Preparation method and application of paeonol and its derivatives

Abstract

The application discloses a preparation method and application of a paeonol derivative, and relates to the technical field of biomedicine. The paeonol derivative has the chemical structure as shown in the following formula: or. The paeonol extraction preparation method is simple, and the extraction rate of the paeonol is high. The prepared paeonol derivative has more excellent antioxidant activity, and the anti-inflammatory effect is enhanced, the anti-platelet aggregation effect is better, and the paeonol derivative also has good inhibitory activity on the occurrence and development of diabetic retinopathy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a preparation method and application of paeonol and its derivatives. BACKGROUND

[0002] China is rich in traditional Chinese medicine resources. Potential anti-inflammatory and immune chemical components are screened from traditional Chinese medicines as lead compounds. The lead compounds are reasonably designed and optimized by using principles of medicinal chemistry, and then a treatment drug for rheumatoid arthritis with better efficacy, less side effects, high bioavailability, good stability and long half-life is screened.

[0003] The effective component paeonol in medicinal material cortex moutan has multiple pharmacological activities such as anti-inflammatory, anti-tumor, antibacterial, antioxidant, anti-arrhythmia, anti-arteriosclerosis, anti-depression, anti-allergy, and blood sugar reduction. However, paeonol has disadvantages such as volatility and metabolism, and its clinical application is limited to a certain extent. In order to improve the stability of paeonol and further improve its bioavailability and pharmacological activity, paeonol can be chemically modified. At present, paeonol is approved by the China Food and Drug Administration for clinical treatment of inflammation, including various dosage forms such as tablets, injections, ointments, and adhesives. However, compared with other non-steroidal anti-inflammatory drugs such as celecoxib, ibuprofen, and indomethacin, the clinical application of paeonol is not widespread. Among the various dosage forms of paeonol, paeonol ointment is used for treating dermatitis, but has adverse reactions such as skin irritation and redness, and other dosage forms do not have good therapeutic effect. Based on the above reasons, it is of great significance to design, synthesize, optimize and obtain derivatives with better activity than paeonol based on paeonol as the mother nucleus. SUMMARY

[0004] The present application aims to provide a preparation method and application of paeonol and its derivatives. The extraction and preparation method of paeonol is simple, and the extraction rate of paeonol is high. The prepared paeonol derivatives have more excellent antioxidant activity, and the anti-inflammatory effect is enhanced, and the anti-platelet aggregation effect is better. At the same time, the paeonol derivatives also have good inhibitory activity on the occurrence and development of diabetic retinopathy.

[0005] The technical scheme adopted by the present application to achieve the above-mentioned purpose is as follows:

[0006] A paeonol derivative, the chemical structure of which is shown in formula I or formula II:

[0007] I or II.The present application synthesizes a class of derivatives by means of chemical modification, using esterification reaction to modify lactate of paeonol or danshensu into the structure of paeonol, and obtains high bioactivity paeonol derivatives by means of the present application, compared with traditional chemical modification based on improving the pharmaceutical properties of paeonol.The paeonol derivatives prepared by the present application have more optimal anti-inflammatory and anti-platelet aggregation activities;and can inhibit retinal neovascularization of diabetic rats by down-regulating the expression of SDF-1 and VEGF in the retinas, have a long-acting time, and have a significantly enhanced inhibitory effect on the occurrence and development of diabetic retinopathy, so that the paeonol derivatives can be used in the preparation of a medicament for treating or preventing diabetic retinopathy, and the application field is developed.In addition, the paeonol derivative modified by lactate of danshensu has a stronger antioxidant effect than paeonol and lactate of danshensu, can inhibit oil oxidation, and is expected to be used as an antioxidant and applied in food.The raw material of the product of the present application is abundant and inexpensive, and the product is safe and low-toxic, and has a strong pharmacological effect, so that the product has a great application prospect in the fields of medicine, food and cosmetics.

[0008] It should be noted that the raw material paeonol required for preparing the paeonol derivative is derived from cortex moutan.

[0009] It should be noted that the extraction method of paeonol includes enzymatic hydrolysis.

[0010] It should be noted that the enzyme used in the enzymatic hydrolysis includes cellulase.

[0011] Further, the extraction method of paeonol includes:

[0012] Pre-treatment: a pulverizer is used to crush cortex moutan to obtain particles with a particle size of 0.4-0.6 mm; the cortex moutan powder is added into vacuum-dried liquid 1-butyl-3-methylimidazole, and stirred at an oil bath temperature of 70-80℃ for 3-5 h, and then cooled to room temperature to obtain a pretreated mixture;

[0013] Enzymatic hydrolysis: cellulase powder is soaked in water with a pH of 4-5 (the ratio of the cellulase powder to the water is 1.5-2 mg: 1 mL), and the pretreated mixture is added, and then mixed thoroughly, and then placed in a water bath shaker at 40-50℃ and shaken at 100-120 r / min for 1.5-3 h, and then filtered through a 0.45 μm filter membrane to obtain an enzymatic hydrolysis solution;

[0014] Purification: the water vapor distillation method is used to collect 1 / 4-1 / 2 of the total weight of the enzymatic hydrolysis solution as a distillate, and the obtained distillate is stored at 2-4℃ for 24-36 h, and then filtered under the above conditions, and then washed with cold water and dried to obtain paeonol.

[0015] It should be noted that the mass ratio of 1-butyl-3-methylimidazole chloride to cortex moutan powder is 6.5-8:1; the mass ratio of cortex moutan powder to enzyme powder is 20:0.1-0.2. The enzyme hydrolysis method is used to extract paeonol from cortex moutan, combined with ion liquid pretreatment auxiliary treatment, which can effectively improve the extraction rate of paeonol. The ion liquid has good solubility to cellulose and can be complexed with metal ions, effectively solving the problem of enzyme hydrolysis effect inhibition caused by too high content of hemicellulose and metal ions in cortex moutan, improving the shielding phenomenon caused by lignin and hemicellulose, so that the cellulase can better play the enzyme hydrolysis effect, and the target substance in the medicinal material can be better dissolved, and the dissolution efficiency is improved.

[0016] The preparation method of the paeonol derivative includes: preparing the paeonol derivative by esterification reaction of paeonol and hemipterine or danshensu lactic acid.

[0017] More preferably, water crystal orchid glycoside is added in the enzyme hydrolysis process, and the addition amount is 0.01-0.2 mg / mL. The water crystal orchid glycoside is added in the enzyme hydrolysis process, which can significantly enhance the enzyme activity of cellulase, and then improve the enzyme hydrolysis efficiency, so that the paeonol is better dissolved from cortex moutan, and the extraction rate of paeonol is effectively improved. At the same time, the water crystal orchid glycoside and the hemipterine modified paeonol derivative are compounded and used, the water crystal orchid glycoside plays a synergistic effect, which can effectively improve the inhibition effect of the paeonol derivative on the expression of SDF-1 and VEGF in the retinal of diabetic rats, and then improve the inhibition activity of the paeonol derivative on the occurrence and development of diabetic retinopathy.

[0018] Further, the preparation method of the paeonol derivative is specifically:

[0019] The hemipterine or danshensu lactic acid is dissolved in dichloromethane (solid-liquid ratio is 0.06-0.09 g:1 mL), 4-dimethylaminopyridine is added, and SO2Cl2 / dichloromethane (v / v, 1:1.8-2.4) mixed solution is dropped into the mixture under stirring at room temperature, the dropping is completed within 0.5-1 h, the temperature is increased to 70-80℃, and the reflux reaction is carried out for 2-4 h, then SO2Cl2 and dichloromethane are removed by evaporation under reduced pressure, and the remaining product is extracted with dichloromethane to obtain the intermediate product A;

[0020] The paeonol, dichloromethane and triethylamine are mixed, and the intermediate product A is slowly added under ice bath, and the reaction is carried out under stirring at room temperature for 2-4 h; then the mixture is washed with water, extracted with dichloromethane for 2-4 times, the organic phases are combined, dried with anhydrous sodium sulfate, and the solvent is removed by evaporation under reduced pressure to obtain an oil, and the oil is separated and purified by column chromatography to obtain the paeonol derivative.

[0021] It is to be noted that the amount of 4-dimethylamino pyridine added is 6-8% of the mass of the leucodin or danshensu lactic acid; the solid-liquid ratio of the leucodin or danshensu lactic acid to SO2Cl2 is 0.3-0.5g:1mL; the solid-liquid ratio of the paeonol to dichloromethane is 0.05-0.08g:1mL; the mass ratio of the triethylamine to the paeonol is 1.2-1.5:1; and the molar ratio of the intermediate product A to the paeonol is 1.2-1.3:1.

[0022] The application further discloses a use of the compound shown in the formula I or the formula II in preparation of a medicament, food or cosmetic.

[0023] It is to be noted that the product efficacy includes antioxidant, anti-inflammatory and anti-platelet aggregation activities.

[0024] The application further discloses a use of the compound shown in the formula I or the formula II in preparation of a drug for diabetic retinopathy.

[0025] Compared with the prior art, the application has the following beneficial effects:

[0026] The application adopts esterification reaction to modify the leucodin or danshensu lactic acid into the structure of the paeonol to synthesize a derivative, which has more excellent anti-inflammatory and anti-platelet aggregation activities; and can inhibit the retinal neovascularization of diabetic rats by down-regulating the expression of SDF-1 and VEGF in the retinas, has a long-acting time, and has a significantly enhanced inhibitory effect on the occurrence and development of diabetic retinopathy. The paeonol derivative modified by the danshensu lactic acid has a stronger antioxidant effect than the paeonol and the danshensu lactic acid, and can inhibit the oxidation of oil and fat. In addition, the application adds the step of adding the coptis deltoidea in the enzymolysis step in the extraction process of the paeonol, can significantly enhance the enzyme activity of the cellulase, and then enhance the enzymolysis efficiency, so that the paeonol is better dissolved from the peony bark, and the extraction rate of the paeonol is effectively improved. Meanwhile, the coptis deltoidea and the paeonol derivative modified by the leucodin are used in combination, the coptis deltoidea plays a synergistic role, and can effectively improve the inhibitory activity of the paeonol derivative on the occurrence and development of DR. The product of the application has abundant raw material sources, is cheap, safe and low-toxic, has a strong pharmacological effect, and has a great application prospect in the fields of medicine, food and cosmetics.

[0027] Therefore, the application provides a preparation method and application of the paeonol and the derivative thereof, the paeonol is prepared by a simple extraction method, and the extraction rate of the paeonol is high; the prepared paeonol derivative has more excellent antioxidant activity, enhanced anti-inflammatory effect and better anti-platelet aggregation effect; and the paeonol derivative also has good inhibitory activity on the occurrence and development of diabetic retinopathy. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1The UV spectrum scanning results of the extracted and purified paeonol in Example 2 of the present application and a standard sample (A-standard sample, B-paeonol extracted and purified in Example 2 of the present application) are shown in the following table.

[0029] Figure 2 The mRNA relative expression level test results in Test Example 3 of the present application are shown in the following table. DETAILED DESCRIPTION

[0030] The technical solutions of the present application are described in further detail below in combination with the specific embodiments and the accompanying drawings:

[0031] Example 1

[0032] Extraction of paeonol:

[0033] Pre-treatment: The cortex moutan was crushed by a pulverizer to obtain particles with a particle size of 0.55 mm; the cortex moutan powder was added into vacuum-dried liquid 1-butyl-3-methylimidazole (the mass ratio of the cortex moutan powder to the liquid 1-butyl-3-methylimidazole was 7.3:1), and the mixture was stirred at an oil bath temperature of 76℃ for 4.5 h, and then cooled to room temperature to obtain a pre-treated mixture;

[0034] Enzymatic hydrolysis: The cellulase powder was soaked in water with a pH of 4.4 (the solid-liquid ratio was 1.81 mg:1 mL), and the pre-treated mixture was added (the mass ratio of the cortex moutan powder to the cellulase powder was 20:0.16), and then the mixture was fully mixed and placed in a water bath shaker at 48℃ and shaken at 100 r / min for 2.5 h, and then filtered through a 0.45 μm filter membrane to obtain an enzymatic hydrolysate;

[0035] Purification: The water vapor distillation method was used to collect a distillate accounting for 1 / 3 of the total weight of the enzymatic hydrolysate, and the obtained distillate was stored at 4℃ for 24 h, and then filtered under the same conditions, and the obtained crystalline was paeonol.

[0036] Example 2

[0037] Extraction of paeonol:

[0038] Pre-treatment: The cortex moutan was crushed by a pulverizer to obtain particles with a particle size of 0.55 mm; the cortex moutan powder was added into vacuum-dried liquid 1-butyl-3-methylimidazole (the mass ratio of the cortex moutan powder to the liquid 1-butyl-3-methylimidazole was 7.3:1), and the mixture was stirred at an oil bath temperature of 76℃ for 4.5 h, and then cooled to room temperature to obtain a pre-treated mixture;

[0039] Enzymatic hydrolysis, taking the cellulase powder with pH 4.4 water immersion (solid-liquid ratio of 1.81 mg: 1 mL), adding the water crystal orchid glycoside (the amount of addition is 0.11 mg / mL), and adding the pretreated mixed solution (the mass ratio of cortex moutan powder and enzyme powder is 20:0.16), after mixing, put into the water bath oscillator at 48℃, shake at 100 r / min for 2.5 h, and then filter through 0.45 μm membrane to obtain the enzymatic hydrolysate;

[0040] Purification, taking the water vapor distillation method to collect 1 / 3 of the total weight of the enzymatic hydrolysate, and then placing the obtained distillate at 4℃ for 24 h, filtering under the condition, washing with cold water, and drying to obtain paeonol.

[0041] Example 3:

[0042] Preparation of paeonol derivative:

[0043] Taking the semi-leafed solute and dissolving it in dichloromethane (solid-liquid ratio of 0.072 g: 1 mL), adding 4-dimethylaminopyridine, and then adding SO2Cl2 / dichloromethane (v / v, 1:2.1) mixed solution dropwise under stirring at room temperature, adding dropwise within 0.8 h, increasing the temperature to 76℃, and refluxing for 3.5 h, and then removing SO2Cl2 and dichloromethane under reduced pressure, and then extracting the remaining product with dichloromethane to obtain intermediate product A;

[0044] Taking paeonol (prepared in example 1), dichloromethane and triethylamine, and then slowly adding the above intermediate product A under ice bath, and then stirring at room temperature for 3 h; and then washing with water, and then extracting 4 times with dichloromethane, and then drying the combined organic phase with anhydrous sodium sulfate, and then removing the solvent under reduced pressure to obtain an oil, and then separating and purifying the oil by column chromatography to obtain paeonol derivative; the chemical structure is as follows:

[0045] In the preparation process, the amount of 4-dimethylaminopyridine added is 7.2% of the mass of semi-leafed solute; the solid-liquid ratio of semi-leafed solute and SO2Cl2 is 0.36 g: 1 mL; the solid-liquid ratio of paeonol and dichloromethane is 0.064 g: 1 mL; the mass ratio of triethylamine and paeonol is 1.38:1; and the molar ratio of intermediate product A and paeonol is 1.24:1.

[0046]

[0047] 1 H NMR (400MHz, CDCl3) δ ppm7.91 (d, 1H, Ar-H), 7.53 (d, 1H, Ar-H), 6.98 (dd, 1H, Ar-H), 4.77 (m, 1H, N-CH), 4.50, 4.41 (dd, 2H, N-CH2), 3.92, 2.59, 2.40 (s, 9H, -CH3), 3.19, 2.89 (m, 2H, S-CH2). Molecular formula: C 15 H 17 N7O5S, HRMS (ESI): m / z [M+H]+, 323.1058.

[0048] Example 4:

[0049] The preparation of the Danshensu derivative is different from that of Example 3 in that Danshensu lactic acid is used instead of HPLN. The chemical structure is as follows:

[0050]

[0051] 1 H NMR (400 MHz, CDCl3) δ ppm 7.96, 7.58, 6.92 (3H, Ar-H), 6.99, 6.81, 6.75 (3H, Ar-H), 4.58 (m, 1H, -CH), 3.19, 2.94 (dd, 2H, -CH2), 3.90, 2.63 (s, 6H, -CH3). Molecular formula: C 18 H 18 O7, HRMS (ESI): m / z [M+H]+, 346.1157.

[0052] Comparative Example 1:

[0053] The extraction of Danshensu is different from that of Example 1 in that the pretreatment process does not add 1-butyl-3-methylimidazole chloride.

[0054] Comparative Example 2:

[0055] The extraction of Danshensu is different from that of Example 1 in that the pretreatment process does not add 1-butyl-3-methylimidazole chloride.

[0056] The powder of Paeonia suffruticosa was crushed by a pulverizer to obtain particles with a particle size of 0.55 mm; the Paeonia suffruticosa powder was placed in a Soxhlet extractor, and ethanol was added according to a solid-liquid ratio of 20 mg: 1 mL, and extracted at 95°C for 8 h until the reflux liquid was colorless; the extraction liquid was combined and filtered through a 0.45 μm filter membrane for use.

[0057] Comparative Example 3:

[0058] Ionic liquid-assisted extraction method:

[0059] The cortex of Paeonia suffruticosa was ground by a grinder to obtain particles with a particle size of 0.55 mm; the cortex powder was added into vacuum-dried liquid 1-butyl-3-methylimidazole chloride (the mass ratio of the cortex powder to the liquid 1-butyl-3-methylimidazole chloride was 7.3:1), and stirred at 76°C in an oil bath for 8 h, and then cooled to room temperature to obtain a pretreated mixture; distilled water was added, and continuously mechanically stirred for 8 min, then filtered, and the filter cake was washed with water, and the filtrate and the washing liquid were combined and filtered through a 0.45 μm filter membrane for use.

[0060] Test Example 1

[0061] 1. Determination of the content of paeonol in the extract (or enzyme hydrolysate)

[0062] The extract or enzyme hydrolysate was concentrated by rotary evaporation, and analyzed by high performance liquid chromatography, and the detection wavelength was 274 nm; the chromatographic conditions were as follows: mobile phase: methanol / water (v / v, 55 / 45); flow rate 1.0 mL / min; column temperature: 35°C; column pressure: 10 MPa; sample injection amount: 20 μL.

[0063] The extraction rate of paeonol was calculated according to the following formula:

[0064] Extraction rate (mg / g) = mass of paeonol in the extract (or enzyme hydrolysate) / mass of cortex of Paeonia suffruticosa powder

[0065] The extract or enzyme hydrolysate prepared in Comparative Examples 1 to 3 and Examples 1 to 2 was subjected to the above tests, and the results are shown in Table 1.

[0066] Table 1. Results of the extraction rate test

[0067]

[0068] From Table 1, it can be seen that the extraction rate of paeonol prepared by the extraction method of Example 1 is higher than that of Comparative Examples 1 to 3, indicating that the pretreatment with ionic liquid, followed by enzyme hydrolysis for the extraction of paeonol, can effectively improve the extraction rate of paeonol, and the effect is obviously better than that of the traditional alcohol extraction method. The effect of Example 2 is significantly better than that of Example 1, indicating that the addition of buchnerioidin during enzyme hydrolysis can promote the enzyme hydrolysis activity of cellulase, promote the dissolution of active substances in plant cells, and thus significantly increase the extraction rate of paeonol.

[0069] 2. Determination of enzyme activity promotion

[0070] Preparation of the enzyme solution to be tested:

[0071] Take solid sample enzyme 1 g, dissolve with water, and add 0.05 g of crystal lily glycoside (experimental group), and dilute with water to constant volume in a 50 mL volumetric flask, incubate at 40°C for 3 h; then take 1.5 mL, dilute with water to constant volume in a 10 mL volumetric flask, mix well, and stand for 10 min, and then measure; and set up a control group: no addition of crystal lily glycoside.

[0072] Enzyme activity determination

[0073] Take a 25 mL graduated stoppered test tube, accurately add 2.00 mL of CMC-Na solution prepared with the corresponding pH buffer solution; add 0.5 mL of diluted enzyme solution to be tested, mix well, and cover the stopper; and set up a blank test tube (only add CMC-Na solution); at the same time, place in a (50±0.2) °C water bath, accurately time, and after 30 min, take out, quickly add 3.0 mL of DNS reagent, add 0.5 mL of diluted enzyme solution to be tested in the blank test tube, shake well, and at the same time, place in a boiling water bath, heat for 10 min, then take out, quickly cool to room temperature, and dilute with water to 25 mL; use the blank test tube to set the instrument zero point, at a wavelength of 540 nm on the spectrophotometer, use a 10 mL cuvette, measure the absorbance of the sample solution in the sample tube, and calculate the CMC enzyme activity according to the following formula:

[0074] H=2×A×N

[0075] In the formula, H is the CMC enzyme activity of the sample, u / g; A is the reducing sugar obtained from the standard curve according to the absorbance, mg; and N is the dilution multiple of the enzyme sample.

[0076] The test results are shown in Table 2:

[0077] Table 2 Enzyme activity test results

[0078]

[0079] From Table 2, it can be seen that the enzyme activity of the experimental group cultured with the addition of crystal lily glycoside is higher than that of the control group without the addition, indicating that the addition of gancirsimultaneous acid effectively improves the enzyme activity of cellulase, and applied to the extraction process of active substances of medicinal materials, can effectively promote the dissolution of active substances in plant cells, and thus significantly enhance the extraction rate of active substances. This result is also consistent with the test results of the extraction rate of aformentioned paeonol.

[0080] 3. Ultraviolet spectrophotometer scanning

[0081] Weigh 5 mg of purified paeonol into a 50 mL volumetric flask, dilute to volume with anhydrous ethanol, and transfer 4 mL of the diluted solution to a 100 mL volumetric flask, dilute to volume with anhydrous ethanol, and prepare a 4 μg / mL experimental sample solution. Accurately weigh 5 mg of paeonol standard into a 50 mL volumetric flask, dilute to volume with anhydrous ethanol, and transfer 5 mL of the diluted solution to a 100 mL volumetric flask, dilute to volume with anhydrous ethanol, and prepare a 5 μg / mL standard solution. Using anhydrous ethanol as a control, perform a full-range scan at a wavelength of 200–400 nm.

[0082] The paeonol extracted in Example 2 was subjected to the above tests, and the results are as follows: Figure 1 As shown in the figure, the UV spectrum of paeonol obtained after purification in Example 2 is basically consistent with that of the standard, indicating that the components of paeonol extracted and purified in Example 2 of this invention are consistent with those of the standard. These results demonstrate that the extraction method provided by this invention does not destroy the components of paeonol and can obtain relatively pure paeonol.

[0083] Experimental Example 2:

[0084] 1. Antioxidant activity

[0085] DPPH Scavenging Rate Determination

[0086] Dissolve the sample in anhydrous ethanol (concentration 2 mg / mL), then add 3.9 mL of a solution containing 6 × 10⁻⁶ mg / mL. - 5 A mol / L DPPH solution (prepared with anhydrous ethanol) was thoroughly mixed and placed in a water bath at 37°C for 1 hour. The absorbance of the solution was then measured as A0. Simultaneously, 3.9 mL of DPPH solution was taken, and 0.1 mL of ethanol was added. The absorbance of this solution was then measured as A0. i Clearance rate K = (A i -A0) / A i ×100%.

[0087] Anti-oil oxidation assay

[0088] The sample concentration was prepared as a 1.0 mg / mL ethanol solution. The lard was freshly refined. The oven drying method was used, and the malondialdehyde (MDA) content in the oil sample was measured every 2 days. The MDA content in the oil sample on day 10 was used as the characterization data. The specific procedure was as follows: 1 mL of oil sample was added to 1 mL of 20% trichloroacetic acid and mixed well. After standing for 20 min, 2 mL of 0.28% thiobarbituric acid was added, and the mixture was shaken well and boiled in a water bath for 15 min. After cooling, 5 mL of chloroform was added, and the mixture was allowed to stand. The supernatant was then measured at 538 nm for the OD value. The experimental result was the average of three measurements. The inhibition rate was calculated according to the following formula:

[0089] Inhibition rate % = (OD空白 OD 样品 OD 空白 ×100%

[0090] The paeonol and paeonol derivatives prepared in Comparative Examples 1-3 and Examples 1-4, danshensu lactic acid, and huaishuosu were subjected to the above tests, and the results are shown in Table 3:

[0091] Table 3 Test results of antioxidant activity

[0092]

[0093] As can be seen from Table 3, the DPPH· clearance rate and the inhibition rate on malondialdehyde of the paeonol derivative prepared in Example 4 are obviously higher than those of Examples 1-2 and danshensu lactic acid, and the effect of Example 3 is equivalent to that of Examples 1-2, indicating that the derivative prepared by modifying paeonol with danshensu lactic acid can significantly improve the antioxidant activity of the derivative; and the derivative prepared by modifying paeonol with huaishuosu does not have negative effect on the antioxidant activity of the derivative.

[0094] 2. Anti-platelet aggregation activity determination

[0095] The experimental subjects were male rabbits (body weight 1.8-2.2 kg), and after local anesthesia with procaine, blood was taken from the carotid artery, anticoagulated with 3.8% sodium citrate, and the volume ratio of blood to sodium citrate solution was 9:1. The blood was centrifuged at 780 r / min for 10 min to prepare platelet-rich plasma (PRP). In the determination tube, 365 μL of PRP, drug solution (dissolved in DMSO) and aspirin (ASP, dissolved in DMSO, positive control group) were added to a final concentration of 0.12 mmol / L, and after incubation for 5 min, 5 μL of adenosine diphosphate (ADP) was added, and 1% DMSO was used as a blank. The maximum aggregation rate within 5 min was observed and recorded, and the inhibition rate of platelet aggregation induced by ADP (AIR) was calculated.

[0096] The paeonol, huaishuosu, danshensu lactic acid, and the paeonol derivatives prepared in Examples 3-4 were subjected to the above tests, and the results are shown in Table 4:

[0097] Table 4 Test results of anti-platelet aggregation

[0098]

[0099] From the analysis in Table 4, it can be seen that the inhibitory effect of the paeonol derivatives prepared in Examples 3 and 4 on platelet aggregation is obviously higher than that of paeonol, half leaflet and danshensu lactic acid, indicating that the derivatives prepared by modifying paeonol with half leaflet or danshensu lactic acid can effectively enhance the anti-platelet aggregation activity of the paeonol derivatives; and the derivatives prepared by modifying paeonol with danshensu lactic acid have stronger inhibitory effect on platelet aggregation.

[0100] 3. Anti-inflammatory activity determination

[0101] Xylene-induced mouse ear swelling test

[0102] Drug preparation

[0103] Paeonol, half leaflet, danshensu lactic acid and paeonol derivatives were prepared into ointments, and the content of each was 1%. The ointment base components were the same, including triethanolamine, glycerol, stearic acid, glycerol monostearate and water.

[0104] Test method

[0105] The experimental subjects were 35 healthy male Kunming mice, which were randomly divided into 6 groups, 6 mice in each group. The blank control group and the drug treatment group were respectively prepared, wherein the drug treatment group included: M1 group, paeonol treatment; M2 group, paeonol derivative prepared in Example 3 treatment; M3 group, paeonol derivative prepared in Example 4 treatment; D1 group, half leaflet treatment; D2 group, danshensu lactic acid treatment. The left ear of the mouse was gently cleaned, and then 0.1 mL of xylene was evenly applied to the front and back of the left ear. The right ear was used as a control. After 15 minutes of inflammation induction, the drug was administered. The experimental mice were applied with 0.2 g of ointment containing the drug on the left ear, and the blank control group was applied with 0.2 g of ointment base. One hour after administration, the mice were sacrificed by cervical dislocation, and then ear pieces were taken from the symmetrical parts of both ears using a 6 mm puncher. The mass difference between the left and right ear pieces was the degree of inflammation swelling. The swelling inhibition rate was calculated according to the following formula:

[0106] Swelling inhibition rate % = (average swelling degree of the blank control group - average swelling degree of the experimental group) / average swelling degree of the blank control group × 100%

[0107] The above test results are shown in Table 5:

[0108] Table 5: Swelling inhibition rate test results

[0109] Sample treatment groups Inhibition rate (%) M1 30.4 M2 42.3 M3 50.9 D1 2.7 D2 31.2

[0110] From the analysis in Table 5, it can be seen that the swelling inhibition rate of the paeonol derivatives prepared in Examples 3 and 4 is obviously higher than that of paeonol, half leaflet and danshensu lactic acid, indicating that the derivatives prepared by modifying paeonol with half leaflet or danshensu lactic acid can significantly enhance the anti-inflammatory activity of the paeonol derivatives.

[0111] Test Example 3:

[0112] Evaluation of therapeutic effect on diabetic retinopathy (DR)

[0113] Experimental subjects and grouping

[0114] 45 male SD rats, 7-8 weeks old, weighing 200-220 g; randomly divided into 9 groups, 5 rats in each group. Including normal control Z group, model control D group, drug intervention group, among which the drug intervention group includes: K1 group, paeonol gavage; K2 group, half leaf gavage; K3 group, danshensu lactic acid gavage; K4 group, paeonol derivative prepared in Example 3 gavage; K5 group: paeonol derivative prepared in Example 4 gavage; K6 group, crystal lan glycoside gavage; K7 group, paeonol derivative prepared in Example 3 + crystal lan glycoside (mass ratio 1:0.74), gavage.

[0115] Animal model construction

[0116] The SD rats were fed in the specific pathogen-free level experimental animal room, and fasted for 12 h. The establishment of diabetic rat model: fresh citrate buffer solution (concentration 1%, pH 4.3) was used to dilute STZ (purchased from the market), and was injected intraperitoneally at a dose of 65 mg / kg once, to induce the establishment of diabetic rat model. Note: if the blood glucose of the rat 2 h after meal is <16.7 mmol / L, then re-inject STZ once, the injection dose is 10 mg / kg; 3 days after the second injection, measure the postprandial blood glucose, and define two times of postprandial 2 h blood glucose concentration >16.7 mmol / L as the success of the establishment of diabetic rat model. The normal control group rats were injected intraperitoneally with an equal volume of citrate buffer solution once, and the postprandial blood glucose value was measured 7 days later to ensure that the postprandial 2 h blood glucose value was <16.7 mmol / L.

[0117] Drug intervention method

[0118] After the success of the establishment of the diabetic model for 3 days, the drug intervention group was gavaged with 3 mL of drug solution at a dose of 3 mg / kg per week; the model control group was gavaged with 3 mL of normal saline per week; the normal control group was gavaged with 3 mL of normal saline per week at the corresponding time point. Immediately after drug intervention, sufficient water and food were provided, and ordinary rat feed was fed. The bedding was changed regularly every day to keep the cage dry, prevent infection, and no rats died in each group during the feeding process.

[0119] Index detection

[0120] 5 months later, the rats in each group were fasted for 12 h, and then anesthetized by intraperitoneal injection of 10% chloral hydrate, and sacrificed by cardiac perfusion with 4% paraformaldehyde. The eyeballs were removed. The eyeball wall was cut at 0.5 mm behind the limbus to remove the anterior segment and vitreous body, and the remaining sclera and retina tissue was immersed in 2.5% glutaraldehyde solution for fixation for one week. The eyeball wall was cut into four pieces with the optic disc as the center, and the retina was peeled off under a microscope.

[0121] SDF-1 and VEGF detection: The mRNA expression of SDF-1 and VEGF was detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted by using an RNA extraction kit (purchased from EZ Bioscience, USA) and reverse transcribed into cDNA for subsequent reactions. The reverse transcription kit was also purchased from EZ Bioscience, USA. The primer sequences are shown in Table 6. Real-time quantitative PCR detection was performed in triplicate for each group. The mRNA expression of SDF-1 and VEGF was calculated by 2 -ΔΔCt Data analysis and processing were performed by SPSS 17.0 software.

[0122] Table 6 Primer sequences used in RT-qPCR reactions

[0123] Primer Sequences β-actin-F 5'-ACTGGAAGCGACGATTAC-3' β-actin-R 5'-GGCGACTGAAGCGCCTGCT-3' SDF-1-F 5'-CATCCGGCAGTCAATGCTAA-3' SDF-1-R 5'-GGAATCTGAGCACTGGCGCCTAGG-3' VEGF-F 5'-TCACGCTGCCCAGGGATTCGAT-3' VEGF-R 5'-ATGCAGTTCGCTGACAGCGTAAT-3'

[0124] Result analysis

[0125] The test results are shown in Figure 2 From the analysis of the figure, it can be seen that the mRNA expression of SDF-1 and VEGF after 5 months of intervention of the paeonol derivative prepared in Example 4 is significantly lower than that of paeonol and danshensu lactic acid, indicating that the derivative prepared by modifying paeonol with danshensu lactic acid can significantly enhance the inhibitory effect of the derivative on the expression of SDF-1, and better inhibit the expression of VEGF through the SDF-1 / CXCR-4 axis and a series of signal pathways, effectively reduce the generation of new blood vessels, and have a better inhibitory effect on the occurrence and development of DR. The inhibitory effect of the paeonol derivative prepared in Example 3 on the mRNA expression of SDF-1 and VEGF is improved to a certain extent compared with paeonol, but when it is used in combination with sellowin, the mRNA expression of SDF-1 and VEGF is significantly reduced, indicating that the derivative prepared by modifying paeonol with sellowin can inhibit the expression of SDF-1 and VEGF to a certain extent, and when it is used in combination with sellowin, the inhibitory effect on the expression of SDF-1 and VEGF is significantly enhanced, that is, the addition of sellowin has a synergistic effect on the paeonol derivative modified by sellowin, further improving its inhibitory effect on the occurrence and development of DR.

[0126] The conventional techniques in the above examples are existing techniques known to those skilled in the art, and therefore will not be described in detail here.

[0127] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of the changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing a paeonol derivative, comprising: Paeonol derivatives are prepared by esterification of paeonol and hemifolin. The extraction method of paeonol includes enzymatic hydrolysis, and the enzyme used in the enzymatic hydrolysis includes cellulase. The extraction method of paeonol includes: For pretreatment, the peony bark was pulverized using a pulverizer to obtain particles with a particle size of 0.4~0.6mm; the peony bark powder was added to vacuum-dried liquid 1-butyl-3-methylimidazolium chloride, stirred in an oil bath at 70~80℃ for 3~5h, and cooled to room temperature to obtain the pretreated mixture. Enzymatic hydrolysis: Soak cellulase powder in water with pH 4-5 at a ratio of 1.5-2 mg: 1 mL, add the pretreated mixture, mix thoroughly, place in a water bath shaker at 40-50℃ and shake at 100-120 r / min for 1.5-3 h, and filter through a 0.45 μm filter membrane to obtain the enzymatic hydrolysate; Purification was carried out by collecting 1 / 4 to 1 / 2 of the total weight of the enzyme hydrolysate using steam distillation. The distillate was then refrigerated at 2 to 4°C for 24 to 36 hours, filtered, washed with cold water, and dried to obtain paeonol. The chemical structure of the paeonol derivative is shown in Formula I: I。 2. The method for preparing a paeonol derivative according to claim 1, characterized in that: The mass ratio of 1-butyl-3-methylimidazolium chloride to peony bark powder is 6.5~8:

1.

3. The method for preparing a paeonol derivative according to claim 1, characterized in that: The mass ratio of peony bark powder to enzyme powder is 20:0.1~0.

2.

4. The paeonol derivative prepared by any one of claims 1-3 has the chemical structure shown in Formula I: I。 5. The use of the compound of formula I according to claim 4 in the preparation of pharmaceutical agents, characterized in that: The product's efficacy includes antioxidant, anti-inflammatory, and anti-platelet aggregation activities.

6. Use of the compound of Formula I according to claim 4 in the preparation of a medicament for the prevention and treatment of diabetic retinopathy.

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