Method for extracting and purifying anthocyanin and procyanidine substances

Abstract

The invention relates to a method for extracting and purifying anthocyanidin and proanthocyanidin substances based on gel permeation chromatography, which comprises the following steps: step 1, preparing an extracting solution containing a mixture of anthocyanidin and proanthocyanidin from agricultural products with high content of anthocyanidin substances by using an intermittent vacuum microwave technology, adding into a gel permeation chromatography sample inlet with a photodiode array detector; step 2, eluting and recovering by using eluents of the first fraction and the second fraction in different proportions respectively, and collecting distillate of the first fraction at a 515nm ultraviolet-visible spectrum; and step 3, testing the purity and oxidation resistance of the anthocyanin in the first-fraction elution recovery liquid and the purity and oxidation resistance of the proanthocyanidins in the second-fraction elution recovery liquid to obtain purification conditions. Specific to anthocyanin and procyanidine substances in agricultural products, a special gel chromatographic column is selected for chromatographic fractionation, the effect of separating water-soluble components is good, and the biological activity of molecules can be kept.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical extraction, and in particular relates to a method for extracting and purifying anthocyanidins and proanthocyanidins. Background Art

[0002] Anthocyanidins are polyphenolic red to blue pigments found in many foods. Studies have shown that anthocyanidins are easily absorbed in the body, play a role in the body's antioxidant defense system, participate in the body's cardioprotection and chemoprevention mechanisms, and have multiple functions such as improving vision, preventing obesity and diabetes, and reducing oxidative stress. Proanthocyanidins, commonly known as condensed tannins or oligomeric proanthocyanidins, are polymers with multiple biological activities such as antioxidant, anti-inflammatory and anti-tumor. Anthocyanidins can be obtained by hydrolysis of proanthocyanidins. Water-soluble anthocyanidins and proanthocyanidins are widely found in agricultural products with corn, rice, soybeans, grains, tomatoes, eggplants, peppers, blueberries, blue indigo fruit, grape seeds, grape skins, sea buckthorn, rose hips, etc. as the main raw materials.

[0003] Gel Permeation Chromatography (GPC) is a method for separating, purifying and analyzing mixtures. An organic solvent is eluted through a porous bead column at a steady rate, and a small amount of the sample of interest is introduced into the solvent at the head of the column. The components pass through the column at different rates and are separated. An important characteristic of the column filler is that the molecules are separated mainly according to their size. The most obvious material with different molecular sizes is a polydisperse polymer, and the instrument used for gel permeation chromatography is mainly a GPC instrument. The GPC instrument mainly consists of the following key parts: a pump, which is used to push the solvent through the entire system; an inlet, which is responsible for introducing the sample to be tested into the chromatographic column; a chromatographic column, which contains a stationary phase; one or more detectors, which are used to detect the components flowing out of the chromatographic column; and control software, which regulates the various components of the instrument, calculates and displays the final results. GPC can well separate the components in a polymer mixture, and has a wide range of applications in the separation and purification of natural polymers such as lignin, proteins and polysaccharides, and can effectively separate oligomers and small molecules.

[0004] Currently, the commonly used extraction methods for anthocyanins and proanthocyanidins include solvent extraction, ultrasound-assisted extraction and microwave-assisted extraction. Traditional organic solvent extraction has problems such as environmental pollution, safety risks and solvent residues. Its flammable and explosive characteristics increase operational risks and may remain in the final product, posing a threat to consumer health.

[0005] Microwaves are electromagnetic waves with a frequency of 300MHz to 300GHz and a wavelength of 1mm to 1m, which is between infrared and radio waves and belongs to high-frequency electromagnetic waves. Microwave heating basically belongs to dipole polarization heating. Due to the heterogeneity of the components of agricultural products containing anthocyanins and proanthocyanidins, microwaves penetrate into the material and interact with the polar molecules of the material. After the polar molecules (such as water molecules) in the material absorb microwave energy, they change their original molecular structure and collide with each other frequently, generating a large amount of frictional heat, thereby generating many hot spots in the material. The water molecules on the hot spots in the material absorb heat and evaporate, generating a "steam explosion effect" inside the material, breaking its structure, promoting cell rupture, and allowing anthocyanins and proanthocyanidins to better seep into the extract, thereby increasing the yield. At the same time, the non-thermal effect of the electromagnetic field generated by microwave radiation can also accelerate the destruction of the structure of agricultural product materials.

[0006] Microwave-assisted extraction has the advantages of reducing production costs, shortening the process flow, and not polluting the environment. However, this simple extraction technology still has obvious shortcomings: due to the very fast heating speed of microwaves, the internal temperature of the material rises instantaneously during the processing of agricultural products, which can easily cause overheating or burns to the material. The material temperature generally exceeds 100°C, affecting the biological activity of anthocyanins and proanthocyanidins. For this reason, the liquid-to-solid ratio used in microwave extraction technology is 40-100:1, the water consumption is too large, and the corresponding cost increases.

[0007] Commonly used methods for purifying anthocyanins and proanthocyanidins include macroporous resin purification, solid phase extraction, preparative high performance liquid chromatography, membrane separation, high-speed countercurrent chromatography, etc. Macroporous resin purification has the advantages of easy use, simple facilities, low cost, etc. This method is the main method for purifying anthocyanins, but the purity of the product is relatively low, generally around 80%. Solid phase extraction has the advantages of short separation time, simple operation, and low air pollution, but because the number of separated samples is small, it is not suitable for large-scale application. Preparative high performance liquid chromatography, membrane separation, and high-speed countercurrent chromatography have the advantages of rapidity, high sensitivity, and high purity, but the composition of the separation solvent is relatively complex and expensive, the cost of machinery and equipment is relatively high, and the experimental conditions are relatively limited.

[0008] Therefore, a new method for extracting and purifying anthocyanins and proanthocyanidins is urgently needed. Summary of the invention

[0009] The purpose of the present invention is to address the problems existing in the above-mentioned prior art and to provide a method for purifying anthocyanidins and proanthocyanidins based on gel permeation chromatography technology.

[0010] The present invention discloses a method for purifying anthocyanidins and proanthocyanidins based on gel permeation chromatography, comprising the following steps:

[0011] Step 1: using intermittent vacuum microwave technology to prepare an extract containing a mixture of anthocyanins and proanthocyanidins from agricultural products with a high content of anthocyanidins, and adding the extract to the injection port of a gel permeation chromatography equipped with a photodiode array detector;

[0012] Step 2: using different ratios of the first fraction and the second fraction eluents for elution and recovery, respectively, collecting the distillate of the first fraction at 515 nm UV-visible spectrum;

[0013] Step 3, testing the purity and antioxidant properties of anthocyanins in the first fraction elution recovery solution, and the purity and antioxidant properties of proanthocyanidins in the second fraction elution recovery solution, to obtain purification conditions;

[0014] Step 4: The collected two fractions are dried under vacuum, and the samples are subjected to multiple fractionation.

[0015] Furthermore, in step 1, the liquid-to-solid ratio is 1:(10-30), and the heating interval time is: heating for 1-5 minutes, interval for 1-5 minutes, repeated 1-10 times, and the temperature is less than 60°C.

[0016] Furthermore, in step one, 10 g of agricultural products with a high content of anthocyanins are used as a substrate, distilled water is added according to a solid-liquid ratio of 1:10, and the mixture is subjected to reduced pressure microwave treatment at a temperature of 60° C. for 1 minute, with an interval of 1 minute, and repeated 5 times, and then filtered to obtain an extract.

[0017] Furthermore, in step 1, the gel permeation chromatography uses a Vantage L chromatographic column filled with gel HW-50F, with an inner diameter of 120 mm×12 mm.

[0018] Furthermore, in step 1, a peristaltic pump was used to adjust the flow rate to 1-5 ml min -1 .

[0019] Furthermore, in step three, the purification conditions of the first fraction of anthocyanins are: elution and collection with a mixture of 40-60 ml of ethanol, water and trifluoroacetic acid, and the volume ratio of ethanol, water and trifluoroacetic acid is (50-55):(45-50):(0.02-0.05).

[0020] Furthermore, in step 3, the purification conditions of the second fraction proanthocyanidins are: elution and collection with 30-50 ml of a mixture of acetone and water, and the volume ratio of acetone to water is (40-60): (40-60).

[0021] Beneficial Effects

[0022] The present invention targets sensitive biological molecules, such as anthocyanins and proanthocyanidins in agricultural products, and uses a chromatographic column with low pressure and a special gel having many hydroxyl groups and ether bonds on the surface and good hydrophilicity for chromatographic fractionation. Therefore, it has a better effect on separating water-soluble components, can maintain the biological activity of the molecules, and is convenient for subsequent functional applications of anthocyanins and proanthocyanidins.

[0023] Gel permeation chromatography (GPC) can separate and fractionate the extract, and the photodiode array detector can be used to test the separated liquid. GPC is used in combination with the photodiode array detector (DAD) technology to select the purified eluent according to the acidity, alkalinity, charge or affinity of the anthocyanin and proanthocyanidin polymer molecules. Anthocyanin substances have strong absorption at ultraviolet 515nm and can be recovered at this wavelength, allowing for more accurate separation and purification.

[0024] When intermittent control is used, during the period when microwave heating stops, the internal heat is used to supplement the energy consumed by surface evaporation, thereby reducing the internal temperature of the material and the temperature gradient. At the same time, it provides time for the redistribution of water inside the material, thus effectively preventing overheating, ensuring the biological activity quality of extracted anthocyanins and proanthocyanidins, and reducing the solid-liquid ratio. When microwave vacuum extraction is used, the degree of vacuum determines the evaporation temperature and evaporation rate of water in the material during the treatment process. Reasonable control of the vacuum degree can make effective use of microwaves and prevent the extraction temperature from being too high, which will destroy the biological activity of anthocyanins and proanthocyanidins.

[0025] The method is suitable for laboratory preparation and can also be applied to industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 For the present invention Figure 1 Chromatogram of GPC fractionation of blue lonicera edulis extract sample using HW-50F column.

[0027] Figure 2 It is the negative ion electrospray ionization (ESI) mass spectrum of the blue honeysuckle fraction F2 in the present invention.

[0028] Figure 3 The electron spin resonance spectra of different fractions F1 and F2 in the present invention in scavenging DPPH free radicals (1 is fraction F1, 2 is fraction F2, and 3 is DPPH).

[0029] Figure 4 The electron spin resonance spectra of different extracts in the present invention in scavenging DPPH free radicals are shown. DETAILED DESCRIPTION

[0030] The following combination Figures 1 to 4 This embodiment will be described in detail.

[0031] The present invention discloses a method for purifying anthocyanidins and proanthocyanidins based on gel permeation chromatography, comprising the following steps:

[0032] Step 1: Using intermittent vacuum microwave technology, prepare an extract containing a mixture of anthocyanins and proanthocyanidins from agricultural products with a high content of anthocyanin substances, and add the extract to the injection port of a gel permeation chromatography with a photodiode array detector; the gel permeation chromatography uses a Vantage L chromatographic column filled with gel HW-50F, with an inner diameter of 120 mm×12 mm, and uses a peristaltic pump to adjust the flow rate to 1-5 ml min -1 .

[0033] In intermittent vacuum microwave technology, agricultural products with high content of anthocyanins are treated under the conditions of liquid-to-solid ratio of 1:(10-30), heating interval time of: heating for 1-5 minutes, interval for 1-5 minutes, repeated 1-10 times, and temperature less than 60°C. The content of anthocyanins and proanthocyanidins and the antioxidant activity of the filtrate are tested after filtration. By comparing the conversion rate and antioxidant activity, the optimal conditions for intermittent vacuum microwave extraction are obtained.

[0034] Step 2: using different ratios of the first fraction and the second fraction eluents for elution and recovery, respectively, collecting the distillate of the first fraction at 515 nm UV-visible spectrum;

[0035] Step 3: Test the purity and antioxidant properties of anthocyanins in the first fraction elution recovery liquid, and the purity and antioxidant properties of proanthocyanidins in the second fraction elution recovery liquid to obtain purification conditions; the purification conditions of the first fraction anthocyanins are: eluting and collecting with a mixture of 40-60 ml of ethanol, water and trifluoroacetic acid, and the volume ratio of ethanol, water and trifluoroacetic acid is (50-55): (45-50): (0.02-0.05). The purification conditions of the second fraction proanthocyanidins are: eluting and collecting with a mixture of 30-50 ml of acetone and water, and the volume ratio of acetone and water is (40-60): (40-60).

[0036] Step 4: The collected two fractions are dried under vacuum, and the samples are subjected to multiple fractionation. Specific implementation method 1

[0038] The steps of this implementation are as follows:

[0039] Step 1: Take 5 g of tomato matrix with known moisture content (by dry weight), grind it with a plant grinder, add distilled water at a solid (by dry weight) to liquid ratio of 1:10, and treat it with a vacuum microwave at 60°C for 2 minutes. After a 2-minute break, repeat the intermittent heating for 5 times, filter, collect the filtrate, and obtain 2 ml of an extract containing a mixture of anthocyanins and proanthocyanidins. Add it to the injection port of a gel permeation chromatography (HLC-8420GPC) with a photodiode array detector (Agilent, G1315B). The gel permeation chromatography uses a Vantage L (120 mm × 12 mm inner diameter) column equipped with gel HW-50F, and use a peristaltic pump to adjust the flow rate to 1 ml min -1 .

[0040] Step 2: The first fraction (F1 anthocyanidins) was eluted with 60 ml of a 55:45:0.05 volume ratio ethanol / water / trifluoroacetic acid mixture, and the distillate was collected at 515 nm UV-visible spectrum. The second fraction (F2 proanthocyanidins) was eluted with 50 ml of a 60:40 volume ratio acetone / water mixture. Specific implementation method 2

[0042] The steps of this implementation are as follows:

[0043] Step 1: Take 10 g of eggplant matrix with known moisture content (by dry weight) and crush it with a plant crusher. Add distilled water at a solid (by dry weight) to liquid ratio of 1:15, vacuum microwave treatment at 50°C for 5 minutes, rest for 3 minutes, repeat intermittent heating 3 times, filter, collect the filtrate, and obtain 5 ml of extract containing a mixture of anthocyanins and proanthocyanidins. Add it to the injection port of a gel permeation chromatography (HLC-8420GPC) with a photodiode array detector (Agilent, G1315B). The gel permeation chromatography uses a Vantage L11 (120 mm × 12 mm inner diameter) column equipped with gel HW-50F, and a peristaltic pump is used to adjust the flow rate to 2 ml min -1 .

[0044] Step 2: The first fraction (F1 anthocyanidins) was eluted with 50 ml of a mixture of ethanol / water / trifluoroacetic acid in a volume ratio of 50:49.05:0.05, and the distillate was collected at 515 nm of the UV-visible spectrum. The second fraction (F2 proanthocyanidins) was eluted with 40 ml of a mixture of acetone / water in a volume ratio of 50:50. Specific implementation method three

[0046] The steps of this implementation are as follows:

[0047] Step 1: Take 6 g of pepper matrix with known moisture content (by dry weight), grind it with a plant grinder, add distilled water at a solid (by dry weight) to liquid ratio of 1:20, and treat it with a vacuum microwave at 55°C for 4 minutes. After a 4-minute break, repeat the intermittent heating 4 times, filter, collect the filtrate, and obtain 3 ml of an extract containing a mixture of anthocyanins and proanthocyanidins. Add it to the injection port of a gel permeation chromatography (HLC-8420GPC) with a photodiode array detector (Agilent, G1315B). The gel permeation chromatography uses a Vantage L (120 mm × 12 mm inner diameter) column equipped with gel HW-50F, and a peristaltic pump is used to adjust the flow rate to 4 ml min -1 .

[0048] Step 2: The first fraction (F1 anthocyanidins) was eluted with 45 ml of a mixture of ethanol / water / trifluoroacetic acid in a volume ratio of 45:54.05:0.05, and the distillate was collected at 515 nm of the UV-visible spectrum. The second fraction (F2 proanthocyanidins) was eluted with 30 ml of a mixture of acetone / water in a volume ratio of 45:55. Specific implementation method four

[0050] The steps of this implementation are as follows:

[0051] Step 1: Take 8 g (by dry weight) of blue indigofruit matrix with known moisture content, grind it with a plant grinder, add distilled water at a solid (by dry weight) to liquid ratio of 1:25, and treat it with a vacuum microwave at 58° C. for 3 minutes. After a 3-minute break, repeat the intermittent heating 5 times, filter, collect the filtrate, and obtain 5 ml of an extract containing anthocyanins and proanthocyanidins. The extract is added to the injection port of a gel permeation chromatography (HLC-8420GPC) with a photodiode array detector (Agilent, G1315B). The gel permeation chromatography uses a Vantage L (120 mm×12 mm inner diameter) column equipped with gel HW-50F, and a peristaltic pump is used to adjust the flow rate to 5 ml min -1 .

[0052] Step 2: The first fraction (F1 anthocyanidins) was eluted with 30 ml of a mixture of ethanol / water / trifluoroacetic acid in a volume ratio of 44:55.05:0.05, and the distillate was collected at 515 nm UV-visible spectrum. The second fraction (F2 proanthocyanidins) was eluted with 35 ml of a mixture of acetone / water in a volume ratio of 43:57.

[0053] Experimental results

[0054] Table 1 shows the purification effect of anthocyanins and proanthocyanidins from blue honeysuckle. Fraction F1 is anthocyanins, and F2 is proanthocyanidins. The purity, degree of polymerization and antioxidant activity are shown in Table 1.

[0055] Table 1

[0056] Fraction purity% Degree of polymerization (mDP) Antioxidant biological activity % F1 94.2 1.5 83.2％ F2 92.3 8 78.8％

[0057] Figure 1 This is a chromatogram recorded at 515 nm during GPC fractionation. It can be observed in the figure that most anthocyanins are eluted in fraction F1, and the purity of anthocyanins in fraction F1 is 94.2% and the degree of polymerization (mDP) is 1.5.

[0058] The minimum response of monomeric anthocyanins observed at 515 nm in fraction F2 was below the detection limit (the peak was close to the baseline), which indicated that the low signal at 515 nm in the separation of F2 was due to the presence of very small amounts of residual monomeric anthocyanins, which could be ignored.

[0059] Figure 2 The negative ion electrospray ionization (ESI) mass spectrum of fraction F2 of Lonicera caerulea isolated solution shows that there are a series of proanthocyanidins ranging from doubly charged tetramer (DP4) to doubly charged octamer (DPA8), and there are no measurable monomers or dimers. The purity of proanthocyanidins in fraction F2 was 92.3% as determined by vanillin.

[0060] Example

[0061] Determination of DPPH free radical scavenging rate by electron paramagnetic resonance

[0062] Using ethanol as solvent, fraction 1 and fraction 2 were prepared into a solution with a mass concentration of 800 mg / L. 1 mL of the sample to be tested was mixed with 1 mL of 0.2 mol / mL DPPH solution, respectively, and then placed into a capillary tube, sealed, and placed in the resonant cavity of the ESR instrument. The spectrum was recorded and the peak area A was calculated. 95% ethanol was used instead of the sample as a blank control. The clearance rate was calculated according to formula (1).

[0063] Clearance rate % = (A control group - A test group) × 100 / A control group (1)

[0064] The electron spin resonance spectra of fraction 1 and fraction 2 in scavenging DPPH free radicals are shown in Figure 3 As shown. In electron paramagnetic resonance, the characteristic absorption intensity of DPPH free radical is between 3460 and 3540G. Figure 3 It can be seen that the clearance rates of fraction 1 and fraction 2 are relatively high. The clearance rates after area integration are shown in Table 2.

[0065] Table 2

[0066] sample Integrated peak area Clearance rate (%) DPPH 68.48 DPPH+ fraction 1 (1+1) 11.50 83.2％ DPPH+ fraction 2 (1+1) 14.52 78.8％

[0067] Comparison of intermittent vacuum microwave extraction with other methods

[0068] As can be seen from Table 3, the intermittent vacuum microwave extraction conditions are relatively mild, the temperature and pressure requirements for the reaction are not very high, the safety is high, and the energy consumption is low; no chemical reagents need to be added, and the reaction time is short, which saves costs accordingly.

[0069] Table 3 Comparison of main parameters of intermittent vacuum microwave extraction and other methods

[0070]

[0071] From Table 3 and Figure 4 It can be seen that the yield of anthocyanins and proanthocyanidins extracted by intermittent vacuum microwave is slightly higher than that by normal pressure microwave during the extraction process; since the intermittent vacuum microwave extraction conditions are milder than normal pressure microwave, the temperature during the extraction process is less than 60°C, which retains the antioxidant activity of anthocyanins and proanthocyanidins to the greatest extent.

[0072] Table 4 Comparison of microwave extraction effects

[0073]

[0074] The above contents of the present invention are only preferred embodiments of the present invention and are not intended to limit the implementation scheme of the present invention. A person skilled in the art can easily make corresponding changes or modifications based on the main concept and spirit of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope required by the claims.

Claims

1. A method for purifying anthocyanidins and proanthocyanidins based on gel permeation chromatography, characterized in that: The following steps are involved: Step 1: using intermittent vacuum microwave technology to prepare an extract containing a mixture of anthocyanins and proanthocyanidins from agricultural products with a high content of anthocyanidins, and adding the extract to a gel permeation chromatography injection port with a photodiode array detector; Step 2: using different ratios of the first fraction and the second fraction eluents for elution and recovery, respectively, collecting the distillate of the first fraction at 515 nm UV-visible spectrum; Step 3, testing the purity and antioxidant properties of anthocyanins in the first fraction elution recovery solution, and the purity and antioxidant properties of proanthocyanidins in the second fraction elution recovery solution, to obtain purification conditions; Step 4: The two collected fractions are dried under vacuum, and the samples are subjected to multiple distillations.

2. The method for purifying anthocyanidins and proanthocyanidins based on gel permeation chromatography technology according to claim 1, characterized in that: In step 1, the intermittent vacuum microwave technology is specifically: Agricultural products with high content of anthocyanins were treated under different liquid-to-solid ratios, heating intermittent times and vacuum degrees. The content and antioxidant activity of anthocyanins and proanthocyanidins in the filtrate were tested after filtration. The optimal conditions for intermittent vacuum microwave extraction were obtained by comparing the conversion rate and antioxidant activity.

3. The method for purifying anthocyanidins and proanthocyanidins based on gel permeation chromatography technology as claimed in claim 2, characterized in that: In step 1, the liquid-to-solid ratio is 1:(10-30), and the heating interval time is: heating for 1-5 minutes, and resting for 1-5 minutes, repeated 1-10 times, and the temperature is less than 60°C.

4. The method for purifying anthocyanidins and proanthocyanidins based on gel permeation chromatography technology as claimed in claim 3, characterized in that: In step 1, 10 g of agricultural products with high anthocyanin content are used as substrates, distilled water is added according to a solid-liquid ratio of 1:10, and the mixture is subjected to reduced pressure microwave treatment at 60° C. for 1 minute, with an interval of 1 minute, and repeated 5 times, and then filtered to obtain an extract.

5. The method for purifying anthocyanidins and proanthocyanidins based on gel permeation chromatography technology according to claim 1, characterized in that: In step 1, the gel permeation chromatography uses a Vantage L chromatographic column filled with gel HW-50F, with an inner diameter of 120 mm×12 mm.

6. The method for purifying anthocyanidins and proanthocyanidins based on gel permeation chromatography technology according to claim 1, characterized in that: In step 1, use a peristaltic pump to adjust the flow rate to 1-5 ml min -1 .

7. The method for purifying anthocyanidins and proanthocyanidins based on gel permeation chromatography according to claim 1, characterized in that: In step 3, the purification conditions of the first fraction of anthocyanins are: elution and collection with a mixture of 40-60 ml of ethanol, water and trifluoroacetic acid, and the volume ratio of ethanol, water and trifluoroacetic acid is (50-55):(45-50):(0.02-0.05).

8. The method for purifying anthocyanidins and proanthocyanidins based on gel permeation chromatography technology according to claim 1, characterized in that: In step 3, the purification conditions of the second fraction of proanthocyanidins are: elution and collection with 30-50 ml of a mixture of acetone and water, and the volume ratio of acetone to water is (40-60): (40-60).

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