted to be raised in a laboratory for 1 week, no abnormity exists in activity, food and excrement, and 56 rats are randomly divided into 6 groups according to a random number table: (1) normal control group: diet regular for 12 weeks; (2) alcoholic fatty liver disease model group: performing intragastric administration with 50% alcohol, 10ml/kg/d,8 weeks; (3) Compound methionine Choline treatment group (10 mg/kg): after 8 weeks of high fat diet, changing into conventional diet for 4 weeks, and adding compound choline methionine (10 mg/kg) into drinking water; (4) resveratrol treatment group (10 mg/kg): changing into regular diet for 4 weeks after high fat diet for 8 weeks, and adding resveratrol (10 mg/kg) into drinking water; (5) Resveratrol A Ring N (CH) 3 ) 2 Group derivative low dose treatment group (2 mg/kg): after 8 weeks of high fat diet, changing into regular diet for 4 weeks, and adding resveratrol A-ring N (CH) into drinking water 3 ) 2 A base derivative (2 mg/kg); (6) Resveratrol A Ring N (CH) 3 ) 2 Group treated with medium dose of derivative (10 mg/kg): changing into regular diet for 4 weeks after 8 weeks of high fat diet, and adding resveratrol A ring N (CH) into drinking water 3 ) 2 A base derivative (10 mg/kg); (7) Resveratrol A Ring N (CH) 3 ) 2 Group derivative high dose treatment group (50 mg/kg): changing into regular diet for 4 weeks after 8 weeks of high fat diet, and adding resveratrol A ring N (CH) into drinking water 3 ) 2 The base derivative (50 mg/kg). Injecting the rats into the abdominal cavity with 2% sodium pentobarbital (45 mg/kg) at the end of 12 weeks, taking blood from the inferior vena cava after anesthesia, centrifuging the blood specimen and detecting serological indexes; taking the left liver leaf, fixing with 10% neutral formalin solution, embedding in paraffin, and slicing for histopathological observation of liver; taking the right lobe of the liver, extracting protein and carrying out WesternBlot detection; taking liver right leaf, freezing, and performing immunofluorescence detection.
4. The experimental contents are as follows:
4.1 general conditions in rats: the activity, diet, coat luster and sleep of the rats were observed.
4.2 body weight, liver index: weighing the weight by a balance, and weighing the liver after the dead anesthesia; liver index (%) = liver weight (g)/body weight (g) × 100%.
4.3 serum TG, TC, HDL-C content: collecting blood from rat femoral artery, collecting blood sample with heparin anticoagulation tube, centrifuging at 4 deg.C for 8 min at 1500 r/min within 30min, collecting supernatant, and freezing at-80 deg.C. Adding physiological saline into the reserved tissue according to the mass ratio of 1. The Elisa kit stored at 4 ℃ was equilibrated at room temperature for 30 minutes, the desired Elisa reaction plate was taken out of the aluminum foil bag, and the remaining reaction plates were sealed with a self-sealing bag and stored at 4 ℃. And arranging blank holes, standard substance holes and sample holes by using a marking pen, wherein the blank holes are vacant, and 50 mu L of standard substances with different samples to be detected and different concentrations are added into the sample holes and the standard substance holes respectively. 100 mu L of detection antibody marked by horseradish peroxidase is respectively added into the sample hole and the standard product hole, and each reaction hole on the Elisa reaction plate is sealed by a sealing plate membrane and then put into a water bath kettle at 37 ℃ for incubation for 60 minutes. The liquid in the reaction well was discarded, and the residual water in the reaction well was patted dry on absorbent paper. 350 mu L of washing solution prepared by distilled water and washing buffer solution according to the volume ratio of 1. 50 μ L of each of the substrates A and B was added to each reaction well, and incubated in an incubator at 37 ℃ for 15 minutes in the absence of light. Adding 50 μ L of stop solution into each reaction well, and measuring the OD value of each well at the wavelength of 450nm set by the microplate reader within 15 minutes. And taking the concentration value of the measured standard product as a vertical coordinate and the OD value as a horizontal coordinate, drawing a standard curve by software and obtaining a linear regression equation. And substituting the OD value of the detection sample into the linear regression equation to calculate the concentration of the detection sample.
4.4 serum ALT, AST content: the method is the same as 4.3.
4.5 serum SOD, MDA content: the method is the same as 4.3.
4.6HE staining: placing the paraffin section in an oven for baking for 1-2 hours at 60 ℃; slicing paraffin into normal xylene, and dewaxing the xylene with ethanol to water; hematoxylin staining for 10 minutes; flushing with running water to remove residual color; 0.7% hydrochloric acid ethanol is differentiated for several seconds; rinsing with running water, and turning the slices blue for about 15 minutes; 7.95% ethanol for 30 seconds; staining with alcoholic eosin for 30 seconds; i95% ethanol for 30 seconds; II95% ethanol for 30 seconds; i100% ethanol for 30 seconds; II100% ethanol for 30 seconds; xylene carbolate for 30 seconds; i xylene for 30 seconds; II xylene for 30 seconds; and (5) sealing the neutral gum.
4.7 immunohistochemistry: paraffin section is dewaxed to water; 3% of H 2 O 2 Incubating for 5-10 minutes at room temperature to eliminate the activity of endogenous peroxidase; washing with distilled water, and soaking in PBS for 2 times, each for 5 min; sealing 5-10% normal goat serum (PBS diluted), incubating at room temperature for 10 min, pouring out serum, and not washing; dripping primary antibody working solution, and incubating at 37 ℃ for 1 hour or overnight at 4 ℃; PBS washing, 3 times, each time for 5 minutes; dripping a proper amount of biotin-labeled secondary antibody working solution, and incubating for 10 minutes at 37 ℃; PBS washing, 3 times, each time for 5 minutes; dropwise adding a proper amount of horseradish enzyme or alkaline phosphatase labeled streptavidin working solution, and incubating for 10 minutes at 37 ℃; PBS washing, 3 times, each time for 5 minutes; developing with developer for 3 min (DAB or NBT/BCIP), washing with tap water, re-dyeing, dewatering, transparentizing, and sealing.
4.8 WesternBlot: to the tissue homogenate were added PMSF (1The mixed homogenate of the tissues was added and the electric homogenizer was adjusted to 30 times/mim, then homogenized 50 times clockwise and 50 times counterclockwise. The tissue suspension is sucked into a 5mL centrifugal tube, 1/3 volume of Buffer solution is added, and the mixture is shaken and mixed uniformly. Then placing the mixture on a test tube rack, boiling the mixture in water bath for 10 minutes, then carrying out ultrasonic oscillation for 20 times at 20 kHz, centrifuging the mixture, taking supernate at 4 ℃/12000g/5 minutes, and then storing the supernate in a centrifuge tube at-80 ℃. The BSA gradient solution was prepared by first preparing 10. Mu.g/. Mu.L BSA buffer solution, and then diluting the buffer solution with double distilled water in a gradient manner to obtain a standard protein, wherein the dilution concentrations were 0. Mu.g/. Mu.L, 0.2. Mu.g/. Mu.L, 0.4. Mu.g/. Mu.L, 0.6. Mu.g/. Mu.L, 0.8. Mu.g/. Mu.L, and 1.0. Mu.g/. Mu.L, in this order. The protein supernatant centrifuge tube was taken out from a-80 ℃ freezer and placed on ice, after slow dissolution 2. Mu.L of protein sample was aspirated and 18. Mu.L of double distilled water was added, and mixed, shaken and mixed well. First, 3 parallel wells were set on a 96-well plate, and then the mixed protein sample and BSA gradient buffer solution were mixed and shaken as described above at a volume of 5. Mu.L/well. Taking 50 parts of the solution A and 1 part of the solution B in the kit, mixing the solution A and the solution B uniformly to prepare a working solution, quickly adding 95 mu L of the working solution into each hole of a 96-hole plate, putting the 96-hole plate into an incubator, and incubating for 30 minutes at 37 ℃. And setting the spectrophotometer at a 562nm light wave band wave, measuring the absorbance value, drawing a standard protein curve, and calculating to obtain the protein concentration value of the sample. Taking 1 part of bromophenol blue and 3 parts of beta-mercaptoethanol, mixing, shaking and uniformly mixing. Diluting the mixed solution by 10 times with double distilled water, boiling in a water bath for 10 minutes, and shaking and mixing uniformly. Adding 10% separation gel into the gel plate, standing at room temperature, pouring out the water layer after the gel is solidified, and sucking with filter paper. Then 5% concentrated glue is added for standing at room temperature, and the mixture is solidified for later use. And (2) placing the rubber plate in an electrophoresis tank for fixation, slowly adding electrophoresis liquid along the electrophoresis tank, fully and uniformly mixing the sample, adding a protein sample in a lane of the electrophoresis tank by using a microsyringe, performing electrophoresis for 30 minutes by using 80V constant voltage until the sample runs to the separation rubber, performing electrophoresis separation by using 120V constant voltage, and determining the electrophoresis time according to the molecular weight of the target protein. The marking pen marks the nitrocellulose membrane, then the nitrocellulose membrane is soaked in the pretreated membrane transferring working solution, the separation gel is peeled off and transversely cut according to the molecular weight range of the target protein, and the fibrous sponge pad → the 3 layers of filter paper → the rubber plate → is sequentially placed from the cathode to the anodeNitrocellulose membrane → 3 layers of filter paper → a fibrous sponge pad. The materials are stacked into a trapezoid shape and then placed into a membrane transferring groove which is incubated at 0 ℃ in advance, membrane is transferred at a constant current of 252mA, and the membrane transferring time is determined according to the molecular weight of the target protein and is generally controlled to be 1h. After the completion of the membrane transfer, the nitrocellulose membrane was taken out from the membrane transfer tank, and washed with PBS 2 times for 5 minutes each. Then, 100. Mu.L of 10% goat serum or 2% bovine serum albumin was used for immersion at room temperature for 10 minutes, and the solution was washed 3 times with PBS for 5 minutes each. Diluting the concentrated solution at a proper ratio to 1cm 2 40 μ L of primary antibody was added dropwise. Incubating at 37 ℃ for 1-2 h or overnight at 4 ℃, washing with a washing buffer solution for 3 times, washing for 10 minutes each time → taking out the nitrocellulose membrane the next day and recovering the primary antibody, adding 0.02% Tween 20 into the TBS buffer solution, rinsing for 3 times, rinsing for 5 minutes each time, sealing the freshness protection package, diluting the concentrated solution at a proper ratio, and keeping the concentration for 1cm 2 40 μ L of biotinylated secondary antibody was added dropwise, incubated at 37 ℃ for 1h or at 4 ℃ overnight, washed with PBS for 5 min and repeated 3 times. The nitrocellulose membrane was removed and the secondary antibody recovered, and 0.02% tween 20 was added to the TBS buffer and rinsed 3 times for 5 minutes each. After rinsing, rinse 3 times 5 minutes each with TBS buffer. The nitrocellulose membranes were scanned at a wavelength of 700nm light wave, respectively.
4.9 immunofluorescence: incubating frozen sections with antibodies, washing with PBS for 3 times, each time for 5 minutes; diluting primary antibody according to corresponding proportion, dripping 50 μ L of primary antibody into each hole, incubating overnight at 4 ℃, recovering the primary antibody, washing with PBS, and multiplying by 3 times for 5 minutes; dripping fluorescent secondary antibody of corresponding species, incubating for 1 hour at room temperature in a dark place, removing the secondary antibody, and washing with PBS for 3 times, 5 minutes each time; adding the diluted second primary antibody dropwise, 50 mu L/hole, and incubating overnight at 4 ℃; primary antibody is recovered, PBS is washed for 3 times, and each time lasts for 5 minutes; and (3) dropwise adding fluorescent secondary antibody of the corresponding species, incubating for 1 hour at room temperature in a dark place, removing the secondary antibody, washing with PBS for 3 times, and dropwise adding a small amount of PBS every 5 minutes to cover the cells. 0.1% TritonX-100 was added to the PBS buffer, and the 96-well plate was washed 3 times for 5 minutes each, and then PBS containing 1 times of Hoechst dilution was added and left for 10 minutes in the dark. 0.1 percent TritonX-100 is added into PBS buffer solution, the 96-hole culture plate is washed for 3 times, each time is 3 minutes, 100 mu L of fluorescence-resistant quenching mounting solution is dripped on a glass slide and mounted by a cover slip, the glass slide is kept at the room temperature for 1 minute, and then the horizontal position is kept and placed in a refrigerator at 4 ℃ and is kept away from light. After 24 hours, the cell morphology and the staining condition can be observed under a fluorescence microscope or a laser confocal microscope.
5. The statistical method comprises the following steps: all data are expressed as means ± standard deviation (± s). Differences between groups were compared using ANOVA and Newman-Student multiple comparisons; the analysis of t test is completed by SPSS 13.0 statistical software, and the difference is considered to be significant when P on two sides is less than 0.05.
6. Results
6.1 general case: (1) The rats in the normal control group are full of energy, flexible and well-moving, have normal diet, clean and tidy fur and slowly increase the weight; (2) The alcoholic fatty liver disease model group has large food consumption, sleepiness, yellowish hair, larger individuals, faster weight increase than the normal group, listlessness, reduced activity and messy fur; (3) The compound methionine choline treatment group and the resveratrol treatment group slightly improve or do not obviously improve the symptoms; (4) Resveratrol A Ring N (CH) 3 ) 2 Low dose treatment group of resveratrol A ring N (CH) based derivatives 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of the derivatives 3 ) 2 The symptoms of the basal derivative high-dose treatment group are obviously improved, and the improvement degree is gradually increased along with the increase of the dose.
6.2 body weight, liver index: the results are shown in Table 1 and FIGS. 3-5, FIG. 3 is a graph of the effect of different experimental groups on the quality of the initial body, wherein ap is compared to the normal control group<0.01; b P compared with alcoholic fatty liver disease model group<0.01; cp compared to Compound methionine Choline treatment group<0.01; dp compared to resveratrol treatment group<0.01. FIG. 4 is a graph of the effect of different experimental groups on end body mass, wherein ap is compared to a normal control group<0.01; b P compared with alcoholic fatty liver disease model group<0.01; cp compared to Compound methionine Choline treatment group<0.01; dp compared to resveratrol treatment group<0.01. FIG. 5 is a graph of the effect of different experimental groups on liver index, where ap is compared to a normal control group<0.01; b P in comparison with the alcoholic fatty liver disease model group<0.01; cp compared to Compound methionine Choline treatment group<0.01；Compared with resveratrol-treated group, dp<0.01. As can be seen from Table 1 and FIGS. 3 to 5, (1) compared with the normal control group, there was no significant difference in the initial body mass of the rats in the other groups (P > 0.01); (2) Resveratrol A Ring N (CH) compared to alcoholic fatty liver disease model group 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of derivatives 3 ) 2 The terminal body mass and the liver index of rats in the high-dose treatment group of the derivative are obviously reduced (P)<0.01 ); (3) Resveratrol A Ring N (CH) compared to resveratrol treatment group 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of derivatives 3 ) 2 The end body mass and the liver index of rats in a high-dose treatment group of the derivative are obviously reduced (P)<0.01)。
Table 1 rat body weight, liver index (n =8,
)
note: aP <0.01 compared to normal control; bP <0.01 compared to alcoholic fatty liver disease model group; cP <0.01 compared to the compound methionine choline treatment group; dP <0.01 compared to resveratrol treatment group.
6.3 serum TG, TC, HDL-C content: the results are shown in Table 2 and FIGS. 6-8, FIG. 6 is the effect of different experimental groups on the content of TG in serum, and ap is compared with normal control group<0.01; b P compared with alcoholic fatty liver disease model group<0.01; cp compared to Compound methionine Choline treatment group<0.01; dp compared to resveratrol treatment group<0.01. FIG. 7 is a graph showing the effect of different experimental groups on serum TC content, ap, in comparison to a normal control group<0.01; b P in comparison with the alcoholic fatty liver disease model group<0.01; cp compared to Compound methionine Choline treatment group<0.01; compared with resveratrol-treated group, dp<0.01. FIG. 8 is a graph showing the effect of different experimental groups on the serum HDL-C content, ap, compared to a normal control group<0.01; model for treating alcoholic fatty liver diseaseType group comparison, bp<0.01; cp compared to Compound methionine Choline treatment group<0.01; compared with resveratrol-treated group, dp<0.01. As is clear from Table 2 and FIGS. 6 to 8, (1) resveratrol A-Ring N (CH) in comparison with the alcoholic fatty liver disease model group 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of the derivatives 3 ) 2 The TG and TC of rats in a high-dose treatment group of the derivative are obviously reduced, and HDL-C is obviously increased (P)<0.01 ); (2) Resveratrol A Ring N (CH) compared to resveratrol treatment group 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of derivatives 3 ) 2 The TG and TC of rats in the high-dose treatment group of the derivative are both obviously reduced, and HDL-C is obviously increased (P)<0.01)。
Table 2 serum TG, TC, HDL-C content (n =8,
)
note: aP <0.01 compared to normal control; bP <0.01 compared to alcoholic fatty liver disease model group; cP <0.01 compared to the compound methionine choline treatment group; dP <0.01 compared to resveratrol treatment group.
6.4 serum ALT, AST content: the results are shown in Table 3 and FIGS. 9-10, and FIG. 9 shows the effect of different experimental groups on serum ALT content, compared with normal control group, ap<0.01; b P compared with alcoholic fatty liver disease model group<0.01; cp compared to Compound methionine Choline treatment group<0.01; dp compared to resveratrol treatment group<0.01. FIG. 10 is a graph showing the effect of different experimental groups on serum AST levels, ap, compared to a normal control group<0.01; b P in comparison with the alcoholic fatty liver disease model group<0.01; cp compared to Compound methionine Choline treatment group<0.01; compared with resveratrol-treated group, dp<0.01. As can be seen from table 3 and fig. 9 to 10: (1) Resveratrol A Ring N (CH) compared with alcoholic fatty liver disease model group 3 ) 2 Low dose treatment group of resveratrol A ring N (CH) based derivatives 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of the derivatives 3 ) 2 The ALT and AST of the rats in the high-dose treatment group of the derivative are both obviously reduced (P)<0.01 ); (2) Resveratrol A Ring N (CH) compared to resveratrol treatment group 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of the derivatives 3 ) 2 The ALT and AST of the rats in the high-dose treatment group of the derivative are obviously reduced (P)<0.01)。
Table 3 serum ALT, AST content (n =8,
)
note: ap <0.01 compared to normal control group; bp <0.01 compared to the alcoholic fatty liver disease model group; cp <0.01 compared to cotinamide choline treatment group; dp <0.01 compared to resveratrol treatment group.
6.5 serum SOD, MDA content: the results are shown in Table 4 and FIGS. 11-12, and FIG. 11 shows the effect of different experimental groups on serum SOD content, compared with normal control group, ap<0.01; b P compared with alcoholic fatty liver disease model group<0.01; cp compared to Compound methionine Choline treatment group<0.01; dp compared to resveratrol treatment group<0.01. FIG. 12 is a graph showing the effect of different experimental groups on the MDA content in serum, compared to a normal control group, ap<0.01; b P compared with alcoholic fatty liver disease model group<0.01; cp compared to Compound methionine Choline treatment group<0.01; dp compared to resveratrol treatment group<0.01. As can be seen from table 4 and fig. 11 to 12: (1) Compared with alcoholic fatty liver disease model group, resveratrol treatment group, resveratrol A ring N (CH) 3 ) 2 Resveratrol A Ring N (CH) in Low dose treatment group based on derivatives 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of derivatives 3 ) 2 High dose treatment group of the group derivativeThe SOD and MDA of the mouse are obviously increased (P)<0.01 ); (2) Compared with resveratrol treatment group, SOD and MDA are in resveratrol A ring N (CH) 3 ) 2 There were no differences between the dose groups of the base derivatives (P > 0.01). The key points are as follows: resveratrol has good antioxidation, but has no good intervention effect on alcoholic fatty liver; resveratrol A Ring N (CH) 3 ) 2 The derivative has good antioxidation, but has good intervention effect on alcoholic fatty liver. In particular, resveratrol A Ring N (CH) 3 ) 2 The intervention effect of the base derivative on alcoholic fatty liver is not completed through antioxidation.
Table 4 serum SOD, MDA content (n =8,
)
note: ap <0.01 compared to normal control group; bp <0.01 compared to alcoholic fatty liver disease model group; cp <0.01 compared to cotinamide choline treatment group; dp <0.01 compared to resveratrol treatment group.
6.6HE staining: the results are shown in fig. 13, and fig. 13 shows the influence of different experimental groups on the structure under the liver microscope (400 ×), and it can be seen that, (1) the hepatic cells of the rat in the alcoholic fatty liver disease model group have obvious balloon-like changes, and the fatty degeneration area is obviously higher than that of the normal control group; (2) The resveratrol treatment group has no obvious inhibition effect on ballooning change of the liver cells, and the fat change area is not obviously reduced; (3) Resveratrol A Ring N (CH) 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of the derivatives 3 ) 2 The liver cells of rats treated by high dose of the derivative have obvious balloon-like changes, and the fatty degeneration area is obviously higher than that of rats of alcoholic fatty liver disease model groups.
6.7 immunohistochemistry: the results are shown in FIG. 14, FIG. 14 for liver NHE-1 expression immune of different experimental groupsThe influence of histochemistry (400 x) shows that (1) the yellow-brown staining particles of the rat liver cells in the alcoholic fatty liver disease model group are obviously increased, and the expression of NHE1 is obviously higher than that in the normal control group; (2) The resveratrol treatment group does not inhibit the increase of yellow-brown staining particles of liver cells, and the NHE1 expression is not obviously different from that of an alcoholic fatty liver disease model group; (3) Resveratrol A Ring N (CH) 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of the derivatives 3 ) 2 The high-dose treatment group of the derivative obviously inhibits the increase of yellow-brown stained granules of the liver cells, and the NHE1 expression is obviously different from that of an alcoholic fatty liver disease model group.
6.8 WesternBlot: the results are shown in FIGS. 15-16, FIG. 15 is a graph of the effect of different experimental groups on hepatic NHE-1 expression of WesternBlot (400 ×), wherein (1) is a normal control group; (2) alcoholic fatty liver disease model group; (3) compound methionine choline treatment group; (4) resveratrol treatment group; (5) Resveratrol A Ring N (CH) 3 ) 2 A group of low dose treatments of the derivative; (6) Resveratrol A Ring N (CH) 3 ) 2 A medium dose treatment group of the derivative; (7) Resveratrol A Ring N (CH) 3 ) 2 Group treated with high dose of the base derivative. FIG. 16 is a statistical plot of the effect of different experimental groups on hepatic NHE-1 expression of WesternBlot, as compared to normal control group, ap<0.01; b P compared with alcoholic fatty liver disease model group<0.01; cp compared to Compound methionine Choline treatment group<0.01; dp compared to resveratrol treatment group<0.01. As can be seen from fig. 15 to 16: (1) The gray level of the hepatic cells of the rats in the alcoholic fatty liver disease model group is obviously increased, and the expression of NHE1 is obviously higher than that of a normal control group; (2) The resveratrol treatment group does not inhibit the gray scale increase of liver cells, and the NHE1 expression is not obviously different from that of the alcoholic fatty liver disease model group; (3) Resveratrol A Ring N (CH) 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of derivatives 3 ) 2 The high-dose treatment group of the derivative obviously inhibits the gray scale increase of liver cells, and the NHE1 expression is obviously different from the alcoholic fatty liver disease model group.
6.9 immunofluorescence: the results are shown in FIG. 17, and FIG. 17 shows the effect of different experimental groupings on liver PI3K and STAT immunofluorescence (400)X), wherein (1) is a normal control group; (2) alcoholic fatty liver disease model group; (3) compound methionine choline treatment group; (4) resveratrol treatment group; (5) Resveratrol A Ring N (CH) 3 ) 2 A group of low dose treatments of the derivative; (6) Resveratrol A Ring N (CH) 3 ) 2 A medium dose treatment group of the derivative; (7) Resveratrol A Ring N (CH) 3 ) 2 Group treated with high dose of the base derivative. As can be seen from fig. 17: (1) The rat liver cells PI3K and STAT of the alcoholic fatty liver disease model group are obviously enhanced in fluorescence, and the expression is obviously higher than that of a normal control group; (2) The fluorescence enhancement of PI3K and STAT is not inhibited in the resveratrol treatment group, and the expression of the resveratrol treatment group is not obviously different from that of an alcoholic fatty liver disease model group; (3) Resveratrol A Ring N (CH) 3 ) 2 Resveratrol A Ring N (CH) in Medium dose treatment group of the derivatives 3 ) 2 The high-dose treatment group of the derivative obviously inhibits the fluorescence enhancement of the PI3K and STAT of the liver cells, and the expression of the derivative is obviously different from that of an alcoholic fatty liver disease model group.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that modifications and adaptations can be made by those skilled in the art without departing from the principle of the present invention, and should be considered as within the scope of the present invention.
Claims (10)
1. Resveratrol A ring N (CH) 3 ) 2 A group derivative having the structure shown in formula I:
2. resveratrol A ring N (CH) as in claim 1 3 ) 2 The preparation method of the derivative is characterized by comprising the following steps:
mixing diethyl phosphite, an organic solvent, naH and 4-methoxybenzyl bromide to carry out debromination reaction to obtain a compound with a structure shown in a formula 1;
mixing 4-dimethylaminosalicylaldehyde, an organic solvent, chloromethyl methyl ether and diisopropylethylamine for substitution reaction to obtain a compound with a structure shown in a formula 2;
mixing a compound with a structure shown in a formula 1, a compound with a structure shown in a formula 2, an organic solvent and NaH for condensation reaction to obtain a compound with a structure shown in a formula 3;
mixing the compound with the structure shown in the formula 3, tert-butyl alcohol and pyridinium p-toluenesulfonate, and refluxing to obtain resveratrol A ring N (CH) with the structure shown in the formula I 3 ) 2 A derivative of phenyl;
3. the method according to claim 2, wherein the debromination reaction is carried out for 1 to 3 hours.
4. The method according to claim 2, wherein the time for the substitution reaction is 10 to 30min.
5. The process according to claim 2, wherein the condensation reaction is carried out at a temperature of 0 to 100 ℃ for a period of 1 to 3 hours.
6. The method according to claim 2, wherein the reflux temperature is 90-100 ℃ and the reflux time is 1-3 hours.
7. The production method according to claim 2, wherein the molar ratio of the compound having the structure represented by formula 3 to the pyridinium p-toluenesulfonate is 1.
8. Resveratrol A ring N (CH) as in claim 1 3 ) 2 Derivatives of the compounds of formula (I) or the process of any one of claims 2 to 7Resveratrol A ring N (CH) prepared by the method 3 ) 2 The application of the derivative in preparing medicines for treating alcoholic fatty liver is provided.
9. The use of claim 8, wherein the medicament for treating alcoholic fatty liver disease comprises an effective amount of resveratrol A ring N (CH) having a structure shown in formula I 3 ) 2 The derivative, the pharmaceutically acceptable salt and pharmaceutically acceptable carriers, auxiliary materials, excipients and diluents.
10. The use of claim 8, wherein the medicament for treating alcoholic fatty liver disease is in the form of tablet, injection, capsule, granule, pill, powder, oral liquid, sustained release preparation, controlled release preparation or nano-preparation pharmaceutically acceptable dosage form.
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| WO1993023357A1 (en) * | 1992-05-21 | 1993-11-25 | Research Corporation Technologies, Inc. | Stilbene derivatives as anticancer agents |
| CN104147026A (en) * | 2014-08-20 | 2014-11-19 | 安树君 | New application of resveratrol derivative |
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| CA2432640A1 (en) * | 2000-12-21 | 2002-06-27 | Cancer Research Technology Limited | Substituted stilbenes, their reactions and anticancer activity |
| US8980954B2 (en) * | 2006-04-25 | 2015-03-17 | Stc.Unm | Substituted cis- and trans-stilbenes as therapeutic agents |
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| WO1993023357A1 (en) * | 1992-05-21 | 1993-11-25 | Research Corporation Technologies, Inc. | Stilbene derivatives as anticancer agents |
| CN104147026A (en) * | 2014-08-20 | 2014-11-19 | 安树君 | New application of resveratrol derivative |
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| Synthesis and biological evaluation of a library of resveratrol analogues as inhibitors of COX-1, COX-2 and NF-κB;Soo Sung Kang等;《Bioorganic & Medicinal Chemistry》;20080516;第1044-1054页 * |
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