CN111039912B

CN111039912B - Method for extracting flavonoid compounds in wild rice by using deep eutectic solvent

Info

Publication number: CN111039912B
Application number: CN201911158495.XA
Authority: CN
Inventors: 谭家能; 窦玉青; 张忠锋; 闫宁; 刘新民
Original assignee: Tobacco Research Institute of CAAS
Current assignee: Tobacco Research Institute of CAAS
Priority date: 2019-11-22
Filing date: 2019-11-22
Publication date: 2021-07-23
Anticipated expiration: 2039-11-22
Also published as: CN111039912A

Abstract

The invention relates to the technical field of natural substance extraction, in particular to a method for extracting flavonoids compounds in wild rice by utilizing a deep eutectic solvent. The invention provides a method for extracting flavonoids compounds in wild rice by using a deep eutectic solvent, which comprises the following steps: pretreating wild rice to obtain wild rice powder; extracting the wild rice powder by using a deep eutectic solvent, and then carrying out solid-liquid separation to obtain a flavone extracting solution; carrying out liquid chromatography separation on the flavone extracting solution to obtain a flavonoid compound; the deep eutectic solvent comprises choline chloride, a hydrogen bond donor and water; the hydrogen bond donor includes 1, 4-butanediol, glycerol, or lactic acid. The deep eutectic solvent adopted by the invention is nontoxic, biodegradable, green and environment-friendly, and can be recycled, thereby greatly reducing the extraction cost. The method provided by the invention has high extraction efficiency and low detection limit on the flavonoid compounds in the wild rice.

Description

Method for extracting flavonoid compounds in wild rice by using deep eutectic solvent

Technical Field

The invention relates to the technical field of natural substance extraction, in particular to a method for extracting flavonoids compounds in wild rice by utilizing a deep eutectic solvent.

Background

The Zizania latifolia is a caryopsis of Zizania of Gramineae, subfamily Oryza, and is a full-grain with rich nutrition, and its edible history in China is at least 3500 years. It mainly grows in diving swamps, ponds, swamps, paddy field sides and shallow water areas of lakes at the edges of lakes; the distribution is more in northeast, north China, south China, southwest and the like. The wild rice contains various phytochemicals such as protein, minerals, vitamins, phenols, saponins, phytosterols and anthocyanins. The wild rice has rich biological activity functions including antioxidation, anti-inflammation, anti-obesity and anti-allergy, and has attracted wide attention of researchers at home and abroad.

At present, flavonoids separated from wild rice mainly comprise quercetin, catechin and procyanidine, can improve insulin sensitivity and blood sugar homeostasis, inhibit proliferation of tumor cells and show antioxidant activity. At present, the extraction method of the flavonoid compounds in the wild rice is mainly an organic solvent extraction method, and the adopted organic solvent mainly comprises methanol, ethanol, acetone or chloroform. However, in the extraction process, the organic solvent is toxic, low in vapor pressure, volatile and flammable, and easily causes pollution to the environment.

Disclosure of Invention

The deep eutectic solvent adopted by the invention is non-toxic, biodegradable, recyclable, environment-friendly, high in extraction efficiency and low in detection limit, and can be recycled and reused.

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

the invention provides a method for extracting flavonoids compounds in wild rice by using a deep eutectic solvent, which comprises the following steps:

pretreating wild rice to obtain wild rice powder;

extracting the wild rice powder by using a deep eutectic solvent, and then carrying out solid-liquid separation to obtain a flavone extracting solution;

carrying out liquid chromatography separation on the flavone extracting solution to obtain a flavonoid compound;

the deep eutectic solvent comprises choline chloride, a hydrogen bond donor and water;

the hydrogen bond donor comprises 1, 4-butanediol, glycerol or lactic acid;

preferably, the particle size of the wild rice powder is 40-100 meshes.

Preferably, the molar ratio of choline chloride to hydrogen bond donor in the deep eutectic solvent is 1: (0.5 to 7);

the content of water in the deep eutectic solvent is 10-50 wt%.

Preferably, the material-to-liquid ratio of the wild rice powder to the deep eutectic solvent is 10-100 mg/mL.

Preferably, the extraction mode is ultrasonic extraction.

Preferably, the power of ultrasonic extraction is 120-600W, and the frequency is 20-25 kHz.

Preferably, the extraction temperature is 30-60 ℃ and the extraction time is 1-20 min.

Preferably, the method further comprises the step of diluting the phenolic acid extract with methanol and filtering the diluted phenolic acid extract before the liquid chromatography separation.

Preferably, the volume ratio of the phenolic acid extract extracting solution to the methanol is 1 (1-4); the pore diameter of the filter membrane for filtration is 0.22 μm.

Preferably, the conditions of the liquid chromatographic separation include: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetonitrile-acetic acid mixed solution, and the volume fraction of the acetic acid is 0.1%; the mobile phase B is an aqueous solution of acetic acid, and the volume fraction of the acetic acid is 0.1%;

gradient elution procedure: the volume percentage of the mobile phase A is increased from 5% to 10% in 0-5 min; 5-7 min, increasing the volume percentage of the mobile phase A from 10% to 20%; the volume percentage of the mobile phase A is increased from 20% to 60% in 7-8 min; the volume percentage of the mobile phase A is increased from 60% to 100% in 8-9 min; the volume percentage of the mobile phase A is reduced from 100% to 5% in 9-10 min; keeping the volume percentage of the mobile phase A at 95% for 10-12 min; the flow rate of the mobile phase is 0.3 mL/min; the sample injection amount is 1 mu L; the column temperature was 25 ℃.

The invention provides a method for extracting flavonoids compounds in wild rice by using a deep eutectic solvent, which comprises the following steps: pretreating wild rice to obtain wild rice powder; extracting the wild rice powder by using a deep eutectic solvent, and then carrying out solid-liquid separation to obtain a flavone extracting solution; carrying out liquid chromatography separation on the flavone extracting solution to obtain a flavonoid compound; the deep eutectic solvent comprises choline chloride, a hydrogen bond donor and water; the hydrogen bond donor includes 1, 4-butanediol, glycerol, or lactic acid. The deep eutectic solvent adopted by the invention has strong polarity, can efficiently break the cell wall of the wild rice and promote the dissolution of the flavonoid compounds of the wild rice, is nontoxic, biodegradable, green and environment-friendly, can not generate secondary pollution, can be recycled, and greatly reduces the extraction cost. The method provided by the invention has high extraction efficiency and low detection limit on the flavonoid compounds in the wild rice.

Drawings

FIG. 1 is a liquid mass spectrum of catechin, procyanidin and quercetin;

FIG. 2 is a diagram showing the DPPH free radical scavenging ability of the flavonoid compounds extracted in examples 1-3;

FIG. 3 is an ABTS free radical scavenging capacity diagram of flavonoids extracted in examples 1-3;

FIG. 4 is a graph showing the amount of total flavonoids extracted from Zizania latifolia L in examples 4 to 8 and comparative example 4;

FIG. 5 is a graph showing the amount of total flavonoids extracted from Zizania latifolia L in examples 9 to 12;

FIG. 6 is a graph showing the amount of total flavonoids extracted from Zizania latifolia L in examples 13 to 17;

FIG. 7 is a graph showing the amount of total flavonoids extracted from Zizania latifolia L in examples 18 to 27.

Detailed Description

pretreating wild rice to obtain wild rice powder;

the hydrogen bond donor includes 1, 4-butanediol, glycerol, or lactic acid.

The invention carries out pretreatment on the wild rice to obtain the wild rice powder.

In the present invention, the pre-treatment preferably includes sequentially pulverizing and drying the zizania latifolia. The pulverization method of the present invention is not particularly limited, and the pulverization method known in the art, specifically, grinding, may be used. In the invention, the particle size of the wild rice powder is preferably 40-100 meshes, and more preferably 60-80 meshes. In the invention, the drying temperature is preferably 30-40 ℃, and more preferably 40 ℃; the drying time is not particularly limited, and the milled wild rice grains are dried to constant weight. In the present invention, the wild rice powder is preferably preserved in the dark at-10 to-20 ℃.

After the wild rice powder is obtained, the wild rice powder is extracted by using a deep eutectic solvent and then subjected to solid-liquid separation to obtain a flavone extracting solution; the deep eutectic solvent comprises choline chloride, a hydrogen bond donor and water; the hydrogen bond donor comprises 1, 4-butanediol, glycerol or lactic acid, more preferably 1, 4-butanediol or glycerol, most preferably 1, 4-butanediol.

In the present invention, the molar ratio of choline chloride and hydrogen bond donor in the deep eutectic solvent is preferably 1: (0.5 to 7), more preferably 1: (1 to 6), most preferably 1: (5.5-6.8). In the invention, the content of water in the deep eutectic solvent is 10-50 wt%, more preferably 20-50 wt%, and most preferably 30 wt% or 40 wt%. The water used in the present invention is not particularly limited, and water known in the art may be used, and specifically, deionized water, distilled water, ultrapure water or high-purity water may be used.

In the present invention, the preparation method of the deep eutectic solvent preferably includes the steps of: and (3) carrying out first mixing on choline chloride and a hydrogen bond donor, and then adding water for second mixing to obtain the deep eutectic solvent. In the present invention, the first mixing and the second mixing are preferably performed by stirring; the rotation speed of the stirring and mixing is preferably 200-800 rpm, and more preferably 350-550 rpm; the temperature of the first mixing is preferably 45-80 ℃, more preferably 60-80 ℃, and most preferably 80 ℃; the first mixing time is preferably 30-360 min, and more preferably 60-240 min; the second mixing temperature is preferably 25-35 ℃, and the time is preferably 10-60 min. In the present invention, the deep eutectic solvent is a stable, uniform and transparent liquid. The deep eutectic solvent provided by the invention is an extracting agent with strong polarity, can efficiently break the cell walls of the wild rice and promote the dissolution of the flavonoid compounds in the wild rice, is nontoxic, biodegradable, green and environment-friendly, can not produce secondary pollution, can be recycled, greatly reduces the extraction cost, and has high extraction efficiency and low detection limit on the flavonoid compounds in the wild rice.

In the present invention, the stirring is preferably performed using an IKA magnetic stirrer (IKA, Germany).

In the invention, the material-to-liquid ratio of the wild rice powder to the deep eutectic solvent is preferably 10-100 mg/mL, more preferably 20-100 mg/mL, and most preferably 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL or 80 mg/mL.

In the present invention, the balance used for weighing is preferably a BSA124S-CW electronic balance (sensitivity 0.0001g, Sidorist scientific instruments Beijing, Ltd., China).

In the present invention, the extraction manner is preferably ultrasonic extraction. In the invention, the extraction temperature is preferably 30-60 ℃, more preferably 40-60 ℃, and most preferably 50 ℃; the extraction time is preferably 1-20 min, more preferably 5-15 min, and most preferably 10 min. In the invention, the power of ultrasonic extraction is preferably 120-600W, more preferably 150-500W, and most preferably 200W, 300W or 400W; the frequency of ultrasonic extraction is preferably 20-25 kHz, more preferably 21-25 kHz, and most preferably 22kHz, 23kHz or 24 kHz. In the present invention, the ultrasonic extraction is preferably performed in a KQ-500GVDV type double-pin constant temperature numerical control ultrasonic generator (ultrasonic instruments Co., Ltd., China, Kunshan city).

In the invention, the solid-liquid separation mode is preferably centrifugal separation, and the rotation speed of the centrifugal separation is preferably 2500-3000 rpm, more preferably 2600-3000 rpm, and most preferably 3000 rpm; the time for centrifugal separation is preferably 5-10 min, and more preferably 10 min.

After the flavone extracting solution is obtained, the invention carries out liquid chromatography separation on the flavone extracting solution to obtain the flavonoid compound.

In the present invention, the flavonoid compound preferably includes catechin, procyanidin and quercetin.

In the invention, the flavone extracting solution is preferably diluted by methanol, filtered and then subjected to liquid chromatography separation.

In the invention, the volume ratio of the flavone extracting solution to the methanol is preferably 1 (1-4), more preferably 1 (2-4), and most preferably 1 (3-4). In the present invention, the pore size of the filtration membrane is preferably 0.22. mu.m.

In the present invention, the detection conditions of the liquid chromatography include: the mobile phase A is preferably an acetonitrile-acetic acid mixed solution, and the volume fraction of acetic acid in the acetonitrile-acetic acid mixed solution is preferably 0.1%; the mobile phase B is preferably an aqueous solution of acetic acid, the volume fraction of acetic acid in the aqueous solution of acetic acid is preferably 0.1%; the gradient elution procedure is preferably: gradient elution procedure: increasing the volume percentage of the mobile phase A within 0-5 min from 5% to 10%, increasing the volume percentage of the mobile phase A within 5-7 min from 10% to 20%, increasing the volume percentage of the mobile phase A within 7-8 min from 20% to 60%, increasing the volume percentage of the mobile phase A within 8-9 min from 60% to 100%, decreasing the volume percentage of the mobile phase A within 9-10 min from 100% to 5%, and keeping the volume percentage of the mobile phase A within 10-12 min at 95%; the flow rate is 0.3 mL/min; the sample injection amount is 1 mu L; the temperature of the chromatographic column is 25 ℃; the flow rate is preferably 0.3 mL/min; the sampling amount is preferably 1 mu L; the column is preferably an Acquity UPLC BEH C18 column (50 mm. times.2.1 mm, 1.7 μm) and the column temperature is preferably 25 ℃.

In the present invention, in order to confirm the structure of the 5 separated flavonoids, it is preferable to perform detection by mass spectrometry; further preferably, the separation and detection are carried out by liquid chromatography-mass spectrometry (HPLC-MS), and the instrument used is preferably a Waters liquid Mass spectrometer (Waters Corp., USA).

In the present invention, the detection conditions of the mass spectrum include: the preferred negative ionization ESI (-) is, the mass scanning range is preferably 105-1500 m/z, the capillary voltage is preferably 3000V, the cone hole voltage is preferably 25V, the ion source temperature is preferably 100 ℃, the atomizing gas temperature is preferably 200 ℃, the cone hole gas flow rate is preferably 50L/h, and the atomizing gas flow rate is preferably 400L/h.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

(1) Preparation of deep eutectic solvent

Choline chloride and 1, 4-butanediol are added into a round-bottom flask according to a molar ratio of 1:6, magnetons are added into the round-bottom flask, a magnetic stirring is carried out for 60min at 80 ℃ by adopting an IKA magnetic stirrer to obtain a choline chloride-1, 4-butanediol mixed solution, and 30 wt% of water is added into the mixed solution to obtain a deep eutectic solvent (DES-1).

(2) Pretreatment of wild rice

Grinding fructus Zizaniae Caduciflorae with pulverizer, drying at 40 deg.C until weight is constant, and storing the obtained fructus Zizaniae Caduciflorae powder at-20 deg.C in dark for use.

(3) Analysis of wild rice flour

Weighing 500mg of wild rice powder by adopting a BSA124S-CW electronic balance, adding 10mL of deep eutectic solvent, fully mixing, placing in a KQ-500GVDV type double-spliced constant-temperature numerical control ultrasonic generator, carrying out ultrasonic extraction for 15min under the conditions of 50 ℃, 200W of ultrasonic power and 25kHz of ultrasonic frequency to obtain a solid-liquid mixture, cooling the solid-liquid mixture to room temperature, placing in a centrifugal machine, centrifuging for 10min at 3000rpm to obtain a supernatant fluid, namely a flavonoid compound extracting solution, mixing 1mL of the flavonoid compound extracting solution with 4mL of methanol, and filtering the obtained mixed solution through a 0.22 mu m filter membrane to obtain a brass extracting solution.

(4) HPLC-MS separation and detection

And (3) carrying out HPLC-MS detection on the brass extracting solution.

Wherein, the liquid chromatography conditions are as follows: mobile phase a (0.1% acetic acid in acetonitrile, v/v) and mobile phase B (0.1% aqueous acetic acid, v/v); the gradient elution procedure was as follows: 0-5 min, 5-10% of mobile phase A; 5-7 min, 10-20% of mobile phase A; 7-8 min, 20-60% of mobile phase A; 9-10 min, 100-5% of mobile phase A; 10-12 min, 95% of mobile phase A; the flow rate is preferably 0.3 mL/min; the sampling amount is preferably 1 mu L; the chromatographic column is Acquity UPLC BEH C18 column (50mm × 2.1mm, 1.7 μm), and the chromatographic column temperature is 25 deg.C; the HPLC-MS separation and detection instrument is preferably a Waters liquid phase mass spectrometer (Waters corporation, USA);

mass spectrum conditions: negative ionization ESI (-); the mass scanning range m/z is 105-1500; the capillary voltage is 3000V; the voltage of the taper hole is 25V; the ion source temperature is 100 ℃; the temperature of atomizing gas is 200 ℃; the air flow rate of the taper hole is 50L/h; the atomization airflow rate is 400L/h.

(5) Preparation of Standard solutions

Mixing catechin and methanol to prepare a standard solution with the concentration of 0.5-25 mug/mL;

mixing procyanidine and methanol to prepare a standard solution with the concentration of 0.5-20 mug/mL;

mixing quercetin and methanol to prepare a standard solution with the concentration of 0.5-20 mug/mL;

and (4) refrigerating the standard solution at 4 ℃ for later use.

Liquid mass spectrograms of catechin, procyanidine and quercetin in the flavonoid extract are shown in figure 1, and mass spectrometric parameter results of catechin, procyanidine and quercetin are shown in table 1.

TABLE 1 Mass Spectrometry parameters of Flavonoids

As can be seen from fig. 1 and table 1, the retention times of procyanidin, catechin, and quercetin were 2.65min, 3.2min, and 8.18min, respectively, and the three substances were highly separated; and 3 kinds of flavone parent ions and daughter ions are consistent with the standard substance, and the separation is proved to obtain procyanidine, catechin and quercetin.

Table 2 shows the results of detection of 5 kinds of flavonoids, catechin, procyanidin, and quercetin.

TABLE 2 flavonoid test results

As can be seen from Table 2, the linear range of catechin is 0.5-25 μ g/g, the linear range of procyanidin and quercetin is 0.5-20 μ g/g, the detection limits of catechin, procyanidin and quercetin are 9.747ng/mL, 4.360ng/mL and 0.479ng/mL respectively, the linear range of three flavonoids is wide, the detection limit is low, the linearity is good, R is²All are more than 0.99, the method is reliable and the reproducibility is good.

Examples 2 to 3

The method of example 1 was used to extract flavonoids and the test was conducted, and the differences from example 1 were that the deep eutectic solvents were different, the compositions of the deep eutectic solvents in examples 1 to 3 are shown in table 3, and the extraction amounts of 3 flavonoids, i.e., catechin, procyanidin and quercetin, are shown in table 4.

TABLE 3 compositions of deep eutectic solvents in examples 1-7

Comparative example 1

The extraction of flavonoids and the detection by HPLC-MS were carried out according to the method of example 1, differing from example 1 in that water was used instead of the deep eutectic solvent. The extraction amounts of 3 flavonoids, catechin, procyanidin and quercetin, are shown in table 4.

Comparative example 2

The extraction of flavonoids and their detection by HPLC-MS were carried out as in example 1, with the difference from example 1 that anhydrous ethanol was used instead of the deep eutectic solvent. Table 4 shows the amounts of extracts of 3 kinds of flavonoids, catechin, procyanidin, and quercetin.

Comparative example 3

Flavonoid was extracted according to the method of example 1 and detected by HPLC-MS, except that 70% ethanol aqueous solution (v/v) was used instead of deep eutectic solvent in example 1. Table 4 shows the amounts of extraction of 3 kinds of flavonoids, namely, catechin, procyanidin and quercetin.

TABLE 4 extraction amounts (. mu.g/g) of the flavonoids in examples 1 to 3 and comparative examples 1 to 3

As can be seen from table 4, in the extraction process of catechins, choline chloride: 1, 4-butanediol deep eutectic solvent, choline chloride: glycerol deep eutectic solvent and choline chloride: the extraction effect of the lactic acid deep eutectic solvent is better than that of 70 percent methanol, 70 percent acetone and 70 percent ethanol.

In the extraction process of the procyanidin, choline chloride: 1, 4-butanediol deep eutectic solvent and choline chloride: glycerol deep eutectic solvent and choline chloride: the extraction effect of the lactic acid deep eutectic solvent is better than that of 70 percent methanol, 70 percent acetone and 70 percent ethanol. Choline chloride: the extraction effect of the lactic acid deep eutectic solvent is equivalent to that of 70% ethanol.

In the extraction process of the quercetin, the content of choline chloride: 1, 4-butanediol deep eutectic solvent, choline chloride: glycerol deep eutectic solvent and choline chloride: the extraction effect of the lactic acid deep eutectic solvent is better than that of 70 percent methanol, 70 percent acetone and 70 percent ethanol.

The DPPH radical scavenging ability of the flavonoids extracted in examples 1-3 is shown in FIG. 2. As can be seen from FIG. 2, the DPPH radical clearance rates of the flavonoids extracted in examples 1 to 3 were 89. + -. 3.1%, 75. + -. 2.5% and 46. + -. 1.7%, respectively. The flavonoid compound extracted by the method provided by the invention has excellent DPPH free radical scavenging capacity.

The ABTS free radical scavenging ability of the flavonoids extracted in examples 1-3 is shown in FIG. 3. As can be seen from FIG. 3, the ABTS free radical clearance rates of the flavonoids extracted in examples 1-3 were 85. + -. 2.9%, 67. + -. 2.5% and 43. + -. 1.7%, respectively. The flavonoid compound extracted by the method provided by the invention has excellent capability of eliminating ABTS free radicals.

Examples 4 to 8

Flavonoid compounds were extracted by the method of example 1, and the extraction conditions of examples 4 to 8 are shown in table 5.

Comparative example 4

Flavonoid compounds were extracted by the method of example 1, except that the water content of the deep eutectic solvent was 0% and the extraction conditions of comparative example 4 were as shown in table 5, as compared with example 1.

TABLE 5 extraction conditions for examples 4-8 and comparative example 4

The total flavone extraction amount of the zizania indica rice extracted in examples 4 to 8 and comparative example 4 is shown in fig. 4. As can be seen from FIG. 4, when the water content of the Deep Eutectic Solvent (DES) is increased within the range of 0 wt% to 50 wt%, the total flavone extraction amount in the wild rice tends to increase first and then decrease, and when the water content is 30 wt%, the total flavone extraction amount in the wild rice reaches the maximum value, at which time the total flavone extraction amount in the wild rice is (6.61 + -0.01) mg/g.

Examples 9 to 12

Flavonoid compounds were extracted by the method of example 1, and the extraction conditions of examples 9 to 12 are shown in table 6.

TABLE 6 extraction conditions of examples 9 to 12

The total flavone extraction amount of the wild rice extracted in examples 9 to 12 is shown in fig. 5. As can be seen from fig. 5, when the extraction temperature is increased within the range of 30-50 ℃, the extraction amount of total flavonoids in the zizania indica rice tends to increase, and the extraction amount of total flavonoids in the zizania indica rice reaches the maximum value at 50 ℃, at this time, the extraction amount of total flavonoids in the zizania indica rice is (6.38 ± 0.10) mg/g; and then, the extraction amount of the total flavonoids in the wild rice is slightly reduced with the temperature rise, but the difference is not obvious (p is more than 0.05).

Examples 13 to 17

Flavonoid compounds were extracted by the method of example 1, and the extraction conditions of examples 13 to 17 are shown in table 7.

TABLE 7 extraction conditions of examples 13 to 17

The total flavone extraction amount of the wild rice extracted in examples 13 to 17 is shown in fig. 6. As can be seen from FIG. 6, when the extraction time is increased within the range of 1-10 min, the total flavone extraction amount is in an upward trend, and when the extraction time is 10min, the total flavone extraction amount reaches the maximum value of (7.64 +/-0.10) mg/g; the total flavone extraction decreased slightly with time, but the difference was not significant (p > 0.05).

Examples 18 to 27

Flavonoid compounds were extracted by the method of example 1, and the extraction conditions of examples 18 to 27 are shown in table 8.

TABLE 8 extraction conditions of examples 18 to 27

The total flavone extraction amount of the wild rice extracted in examples 18 to 27 is shown in fig. 7. As can be seen from FIG. 7, the total flavone extraction amount is in an increasing trend when the feed-liquid ratio is in the range of 10-40 mg/mL; when the feed-liquid ratio is increased within the range of 40-100 mg/mL, the extraction amount of the total flavone is in a descending trend. From the aspects of saving the solvent, controlling the extraction cost and the like, the material-liquid ratio is selected to be 40mg/mL, and the total flavone extraction amount is (4.25 +/-0.13) mg/g at the moment.

In conclusion, the deep eutectic solvent adopted by the invention can replace toxic organic solvents such as methanol, chloroform and the like extracted traditionally, effectively reduces the environmental pollution in the extraction process, is green and environment-friendly, greatly reduces the extraction cost, and is suitable for industrial mass production.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for extracting flavonoid compounds in wild rice by using a deep eutectic solvent is characterized by comprising the following steps:

pretreating wild rice to obtain wild rice powder;

diluting the flavone extract with methanol, filtering, and performing liquid chromatography to obtain flavone compounds;

the deep eutectic solvent is choline chloride, a hydrogen bond donor and water; the content of water in the deep eutectic solvent is 10-50 wt%;

the feed-liquid ratio of the wild rice powder to the deep eutectic solvent is 10-100 mg/mL;

the hydrogen bond donor is selected from 1, 4-butanediol, glycerol or lactic acid;

the molar ratio of choline chloride to a hydrogen bond donor in the deep eutectic solvent is 1: (0.5 to 7);

the extraction temperature is 30-60 ℃, and the extraction time is 1-20 min;

the conditions of the liquid chromatographic separation include: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetonitrile-acetic acid mixed solution, and the volume fraction of the acetic acid is 0.1%; the mobile phase B is an aqueous solution of acetic acid, and the volume fraction of the acetic acid is 0.1%;

2. The method according to claim 1, wherein the particle size of the wild rice powder is 40-100 mesh.

3. The method of claim 1, wherein the extraction is by ultrasound extraction.

4. The method according to claim 3, wherein the power of the ultrasonic extraction is 120-600W, and the frequency is 20-25 kHz.

5. The method according to claim 1, wherein the volume ratio of the flavone extract to the methanol is 1 (1-4);

the pore diameter of the filter membrane for filtration is 0.22 μm.