CN114890972B - A method for preparing desmethyl nobiletin using a microwave-assisted deep co-solvent reagent to degrade nobiletin - Google Patents

Abstract

The invention provides a method for preparing desmethyl nobiletin by degrading nobiletin using a microwave-assisted deep co-solvent reagent, and belongs to the technical field of preparation of desmethyl nobiletin. The deep co-soluble reagent formed by choline chloride and oxalic acid used in the present invention has the dual functions of organic solvent and acidic reagent. There are isolated pairs of oxygen atoms in the oxygen atoms of the methoxy group at position 5 of nobiletin and the carbonyl group of the C ring. In the strong acid environment formed by deep mutually soluble reagents, it is easy to form a six-membered structure with proton-hydrogen and electron-donor-oxygen coordination. Ring structure, and the six-membered ring will continue to be hydrolyzed into hydroxyl groups, thereby removing the methoxy group at the 5-position of nobiletin and converting it into desmethyl nobiletin. The invention uses microwaves for degradation, which facilitates hydrogen ions to attack nobiletin, thereby preparing demethylnobiletin in a short time. Compared with the traditional acidolysis method, the method of the present invention has high conversion efficiency and can control the reaction time to about 20 minutes.

Description

A microwave synergistic deep co-solvent reagent degrades nobiletin to prepare desmethyl nobiletin Methods

Technical field

The present invention relates to the technical field of preparation of desmethyl nobiletin, and in particular to a method for preparing desmethyl nobiletin by degrading nobiletin using a microwave-assisted deep co-solvent reagent.

Background technique

Desmethyl nobiletin is a flavonoid that has a variety of stronger active functions than nobiletin, such as antioxidant, anti-inflammatory, anti-cancer cell proliferation, anti-neurodegenerative diseases and prevention of liver fibrosis. However, the conversion rate of nobiletin demethylation under natural conditions is very low and will be affected by factors such as storage temperature, humidity, and microbial distribution, which greatly limits the use of nobiletin in functional foods and drugs. use.

At present, the main method for degrading nobiletin to prepare demethylnobiletin is acidolysis. Luo Shikun and others found that desmethyl nobiletin can be produced by heating nobiletin to reflux for 24 hours under the condition that the volume ratio of concentrated hydrochloric acid to absolute ethanol is 1:9 (Luo Shikun, Wang Jiahuan, Xu Lanying, Long Tao, & Li Shiming. Extraction, isolation and modification of nobiletin from tangerine peel. Food Science, 2014(24), 20-23.); Chu et al. used boron tribromide, boron trichloride, aluminum trichloride and concentrated hydrochloric acid to prepare Methyl nobiletin (Chu H W, Wu H T, Lee Y J. Regioselective hydroxylation of 2-hydroxychalcones by dimethyldioxirane towards polymethoxylatedflavonoids. Tetrahedron, 2004, 60 (11): 2647-2655.). However, hydrochloric acid is a volatile acid with poor operability, and the use of hydrochloric acid and organic reagents can easily corrode production equipment, produce non-degradable acidic waste liquid, pollute the environment, and is not conducive to industrial production. Huang et al. used edible acids such as phosphoric acid, lactic acid and citric acid to replace strong corrosive acids, but the conversion occurred at a high temperature of 175°C (Huang X, KouX, Wang L, et al. Effective hydroxylation of tangeretin from Citrus Peel (Chenpi )by edible acids and its improvement in antioxidant and anti-lipase activities.LWT,2019,116:108469.), and this method requires a long-term high-temperature heating process, high energy consumption and poor safety. Therefore, there is an urgent need for an efficient, energy-saving, green and environmentally friendly method to degrade nobiletin to prepare demethylnobiletin.

Contents of the invention

The object of the present invention is to provide a method for preparing desmethyl nobiletin by degrading nobiletin using a microwave-assisted deep co-solvent reagent, which is highly efficient, green and environmentally friendly and produces no organic or acidic waste liquid during the process.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

The invention provides a method for preparing desmethyl nobiletin by degrading nobiletin using a microwave-assisted deep co-solvent reagent, which includes the following steps:

Mix nobiletin and a deep co-soluble reagent, and degrade the resulting mixture under microwave conditions. After separation, desmethyl nobiletin is obtained; the deep co-soluble reagent is choline chloride and oxalic acid.

Preferably, the molar ratio of choline chloride and oxalic acid is 1:1.

Preferably, the concentration of nobiletin in the deep co-solvent reagent is 1 to 2 mg/mL.

Preferably, the power of the microwave is 750-850W, and the temperature is 100-105°C.

Preferably, the degradation time is 15 to 20 minutes.

Preferably, the separation includes mixing the degradation products obtained by degradation with water, and performing refrigeration and centrifugal separation in sequence.

Preferably, the volume ratio of water to deeply co-soluble reagent is (10-12):1.

Preferably, the refrigeration temperature is 4°C and the time is 3 to 5 hours.

Preferably, after the separation, the liquid obtained is rotary evaporated to obtain a deep co-soluble reagent.

Preferably, the rotary evaporation temperature is 45°C.

The invention provides a method for preparing desmethyl nobiletin by degrading nobiletin with a microwave-assisted deep co-soluble reagent, which includes the following steps: mixing nobiletin and a deep co-soluble reagent, and degrading the resulting mixture under microwave conditions , after separation, desmethyl nobiletin was obtained. The deep co-soluble reagent formed by choline chloride and oxalic acid used in the present invention not only has the solubility of organic solvents, but also has a hydrogen ion concentration higher than strong acids such as hydrochloric acid and sulfuric acid. Therefore, the deep co-soluble reagent has the dual functions of organic solvent and acidic reagent. Function. There are isolated pairs of oxygen atoms in the methoxy group at the 5-position of nobiletin and the oxygen atom in the C ring carbonyl group. In the strong acid environment formed by the deep eutectic reagent, it is easy to form a six-membered proton-hydrogen and electron-donor-oxygen coordination system. Ring structure, and the six-membered ring will continue to be hydrolyzed into hydroxyl groups, thereby removing the 5-position methoxy group of nobiletin and converting it into desmethyl nobiletin. The invention uses microwaves for degradation. The penetration of microwave energy can heat the reactants quickly and evenly, and improve the porosity of the reactants, so that the entire reaction system can be heated to about 100°C in a short time, increasing the polarity of the system. The probability of collision of sexual molecules also increases the intermolecular force in the system, which facilitates hydrogen ions to attack nobiletin, thereby preparing demethylnobiletin in a short time. Compared with the traditional acidolysis method, the method of the present invention has high conversion efficiency and can control the reaction time to about 20 minutes.

The deep eutectic reagent used in the present invention is a new generation of ionic liquid formed by a hydrogen bond donor and a hydrogen bond acceptor, and has excellent properties such as low cost, low toxicity and biodegradability.

The present invention uses a method of microwave synergy with deep co-soluble reagents to degrade nobiletin to prepare demethylnobiletin. Compared with the traditional acidolysis method, it does not require long-term high-temperature heating treatment, is safe to operate, has low energy consumption costs, and does not need to use ethanol, etc. Organic solvents, and there is no need to use highly corrosive inorganic acids and organic solvents such as concentrated hydrochloric acid. The deep co-solvent reagents used can be recycled and reused after separation, achieving no acidic and organic waste liquids in the entire process. The method is green and environmentally friendly, and is a good solution for detoxification. Guidance is provided for the preparation of other desmethylpolymethoxyflavonoids such as methyl nobiletin.

Furthermore, the present invention uses water to precipitate methyl nobiletin. Since water is both a hydrogen bond acceptor and a hydrogen bond donor, the addition of water will destroy the stability of the deep co-soluble reagent and reduce its solubility. Methyl nobiletin has poor water solubility and will precipitate when the stability of the system is destroyed. After collecting the precipitated desmethyl nobiletin, the solution can be rotary evaporated to remove the water and the deep co-soluble reagent can be recovered again. Utilized, so that no acidic waste liquid is produced during the entire reaction process.

Description of the drawings

Figure 1 is the liquid chromatogram of the standard products of nobiletin and desmethyl nobiletin;

Figure 2 is a liquid chromatogram of nobiletin and desmethyl nobiletin in Example 1;

Figure 3 is a liquid chromatogram of nobiletin and desmethyl nobiletin in Example 2;

Figure 4 is a liquid chromatogram of nobiletin and desmethyl nobiletin in Example 3;

Figure 5 is a liquid chromatogram of nobiletin and desmethyl nobiletin in Comparative Example 1.

Detailed ways

The invention provides a method for preparing desmethyl nobiletin by degrading nobiletin using a microwave-assisted deep co-solvent reagent, which includes the following steps:

Mix nobiletin and a deep co-soluble reagent, degrade the resulting mixture under microwave conditions, and after separation, obtain desmethyl nobiletin; the deep co-soluble reagent preferably includes choline chloride and oxalic acid.

In the present invention, unless otherwise specified, the required preparation raw materials are all commercially available products well known to those skilled in the art.

In the present invention, nobiletin and a deep co-soluble reagent are mixed. The present invention has no special limitation on the source of nobiletin, and any commercially available product well known in the art can be used.

In the present invention, the deep co-soluble reagent is choline chloride and oxalic acid; the molar ratio of the choline chloride and oxalic acid is preferably 1:1; the preparation method of the deep co-soluble reagent is preferably chlorine chloride and oxalic acid. Choline and oxalic acid are mixed, placed in a 100°C water bath and stirred until a clear and transparent deep co-soluble reagent is formed; the present invention has no special limit on the stirring rate, and the materials can be mixed evenly according to the process well known in the art. .

In the present invention, the concentration of nobiletin in the deep co-solvent reagent is preferably 1 to 2 mg/mL.

The present invention has no special restrictions on the mixing process of nobiletin and deep co-soluble reagent. The materials can be mixed evenly according to the process well known in the art.

After completing the mixing, the present invention degrades the resulting mixture under microwave conditions. In the present invention, the power of the microwave is preferably 750-850W, more preferably 800W, the temperature is preferably 100-105°C, more preferably 102°C; the degradation time is preferably 15-20min, more preferably 17min .

In the present invention, the microwave digestion instrument used for the microwave is preferably the MARS 6240/50 microwave digestion instrument of the American CEM Company.

After completing the degradation, the present invention separates the obtained products; the separation preferably includes mixing the degradation products obtained by degradation with water, and sequentially performing refrigeration and centrifugal separation; the water is preferably ultrapure water; the water is co-soluble with deep The volume ratio of reagents is preferably (10-12):1, more preferably 11:1. The present invention has no special limitations on the process of mixing the degradation product and water, and it can be carried out according to processes well known in the art. The present invention utilizes ultrapure water to precipitate demethylnobiletin.

In the present invention, the refrigeration temperature is preferably 4°C, and the refrigeration time is preferably 3 to 5 hours.

The present invention has no special limitations on the centrifugal separation process, and it can be carried out according to processes well known in the art.

After completing the centrifugal separation, the present invention preferably washes the obtained precipitate with ultrapure water until neutral and then freeze-dries it to obtain desmethyl nobiletin. The present invention has no special limitations on the washing and freeze-drying processes, and can be carried out according to processes well known in the art.

After completing the centrifugal separation, it is also preferred to perform rotary evaporation of the obtained liquid to collect high-viscosity components to obtain a deep co-soluble reagent. In the present invention, the temperature of the rotary evaporation is preferably 45°C. The present invention removes moisture in the liquid through rotary evaporation; the present invention has no special limit on the time of the rotary evaporation, as long as all the moisture in the liquid can be removed.

The technical solutions in the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

In the following examples, the microwave equipment used is the MARS 6240/50 microwave digestion instrument of the American CEM Company.

Example 1

Mix choline chloride and oxalic acid at a molar ratio of 1:1, place in a 100°C water bath and stir until a clear and transparent deep co-soluble reagent is formed;

Dissolve 20 mg of pure nobiletin in 20 mL of prepared deep co-solvent reagent and mix evenly. Put the resulting mixture into a microwave digestion instrument. The processing conditions are microwave power 750W, microwave temperature 100°C, and processing time 15 minutes to obtain the degradation product. ;

Add 10 times the volume of ultrapure water to the degradation product, refrigerate the resulting mixture at 4°C for 5 hours, and then centrifuge. The resulting precipitate is washed with ultrapure water to neutrality and then freeze-dried to obtain demethylated tangerine peel. white.

Example 2

Mix choline chloride and oxalic acid at a molar ratio of 1:1, place in a 100°C water bath and stir until a clear and transparent deep co-soluble reagent is formed;

Dissolve 40 mg of pure nobiletin in 20 mL of prepared deep co-solvent reagent and mix evenly. Put the resulting mixture into a microwave digestion instrument. The processing conditions are microwave power 800W, microwave temperature 102°C, and processing time 17 minutes to obtain the degradation product. ;

Add 11 times the volume of ultrapure water to the degradation product, refrigerate the resulting mixture at 4°C for 5 seconds, then centrifuge, wash the resulting precipitate with ultrapure water to neutrality, and freeze-dry to obtain demethylated tangerine peel. white.

Example 3

Mix choline chloride and oxalic acid at a molar ratio of 1:1, place in a 100°C water bath and stir until a clear and transparent deep co-soluble reagent is formed;

Dissolve 20 mg of pure nobiletin in 20 mL of prepared deep co-solvent reagent and mix evenly. Put the resulting mixture into a microwave digestion instrument. The processing conditions are microwave power 800W, microwave temperature 105°C, and processing time 20 minutes to obtain the degradation product. ;

Add 12 times the volume of ultrapure water to the degradation product, refrigerate the resulting mixture at 4°C for 5 seconds, then centrifuge, wash the resulting precipitate with ultrapure water until neutral, and freeze-dry to obtain demethylated tangerine peel. white.

Comparative example 1

Traditional acidolysis method: 20 mg nobiletin was catalyzed in 20 mL of concentrated hydrochloric acid with a mass percentage of 37% for 12 hours. The reaction temperature was always maintained at 90°C to obtain the degradation product.

Result detection

1) Mix 1 mg nobiletin standard and 1 mg nobiletin standard in 1 mL methanol, use the resulting mixture as a standard sample, take 500 μL standard sample, and perform high-performance liquid chromatography detection. The process includes:

Take 500 μL of sample, add 500 μL of chromatography grade methanol, mix and pass through a 0.45 μm nylon membrane for later use;

The detection equipment is an Agilent 1260Infinity high performance liquid chromatograph, the chromatographic column model is WatersAtlantis T3 column (2.1×50mm, 1μm), the mobile phase A is 0.1% (v/v) glacial acetic acid, the mobile phase B is acetonitrile, and the flow rate is 0.8 mL/min;

The elution procedure is as follows: 0 to 2 minutes, 5 to 5% B; 2 to 10 minutes, 5 to 30% B; 10 to 12 minutes, 30 to 100% B; 12 to 18 minutes, 100% to 100% B; 18 to 20 minutes, 100～5%B; 20.00～25.00min, 5～5%;

The column temperature was maintained at 25°C, and the UV detection wavelength was 210nm; the results are shown in Figure 1; Figure 1 is the chromatogram of nobiletin standard and demethylnobiletin standard; it can be clearly seen in Figure 1 that nobiletin and nobiletin are Peak time of demethylnobiletin.

2) Use the high-performance liquid chromatography method of the above 1) to detect the conversion status of nobiletin and the production status of demethylnobiletin in Examples 1 to 3: respectively, use nobiletin-deep co-solubilization in Examples 1 to 3 The reagent mixture and the deep co-soluble reagent mixture (i.e., degradation products) after nobiletin degradation were used as detection samples. The results obtained are shown in Figures 2 to 4. At the same time, the degradation products of Comparative Example 1 were tested by high-performance liquid chromatography. The results are shown in the figure. 5. The result analysis is as follows:

As shown in Figure 2, in Example 1, almost all nobiletin was converted into desmethyl nobiletin, the conversion rate was 99.62%, and the processing time was 15 minutes, which is highly efficient. As shown in Figure 3, in Example 2, almost all nobiletin was converted into desmethyl nobiletin, the conversion rate was 99.57%, and the processing time was 17 minutes, which is highly efficient. As shown in Figure 4, in Example 3, almost all nobiletin was converted into desmethyl nobiletin, the conversion rate was 99.65%, and the processing time was 20 minutes, which is highly efficient. In Comparative Example 1, when the traditional acidolysis method was used for reaction for 12 hours, the conversion rate only reached 23.52%, indicating that the method of the present invention can significantly reduce the treatment time and increase the conversion rate.

The above are only preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (8)

1. A method for preparing demethyl nobiletin by degrading nobiletin with microwave synergistic deep-dissolution reagent comprises the following steps:

mixing nobiletin and a deep co-solvent reagent, degrading the obtained mixture under the microwave condition, and separating to obtain the demethylated nobiletin; the deep co-dissolution reagent is choline chloride and oxalic acid; the power of the microwaves is 750-850W, and the temperature is 100-105 ℃; the degradation time is 15-20 min.

2. The method according to claim 1, wherein the molar ratio of choline chloride to oxalic acid is 1:1.

3. the method according to claim 1, wherein the concentration of nobiletin in the deep-solubilizing reagent is 1-2 mg/mL.

4. The method of claim 1, wherein the separating comprises mixing degradation products from the degradation with water, and sequentially refrigerating and centrifuging.

5. The method of claim 4, wherein the volume ratio of water to deep co-solvent agent is (10-12): 1.

6. the method of claim 4, wherein the temperature of the refrigeration is 4 ℃ for 3 to 5 hours.

7. The method of claim 1, further comprising subjecting the resulting liquid to spin evaporation to provide a deep-eutectic reagent after the separating.

8. The method of claim 7, wherein the temperature of the rotary evaporation is 45 ℃.