CN110974911A - Method for extracting phenolic acid compounds from wild rice by using deep eutectic solvent - Google Patents
Method for extracting phenolic acid compounds from wild rice by using deep eutectic solvent Download PDFInfo
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- CN110974911A CN110974911A CN201911157021.3A CN201911157021A CN110974911A CN 110974911 A CN110974911 A CN 110974911A CN 201911157021 A CN201911157021 A CN 201911157021A CN 110974911 A CN110974911 A CN 110974911A
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- eutectic solvent
- deep eutectic
- wild rice
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- 239000002904 solvent Substances 0.000 title claims abstract description 80
- 230000005496 eutectics Effects 0.000 title claims abstract description 79
- 241000746966 Zizania Species 0.000 title claims abstract description 66
- 235000002636 Zizania aquatica Nutrition 0.000 title claims abstract description 65
- -1 phenolic acid compounds Chemical class 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 46
- 238000000605 extraction Methods 0.000 claims abstract description 77
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims abstract description 42
- 235000019743 Choline chloride Nutrition 0.000 claims abstract description 42
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims abstract description 42
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- 150000007965 phenolic acids Chemical class 0.000 claims abstract description 37
- 238000000926 separation method Methods 0.000 claims abstract description 25
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- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 description 11
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- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 2
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention relates to the technical field of natural substance extraction, in particular to a method for extracting phenolic acid compounds in wild rice by utilizing a deep eutectic solvent. The invention provides a method for extracting phenolic acid compounds from 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 phenolic acid extracting solution; carrying out liquid chromatography separation on the phenolic acid extracting solution to obtain a phenolic acid compound; the deep eutectic solvent comprises choline chloride, a hydrogen bond donor and water; the hydrogen bond donor comprises D-fructose, D-glucose, DL-malic acid or urea. 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 phenolic acid compounds in the wild rice.
Description
Technical Field
The invention relates to the technical field of natural substance extraction, in particular to a method for extracting phenolic acid 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, phenolic acid compounds separated from wild rice mainly comprise ferulic acid, sinapic acid, vanillic acid, procyanidine, catechin, quercetin and the like; the extraction method of phenolic acid 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 efficiency of extracting the phenolic acid compounds from the wild rice and low in detection limit.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for extracting phenolic acid compounds from 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 phenolic acid extracting solution;
carrying out liquid chromatography separation on the phenolic acid extracting solution to obtain a phenolic acid compound;
the deep eutectic solvent comprises choline chloride, a hydrogen bond donor and water;
the hydrogen bond donor comprises D-fructose, D-glucose, DL-malic acid or urea.
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 6);
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 extracting solution with methanol and then filtering the phenolic acid extracting solution before the liquid chromatography separation.
Preferably, the volume ratio of the phenolic acid 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 phenolic acid compounds from 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 phenolic acid extracting solution; carrying out liquid chromatography separation on the phenolic acid extracting solution to obtain a phenolic acid compound; the deep eutectic solvent comprises choline chloride, a hydrogen bond donor and water; the hydrogen bond donor comprises D-fructose, D-glucose, DL-malic acid or urea, wherein choline chloride and fructose are both naturally derived molecules, and the ratio of choline chloride: the phenolic acid extracting solution obtained by using the D-fructose as the deep eutectic solvent has higher inoxidizability and good biocompatibility, and the phenolic acid extracting solution can be directly applied to the fields of food and medicines without subsequent purification and separation. 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 wild rice phenolic acid compounds, 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 phenolic acid compounds in the wild rice.
Drawings
FIG. 1 is a liquid phase mass spectrum of 9 phenolic acid compounds;
FIG. 2 is a diagram showing the DPPH radical scavenging ability of the phenolic acid compounds extracted in examples 1 to 4;
FIG. 3 is a diagram of 9 phenolic acid compounds in wild rice extracted in examples 5 to 9 and comparative example 4;
FIG. 4 is a total extraction amount chart of 9 phenolic acid compounds in the wild rice extracted in examples 10 to 13;
FIG. 5 is a total extraction amount chart of 9 phenolic acid compounds in the wild rice extracted in examples 14 to 18;
FIG. 6 is a graph showing the total amount of 9 phenolic acid compounds extracted from the wild rice extracted in examples 19 to 28.
Detailed Description
The invention provides a method for extracting phenolic acid compounds from 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 phenolic acid extracting solution;
carrying out liquid chromatography separation on the phenolic acid extracting solution to obtain a phenolic acid compound;
the deep eutectic solvent comprises choline chloride, a hydrogen bond donor and water;
the hydrogen bond donor comprises D-fructose, D-glucose, DL-malic acid or urea
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 ℃; in the present invention, the drying time is not particularly limited, and the drying time may be set to a 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 subjected to solid-liquid separation after extraction by using a deep eutectic solvent to obtain a phenolic acid extracting solution; the deep eutectic solvent comprises choline chloride, a hydrogen bond donor and water; the hydrogen bond donor comprises D-fructose, D-glucose, DL-malic acid or urea.
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 6), more preferably 1: (1-5), most preferably 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:2, 1:3, 1:4 or 1: 5. 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 a strong-polarity extractant, can efficiently break the cell wall of the wild rice and promote the dissolution of phenolic acid compounds of the wild rice, is nontoxic, biodegradable, free from secondary pollution, green and environment-friendly, can be recycled, greatly reduces the extraction cost, and has high extraction efficiency and low detection limit on the phenolic acid 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 5-20 min, and most preferably 10min or 15 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 phenolic acid extracting solution is obtained, the phenolic acid extracting solution is subjected to liquid chromatography separation to obtain the phenolic acid compound.
In the present invention, the phenolic acid compound preferably includes vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, syringic acid, ferulic acid, sinapic acid, and vanillic acid.
In the invention, the phenolic acid extracting solution is preferably diluted by methanol, filtered and then subjected to liquid chromatography separation.
In the invention, the volume ratio of the phenolic acid 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 conditions of the liquid chromatography 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 is preferably AcquisyUPLC 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 9 isolated phenolic acid compounds, 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 D-fructose are added into a round-bottom flask according to the molar ratio of 1:1, magnetons are added into the round-bottom flask, an IKA magnetic stirrer is adopted to magnetically stir for 180min at the temperature of 80 ℃ to obtain a choline chloride-fructose 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, namely a phenolic acid compound extracting solution, mixing 1mL of the phenolic acid compound extracting solution with 4mL of methanol, and filtering the obtained mixed solution through a 0.22 mu m filter membrane to obtain a solution to be detected.
(4) HPLC-MS separation and detection
And carrying out HPLC-MS detection on the solution to be detected.
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 AcquisyUPLC BEH C18 column (50mm × 2.1mm, 1.7 μm) and the temperature of the chromatographic column 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 flow speed of the taper hole gas mobile phase is 50L/h; the flow rate of the atomizing gas mobile phase was 400L/h.
(5) Preparation of Standard solutions
Mixing vanillin and methanol to prepare a standard solution with the concentration of 0.3-30 mu g/mL;
mixing p-hydroxybenzaldehyde and methanol to prepare a standard solution with the concentration of 0.2-20 mug/mL;
mixing p-hydroxybenzoic acid and methanol to prepare a standard solution with the concentration of 0.2-22 mug/mL;
mixing p-coumaric acid and methanol to prepare a standard solution with the concentration of 0.05-5 mug/mL;
mixing protocatechuic acid and methanol to prepare a standard solution with the concentration of 0.2-20 mug/mL;
mixing syringic acid and methanol to prepare a standard solution with the concentration of 0.02-10 mug/mL;
mixing ferulic acid and methanol to prepare a standard solution with the concentration of 0.5-120 mug/mL;
mixing sinapic acid and methanol to prepare a standard solution with the concentration of 0.5-120 mu g/mL;
mixing vanillic acid and methanol to prepare a standard solution with the concentration of 0.2-20 mu g/mL.
The liquid phase mass spectrum of vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, syringic acid, ferulic acid, sinapic acid and vanillic acid in the phenolic acid compound extract is shown in figure 1, and the mass spectrum parameter results are shown in table 1.
TABLE 1 Mass Spectrometry parameters of nine phenolic acids
As can be seen from fig. 1 and table 1, the retention times of vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, syringic acid, ferulic acid, sinapic acid and vanillic acid are 5.02min, 3.76min, 2.60min, 5.82min, 1.57min, 4.07min, 6.68min, 6.84min and 3.38min, respectively, and the separation degrees of 9 substances are high; and the 9 phenolic acid parent ions and the 9 phenolic acid daughter ions are consistent with the standard substances, which proves that vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, syringic acid, ferulic acid, sinapic acid and vanillic acid are obtained by separation.
The results of testing nine phenolic compounds, vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, syringic acid, ferulic acid, sinapic acid and vanillic acid, are shown in Table 2.
TABLE 2 detection results of nine phenolic acid compounds
As can be seen from Table 2, the polyphenols had a wide linear range and a low detection limit, and the method of the present invention had good linearity, R2All are more than 0.99, the method is reliable and the reproducibility is good.
Examples 2 to 4
The phenolic acid compounds were extracted and tested according to the method of example 1, the difference from example 1 is that the deep eutectic solvent is different, the composition of the deep eutectic solvent in examples 1 to 4 is shown in table 3, and the extraction amounts of nine phenolic acid compounds, i.e., vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, syringic acid, ferulic acid, sinapic acid and vanillic acid, are shown in table 3.
TABLE 3 compositions of deep eutectic solvents in examples 1-4
Comparative example 1
The phenolic compounds were extracted and tested by HPLC-MS according to the method of example 1, except that 70% methanol aqueous solution (v/v) was used instead of the deep eutectic solvent in example 1. The extraction amounts of nine phenolic acid compounds, i.e., vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, syringic acid, ferulic acid, sinapic acid, and vanillic acid, are shown in Table 4.
Comparative example 2
The phenolic compounds were extracted and tested by HPLC-MS according to the method of example 1, except that 70% ethanol aqueous solution (v/v) was used instead of the deep eutectic solvent in example 1. The extraction amounts of nine phenolic acid compounds, i.e., vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, syringic acid, ferulic acid, sinapic acid, and vanillic acid, are shown in Table 4.
Comparative example 3
Phenolic compounds were extracted and tested by HPLC-MS according to the method of example 1, except that 70% acetone aqueous solution (v/v) was used instead of the deep eutectic solvent in example 1. The extraction amounts of 9 phenolic compounds, i.e., vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, syringic acid, ferulic acid, sinapic acid, and vanillic acid, are shown in Table 4.
TABLE 4 extraction amounts (. mu.g/g) of nine phenolic acid compounds in examples 1 to 4 and comparative examples 1 to 3
As can be seen from table 4, in the extraction of vanillin, choline chloride: the extraction effect of the D-fructose deep eutectic solvent is better than that of 70 percent methanol and 70 percent acetone, choline chloride: DL-malic acid deep eutectic solvent and choline chloride: the extraction effect of the urea deep eutectic solvent is better than that of 70 percent acetone.
In the process of extracting the p-hydroxybenzaldehyde, choline chloride: the extraction effect of the DL-malic acid deep eutectic solvent is better than that of 70% methanol, 70% ethanol and 70% acetone; choline chloride: the D-fructose deep eutectic solvent is better than 70 percent methanol.
In the process of extracting the p-hydroxybenzoic acid, choline chloride: the extraction effect of the D-fructose deep eutectic solvent is better than that of 70% methanol, 70% ethanol and 70% acetone, choline chloride: d-glucose deep eutectic solvent, choline chloride: DL-malic acid deep eutectic solvent and choline chloride: the extraction effect of the urea deep eutectic solvent is better than that of 70 percent methanol and 70 percent acetone.
In the process of extracting p-coumaric acid, choline chloride: the extraction effect of the D-fructose deep eutectic solvent is better than that of 70% methanol, 70% ethanol and 70% acetone; choline chloride: the extraction effect of the urea deep eutectic solvent is better than that of 70 percent methanol and 70 percent acetone.
In the process of extracting protocatechuic acid, choline chloride: the extraction effect of the urea deep eutectic solvent is better than that of 70% methanol, 70% ethanol and 70% acetone; choline chloride: the extraction effect of the D-fructose deep eutectic solvent is equivalent to that of 70% ethanol.
In the syringic acid extraction process, choline chloride: the extraction effect of the D-fructose deep eutectic solvent is superior to that of 70% ethanol and 70% acetone, and is equivalent to that of 70% methanol; choline chloride: d-glucose deep eutectic solvent and choline chloride: the extraction effect of the DL-malic acid deep eutectic solvent is better than that of 70% acetone.
In the process of extracting ferulic acid, choline chloride: d-fructose deep eutectic solvent and choline chloride: the extraction effect of the D-glucose deep eutectic solvent is better than that of 70% methanol, 70% ethanol and 70% acetone; choline chloride: the extraction effect of the urea deep eutectic solvent is equivalent to 70% methanol.
In the process of extracting sinapic acid, choline chloride: d-fructose deep eutectic solvent and choline chloride: the extraction effect of the D-glucose deep eutectic solvent is better than that of 70% methanol, 70% ethanol and 70% acetone; choline chloride: the extraction effect of the urea deep eutectic solvent is better than that of 70 percent methanol.
In the process of extracting vanillic acid, choline chloride: the extraction effect of the D-fructose deep eutectic solvent is better than that of 70% methanol, 70% ethanol and 70% acetone; choline chloride: d-glucose deep eutectic solvent and choline chloride: the extraction effect of the DL-malic acid deep eutectic solvent is better than that of 70% ethanol and 70% acetone.
The DPPH radical scavenging ability of the phenolic acid compounds extracted in examples 1 to 4 is shown in FIG. 2. As can be seen from FIG. 2, the DPPH radical clearance rates of the 9 phenolic acid compounds extracted in examples 1-4 are 97.2 + -1.6%, 81 + -1.3%, 54 + -1.2% and 48.6 + -1.5%, respectively, i.e. choline chloride: d-fructose deep eutectic solvent, choline chloride: d-glucose deep eutectic solvent, choline chloride: DL-malic acid deep eutectic solvent or choline chloride: the scavenging activity of the urea deep eutectic solvent to DPPH free radicals is decreased gradually.
Examples 5 to 9
The phenolic acid compounds were extracted according to the method of example 1, and the extraction conditions of examples 5 to 9 are shown in Table 5.
Comparative example 4
The phenolic acid compound was extracted according to 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 are shown in Table 5, as compared with example 1.
TABLE 5 extraction conditions for examples 5 to 9 and comparative example 4
The total extraction amount of 9 phenolic acid compounds in the wild rice extracted in examples 5 to 9 and comparative example 4 is shown in fig. 3. As can be seen from fig. 3, when the water content of the Deep Eutectic Solvent (DES) is increased within the range of 0 wt% to 50 wt%, the total extraction amount of phenolic acids in the wild rice tends to increase first and then decrease, and when the water content is 30 wt%, the total extraction amount of phenolic acids in the wild rice reaches the maximum value, at this time, the total extraction amount of phenolic acids in the wild rice is (4.74 ± 0.03) mg/g.
Examples 10 to 13
The phenolic acid compound was extracted by the method of example 1, and the extraction conditions of examples 10 to 13 are shown in Table 6.
TABLE 6 extraction conditions of examples 10 to 13
The total phenol extraction amount of the wild rice extracted in examples 10 to 13 is shown in fig. 4. As can be seen from fig. 4, when the extraction temperature is increased within the range of 30-50 ℃, the total extraction amount of 9 phenolic acid compounds in the wild rice tends to increase, and the total extraction amount of phenolic acids in the wild rice reaches the maximum value at 50 ℃, at this time, the total extraction amount of phenolic acids in the wild rice is (4.97 ± 0.12) mg/g; and then, the total extraction amount of the phenolic acid in the wild rice is slightly reduced along with the increase of the temperature.
Examples 14 to 18
The phenolic acid compounds were extracted according to the method of example 1, and the extraction conditions of examples 14 to 18 are shown in Table 7.
TABLE 7 extraction conditions of examples 14 to 18
The total extraction amount of 9 phenolic acid compounds in the wild rice extracted in examples 14 to 18 is shown in fig. 5. As can be seen from FIG. 5, the total phenolic acid extraction amount is in an upward trend when the extraction time is increased within the range of 1-15 min, and the maximum total phenolic acid extraction amount reaches (4.83. + -. 0.08) mg/g when the extraction time is 15 min; then, the total extraction amount of phenolic acid is slightly reduced along with the prolonging of time.
Examples 19 to 28
The phenolic acid compound was extracted by the method of example 1, and the extraction conditions of examples 19 to 28 are shown in Table 8.
TABLE 8 extraction conditions of examples 19 to 28
The total phenol extraction amount of the wild rice extracted in examples 19 to 28 is shown in fig. 6. As can be seen from FIG. 6, the feed-to-liquid ratio is in the range of 10-50 mg/mL, and the total extraction amount of phenolic acids of 9 phenolic acid compounds is increasing; when the feed-liquid ratio is increased within the range of 50-100 mg/mL, the total extraction amount of phenolic acid 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 50mg/mL, and the total extraction amount of the phenolic acid is (4.87 +/-0.02) mg/g.
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 (10)
1. A method for extracting phenolic acid compounds from wild rice by using a deep eutectic solvent is characterized by comprising 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 phenolic acid extracting solution;
carrying out liquid chromatography separation on the phenolic acid extracting solution to obtain a phenolic acid compound;
the deep eutectic solvent comprises choline chloride, a hydrogen bond donor and water;
the hydrogen bond donor comprises D-fructose, D-glucose, DL-malic acid or urea.
2. The method according to claim 1, wherein the particle size of the wild rice powder is 40-100 mesh.
3. The process according to claim 1, characterized in that the molar ratio of choline chloride and hydrogen bond donor in the deep eutectic solvent is 1: (0.5 to 6);
the content of water in the deep eutectic solvent is 10-50 wt%.
4. The method according to claim 1 or 3, wherein the stock-to-solution ratio of the wild rice powder to the deep eutectic solvent is 10-100 mg/mL.
5. The method of claim 1, wherein the extraction is by ultrasound extraction.
6. The method of claim 5, wherein the power of the ultrasonic extraction is 120-600W, and the frequency is 20-25 kHz.
7. The method according to claim 1, 5 or 6, wherein the temperature of the extraction is 30-60 ℃ and the time is 1-20 min.
8. The method of claim 1, further comprising, prior to the liquid chromatography, diluting the phenolic acid extract with methanol and filtering.
9. The method according to claim 8, wherein the volume ratio of the phenolic acid extracting solution to the methanol is 1 (1-4);
the pore diameter of the filter membrane for filtration is 0.22 μm.
10. The method of claim 1 or 8, wherein the conditions of the liquid chromatographic separation comprise: 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 ℃.
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