Preparation method of cyanidin-3-O-glucoside-jasmonic acid methyl ester acylated derivative

Abstract

The invention provides a preparation method of a cyanidin-3-O-glucoside-jasmonic acid methyl ester acylated derivative, which comprises the innovativeness of selecting reactants, the characteristics conforming to natural green organic compounds, and the addition of a small amount of cosolvent in a reaction medium, so that the low toxicity is maintained, and the yield of the cyanidin-3-O-glucoside-jasmonic acid methyl ester acylated derivative is improved. By improving the solubility of cyanidin-3-O-glucoside in a reaction medium and further improving the conversion rate, jasmonic acid methyl ester is successfully grafted to cyanidin-3-O-glucoside through a water bath heating oscillation mode and adding an enzyme for catalytic reaction, and a derivative is separated from a solution through semi-preparative liquid chromatography purification. And freeze-drying to obtain a powdery product. Methyl jasmonate is grafted to cyanidin-3-O-glucoside, so that the thermal stability of cyanidin-3-O-glucoside can be enhanced, the fat solubility can be improved, and the application prospect of natural anthocyanin in the fields of food, medicines and the like is further expanded.

Description

Technical Field

[0001] The invention belongs to a chemical modification stabilization method of anthocyanins, and specifically relates to a preparation method of cyanidin-3-O-glucoside-methyl jasmonate acylation derivative. Background Art

[0002] Anthocyanin is a flavonoid compound widely found in berries such as blueberries and blackcurrants and food crops such as purple sweet potatoes and black rice. Anthocyanin gives plants bright colors. Cyanidin-3-O-glucoside is one of many anthocyanins. It has antioxidant, antibacterial, anti-inflammatory, cell protection, nerve nutrition, and lipid-lowering activities. It is also a natural edible pigment. Cyanidin-3-O-glucoside is natural and non-toxic, and has rich active functions, which makes it have great application potential in the fields of food and medicine. However, due to the presence of multiple active hydroxyl groups and a parent nucleus containing positive ions in anthocyanins, its chemical properties are extremely active, which easily causes the color or activity to disappear. At the same time, the presence of multiple hydroxyl groups makes anthocyanins highly hydrophilic, but there are great obstacles in membrane permeability, resulting in low bioavailability. Its application in the food, medicine, cosmetics and other industries is limited, which limits the role of its multiple biological activities.

[0003] In recent years, researchers have used molecular structure modification to adjust the physical and chemical properties of anthocyanins. Molecular structure modification refers to the process of grafting new groups onto anthocyanins through various chemical reactions while maintaining the structure of the anthocyanin precursor unchanged, thereby improving the structural stability. In recent studies, researchers have mostly chosen long-chain fatty acids to modify the structure of anthocyanins, and there are relatively few studies on the use of natural green compounds for modification. Therefore, the development of a natural green organic compound to modify the structure of anthocyanins has broad application prospects. Summary of the invention

[0004] The purpose of the present invention is to provide a method for acylation and structural modification of cyanidin-3-O-glucoside using methyl jasmonate. The cyanidin-3-O-glucoside-methyl jasmonate acylated derivative prepared by the present invention can significantly improve the stability and lipophilicity of anthocyanins.

[0005] The present invention provides a method for preparing a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative, comprising the following technical scheme and steps:

[0006] (1) Dissolve cyanidin-3-O-glucoside in a mixed solution of N,N-dimethylformamide and tert-butyl alcohol, add lipase Novozym 435 and methyl jasmonate, and sonicate for 30 minutes to completely dissolve them.

[0007] (2) Place the solution obtained in step (1) in a shaker at 55°C to 60°C for 24 hours, remove the enzyme by filtration, and terminate the reaction.

[0008] (3) Purify the solution obtained in step (2) by semi-preparative liquid chromatography to obtain an eluent of cyanidin-3-O-glucoside-jasmonate methyl ester acylated derivative

[0009] (4) The collected eluate is concentrated under reduced pressure, and the concentrate is vacuum freeze-dried. The vacuum freeze-drying conditions are a vacuum degree of 0.05 to 0.1 MPa and a temperature of -20 to -40°C, and finally a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative is obtained.

[0010] In the above technical solution, the concentration of cyanidin-3-O-glucoside in step (1) is 1 to 2 mg / ml.

[0011] In the above technical solution, in step (1), the ratio of the mixed solution of N,N-dimethylformamide and tert-butanol is (9:1).

[0012] In the above technical solution, the dosage of Novozym 435 enzyme in step (1) is 10-20 mg / ml.

[0013] In the above technical solution, the molar ratio of cyanidin-3-O-glucoside to methyl jasmonate in step (1) is 1:100 to 1:150.

[0014] In the above technical scheme, the conditions of semi-preparative liquid chromatography in step (3) are that mobile phase A is a formic acid aqueous solution with a volume concentration of 5%, mobile phase B is acetonitrile, and a C18 column is used for gradient elution at a flow rate of 5 mL / min. The gradient program of the mobile phase is as follows: 0-5 min, the concentration of mobile phase B is 5-10%; 5-10 min, 10% B; 10-17 min, 10-24% B; 17-27 min, 24-90% B; 27-30 min, 90% B; 30-35 min, 90-5% B; 35-40 min, 5% B. The injection volume is 1000-2000 μL, and the column temperature is maintained at 40°C. The detection wavelength is 520 nm. The peak at about 21 min is the peak of cyanidin-3-O-glucoside-jasmonate methyl ester acylated derivative.

[0015] A cyanidin-3-O-glucoside-methyl jasmonate acylated derivative prepared by the above preparation method has significantly improved light stability, thermal stability and lipophilicity compared with that before structural modification. The beneficial technical effects of the present invention are embodied in the following aspects:

[0016] (1) In the present invention, methyl jasmonate is grafted onto cyanidin-3-O-glucoside, and the new derivative has significantly improved stability and lipophilicity compared with the derivative before the reaction.

[0017] (2) The present invention uses methyl jasmonate for structural modification. Methyl jasmonate is a natural green organic compound and is more innovative than the commonly used long-chain fatty acid modification.

[0018] (3) The overall toxicity of the present invention is low and the conversion rate is high. Compared with the method using DMF and tetrahydrofuran, the present invention uses a low proportion of DMF and a high proportion of tert-butyl alcohol, which maximizes the conversion rate while maintaining low toxicity. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods:

[0020] Embodiment 1:

[0021] A method for preparing a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative comprises the following steps:

[0022] (1) Dissolve 2 mg of cyanidin-3-O-glucoside in 2 ml of a mixture of N,N-dimethylformamide and tert-butyl alcohol (9:1), add 30 mg of Novozym 435 enzyme and 100 mg of methyl jasmonate, and sonicate for 30 min to completely dissolve it.

[0023] (2) Place the solution obtained in step (1) in a shaker at 55°C for 24 hours, remove the enzyme by filtration, and terminate the reaction.

[0024] (3) The solution obtained in step (2) is purified by semi-preparative liquid chromatography. The conditions of semi-preparative liquid chromatography are as follows: mobile phase A is a 5% volume concentration of formic acid aqueous solution, mobile phase B is acetonitrile, and a C18 column is used for gradient elution at a flow rate of 5 mL / min. The gradient program of the mobile phase is as follows: 0-5 min, the concentration of mobile phase B is 5-10%; 5-10 minutes, 10% B; 10-17 min, 10-24% B; 17-27 min, 24-90% B; 27-30 min, 90% B; 30-35 min, 90-5% B; 35-40 min, 5% B. The injection volume is 1000 μL, and the column temperature is maintained at 40°C. The detection wavelength is 520 nm. The peak at about 21 min is the peak of cyanidin-3-O-glucoside-jasmonate methyl ester acylated derivative, and the eluate is collected.

[0025] (4) The collected eluate is concentrated under reduced pressure, and the concentrate is vacuum freeze-dried. The vacuum freeze-drying conditions are a vacuum degree of 0.05 MPa and a temperature of -40°C, and finally a cyanidin-3-O-glucoside-jasmonate methyl ester acylated derivative is obtained.

[0026] Embodiment 2:

[0027] A method for preparing a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative comprises the following steps:

[0028] (1) Dissolve 10 mg of cyanidin-3-O-glucoside in 10 ml of a mixed solution of N,N-dimethylformamide and tert-butyl alcohol (9:1), add 110 mg of Novozym 435 enzyme and 500 mg of methyl jasmonate, and sonicate for 30 min to completely dissolve it.

[0029] (2) Place the solution obtained in step (1) in a shaker at 55°C for 24 hours, and remove the enzyme by filtration to terminate the reaction.

[0030] (3) The solution obtained in step (2) is purified by semi-preparative liquid chromatography. The conditions of semi-preparative liquid chromatography are as follows: mobile phase A is a 5% volume concentration of formic acid aqueous solution, mobile phase B is acetonitrile, and a C18 column is used for gradient elution at a flow rate of 5 mL / min. The gradient program of the mobile phase is as follows: 0-5 min, the concentration of mobile phase B is 5-10%; 5-10 min, 10% B; 10-17 min, 10-24% B; 17-27 min, 24-90% B; 27-30 min, 90% B; 30-35 min, 90-5% B; 35-40 min, 5% B. The injection volume is 1000 μL, and the column temperature is maintained at 40°C. The detection wavelength is 520 nm. The peak at about 21 min is the peak of cyanidin-3-O-glucoside-jasmonate methyl ester acylated derivative, and the eluate is collected. (4) The collected eluate is concentrated under reduced pressure, and the concentrate is vacuum freeze-dried. The vacuum freeze-drying conditions are a vacuum degree of 0.05 MPa and a temperature of -40°C, and finally a cyanidin-3-O-glucoside-jasmonate methyl ester acylated derivative is obtained.

[0031] Embodiment 3:

[0032] A method for preparing a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative comprises the following steps:

[0033] (1) Dissolve 20 mg of cyanidin-3-O-glucoside in 20 ml of a mixed solution of N,N-dimethylformamide and tert-butyl alcohol (9:1), add 230 mg of Novozym 435 enzyme and 1000 mg of methyl jasmonate, and sonicate for 30 min to completely dissolve it.

[0034] (2) The solution obtained in step (1) was placed in a shaker at 60°C for 24 hours, and the enzyme was removed by filtration to terminate the reaction.

[0035] (3) The solution obtained in step (2) is purified by semi-preparative liquid chromatography. The conditions of semi-preparative liquid chromatography are as follows: mobile phase A is a 5% volume concentration of formic acid aqueous solution, mobile phase B is acetonitrile, and a C18 column is used for gradient elution at a flow rate of 5 mL / min. The gradient program of the mobile phase is as follows: 0-5 min, the concentration of mobile phase B is 5-10%; 5-10 min, 10% B; 10-17 min, 10-24% B; 17-27 min, 24-90% B; 27-30 min, 90% B; 30-35 min, 90-5% B; 35-40 min, 5% B. The injection volume is 2000 μL, and the column temperature is maintained at 40°C. The detection wavelength is 520 nm. The peak at about 21 min is the peak of cyanidin-3-O-glucoside-jasmonate methyl ester acylated derivative, and the eluate is collected.

[0036] (4) The collected eluate is concentrated under reduced pressure, and the concentrate is vacuum freeze-dried. The vacuum freeze-drying conditions are a vacuum degree of 0.1 MPa and a temperature of -35°C, and finally a cyanidin-3-O-glucoside-jasmonate methyl ester acylated derivative is obtained.

[0037] Example 4: Determination of thermal stability of cyanidin-3-O-glucoside-methyl jasmonate acylated derivatives. Cyanidin-3-O-glucoside and derivatives were dissolved in 0.01M hydrochloric acid aqueous solution (pH=2.1) to prepare a solution with absorbance between 0.8 and 1.0, and placed in a constant temperature water bath at 65, 75 and 85°C to study the effect of temperature on the color stability of anthocyanin solution. The absorbance at 520nm was measured every 2h using a UV-visible spectrophotometer, the absorbance was converted to concentration, and the thermal degradation kinetics were analyzed in combination with thermodynamic formulas.

[0038] The Ea value of cyanidin-3-O-glucoside is 45-55 kJ mol –1 , the Ea value of the derivative obtained in implementation case 1-3 will be reduced by 5% to 10%.

[0039] Embodiment 5:

[0040] The lipophilicity of cyanidin-3-O-glucoside and derivatives was evaluated by measuring the n-octanol / water partition coefficient (log P). The saturated n-octanol and water phases were separated after presaturation with 2% hydrochloric acid aqueous solution and n-octanol (1:3, v / v) for at least 24 hours. 1 mg of the reaction product and cyanidin were dissolved in 1 mL of saturated n-octanol, and their absorbance (A) was measured at a wavelength of 520 nm. 0 ). Then, 1 mL of saturated acidified water was added and the mixture was shaken vigorously for 1 h. Finally, the mixture was centrifuged at 4°C, 12,000 rpm for 1 h, and the absorbance (Ax) of the upper n-octanol solution was measured at a wavelength of 520 nm. The log P value was calculated using the following formula:

[0041] log P = log(Ax / A 0 -Ax)

[0042] The log P value of cyanidin-3-O-glucoside was measured to be -1.2 to -1.4, and the logP value of the derivative obtained in Example 1-3 was 0.4 to 0.6.

Claims

1. A method for preparing a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative, the method comprising the following steps: (1) Dissolve cyanidin-3-O-glucoside in a DMF-tert-butyl alcohol mixed solution, add Novozym435 enzyme and methyl jasmonate, and sonicate for 30 minutes to completely dissolve it. (2) Place the solution obtained in step (1) in a shaker at 55°C to 60°C for 24 hours, and remove the enzyme by filtration to terminate the reaction. (3) Purify the solution obtained in step (2) by semi-preparative liquid chromatography to obtain an eluent of cyanidin-3-O-glucoside-jasmonate methyl ester acylated derivative (4) The collected eluate is concentrated under reduced pressure, and the concentrate is vacuum freeze-dried. The vacuum freeze-drying conditions are a vacuum degree of 0.05 to 0.1 MPa and a temperature of -20°C to -40°C, and finally a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative is obtained.

2. The method for preparing a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative according to claim 1, characterized in that: In step (1), the concentration of cyanidin-3-O-glucoside is 1-2 mg / ml.

3. According to the method for preparing a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative according to claim 1, the ratio of the DMF-tert-butanol mixed solution in step (1) is (9:1).

4. The method for preparing a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative according to claim 1, wherein the amount of Novozym435 enzyme used in step (1) is 10-20 mg / ml.

5. The method for preparing a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative according to claim 1, wherein the molar ratio of cyanidin-3-O-glucoside to methyl jasmonate in step (1) is 1:100 to 1:

150.

6. The method for preparing a cyanidin-3-O-glucoside-jasmonate methyl ester acylated derivative according to claim 1, wherein the conditions of the semi-preparative liquid chromatography in step (3) are that the mobile phase A is a formic acid aqueous solution with a volume concentration of 5%, the mobile phase B is acetonitrile, and a C18 column is used for gradient elution at a flow rate of 5 mL / min. The gradient program of the mobile phase is as follows: 0-5 min, the concentration of mobile phase B is 5-10%; 5-10 min, 10% B; 10-17 min, 10-24% B; 17-27 min, 24-90% B; 27-30 min, 90% B; 30-35 min, 90-5% B; and 35-40 min, 5% B.

7. The method for preparing a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative according to claim 1, characterized in that: The Ea value of cyanidin-3-O-glucoside is 45-55 kJ mol–1, and the Ea value of the prepared derivative will be reduced by 5% to 10%.

8. The method for preparing a cyanidin-3-O-glucoside-methyl jasmonate acylated derivative according to claim 1, characterized in that: The log P value of cyanidin-3-O-glucoside is -1.2 to -1.4, and the log P value of the prepared derivative is 0.4 to 0.6.