A method for extracting proanthocyanidins from peanut skins

Abstract

The present invention discloses a method for extracting proanthocyanidins from peanut skins. The peanut skins are dried, pulverized, and defatted, and the defatted peanut skin powder is extracted with an aqueous solution of benzyl ether reagents. After centrifugation, concentration, purification, re-concentration, and freeze-drying, a proanthocyanidin dry powder is obtained. By using an aqueous solution of benzyl ether reagents as the extraction solution and combining ultrasonic assistance for the extraction of proanthocyanidins from peanut skins, the extraction conditions are mild, the extraction rate is higher, the obtained product has high purity and is more stable, solving the problems of low extraction rate of proanthocyanidins, large consumption of organic reagents, long extraction time, and easy destruction during the extraction process in the traditional solution extraction method.

Description

Technical Field

[0001] The present invention belongs to the field of material extraction processes, and relates to a method for extracting active ingredients from natural plants, and particularly to a method for extracting proanthocyanidins from peanut skins. Background Art

[0002] Peanut skins are the inner seed coats of peanuts, accounting for about 2.6% of the weight of the whole peanut seeds. Because of their astringent taste, as a by-product of peanut processing, most of them are used as feed, and even a large part is directly discarded, with low overall utilization rate and low economic value. However, peanut skins contain various nutrients, such as about 25% crude fat, about 18% protein, about 13% crude fiber, and various trace elements such as calcium, phosphorus, iron, copper, zinc, and manganese. In addition, peanut skins also contain various special substances, such as proanthocyanidins, p-coumaric acid, quinic acid, caffeic acid, gallic acid, resveratrol, etc.

[0003] Among them, proanthocyanidins are a general term for all colorless compounds derived from flavonoids in the presence of inorganic acids isolated from plants. Their biological activity effects include antioxidant activity, antimutagenic activity, enzyme inhibition activity, anticarcinogenic agent effects, and scavenging of human free radicals, etc., showing effects such as anti-myocardial ischemia, regulating blood lipids, and protecting the skin, and are widely used in the fields of food, medicine, health products, and cosmetics. Due to the above characteristics of proanthocyanidins, they are sensitive to environmental conditions such as light, heat, and pH during the extraction process, and specific processes are required to improve the extraction rate and prevent their oxidation.

[0004] Because proanthocyanidins are soluble in water or polar solutions, water extraction is often used during extraction. However, due to the unsatisfactory effect, a complex solvent is often used for extraction. For example, in the Chinese invention patent with the document number CN102911150B, "Production process for preparing proanthocyanidins from peanut skins", the crushed peanut skins are extracted with a complex solvent of water, ethanol, acetone, and ethyl acetate, and then through plate and frame filtration, ceramic membrane filtration, chromatography, elution, and then concentration and freeze-drying to obtain proanthocyanidin powder. The yield of this method is about 2%, and the purity is 95%. In recent years, people have continued to study the extraction and purification steps of peanut skins to further improve the purity. For example, in the Chinese invention patent application with the document number CN114524793A, "A method for extracting and purifying proanthocyanidins from peanut skins", after degreasing peanut skins with petroleum ether, extraction is carried out in an alcohol solution, and after ultrasonic and microwave synergistic treatment, extraction is carried out, and after chromatography purification, concentration, and then freeze-drying to obtain proanthocyanidin powder. This process can increase the purity of proanthocyanidins to 98%, and the antioxidant activity of the obtained proanthocyanidins is tested, but the extraction rate or yield of this method is not disclosed. Summary of the Invention

[0005] The object of the present invention is to provide a method for extracting proanthocyanidins from peanut skins to improve the extraction rate and purity of proanthocyanidins.

[0006] The technical solution adopted by the present invention is to provide a method for extracting proanthocyanidins from peanut skins. The peanut skins are dried, crushed, and defatted. The key lies in using an aqueous solution of benzethonium reagents to extract the defatted peanut skin powder. After centrifugation, primary concentration, purification, secondary concentration, and freeze-drying, proanthocyanidin dry powder is obtained.

[0007] Further, the above extraction is to mix the defatted peanut skin powder with an aqueous solution of benzethonium reagents at a mass-to-volume ratio of 1 g: 10 ml - 60 ml to prepare a peanut skin solution, and then perform ultrasonic extraction at a temperature of 30°C - 50°C for 30 min - 70 min with an ultrasonic power of 300 w - 500 w.

[0008] Further, the concentration of the above aqueous solution of benzethonium reagents is that the weight-to-volume ratio of benzethonium reagents to the aqueous solution is 0.5% - 3%, and the pH is 4.0 - 5.0.

[0009] Further, the above benzethonium reagents include Brij78, Brij76, Brij35, Brij56, and Brij58.

[0010] Even further, the above drying is to dry the peanut skins at 45°C - 55°C for 4 h - 5 h; the above crushing means crushing the dried peanut skins to a particle size not higher than 60 mesh.

[0011] Even further, the above defatting is to place the crushed peanut skin powder in an organic solvent and soak it for 24 h. The organic solvent used is n-hexane or petroleum ether.

[0012] Even further, the above centrifugation means centrifuging the peanut skin solution at 5000 rpm / min - 8000 rpm / min for 10 min - 15 min, and taking the supernatant for subsequent operations.

[0013] Even further, the above primary concentration means concentrating the supernatant after centrifugation with a rotary evaporator to obtain a primary concentrated solution containing proanthocyanidins from peanut skins.

[0014] Even further, the above purification is to separate and elute the primary concentrated solution obtained after primary concentration with macroporous resin to obtain an eluate; the macroporous resin is one of S-8, LX-8, AB-8, and D-101.

[0015] Further, the above-mentioned reconcentration and freeze-drying are carried out by reconcentrating the eluate obtained after purification with a rotary evaporator and then freeze-drying at -75°C to -85°C for 45h to 50h to obtain the peanut skin proanthocyanidin dry powder.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] In the present invention, an aqueous solution of benzethonium reagent is used as the extraction solution, and ultrasonic assistance is combined to extract proanthocyanidins from peanut skin. Ultrasonic waves can fully destroy cell membranes and enable the substances inside peanut skin cells to be fully dissolved. Benzethonium reagents are a series of high-carbon fatty alcohol polyoxyethylene ether compounds that do not produce ions in aqueous solutions, have high stability, good compatibility, low toxicity, are not easily affected by electrolytes, have hydrophilic and hydrophobic groups, belong to non-ionic surfactants, and are often used as solubilizers, dispersants, emulsifiers, etc. The use of benzethonium aqueous solution for the extraction of proanthocyanidins from peanut skin in the present invention not only realizes the reuse of peanut skin, reduces resource waste, and realizes the high-value development and utilization of peanut skin, but also improves the extraction rate and stability of proanthocyanidins, reduces the extraction cost, and has no pollution to the environment.

[0018] When equal to or greater than the critical micelle concentration, benzethonium reagents will form spherical micelles in aqueous solutions. Their hydrophobic parts aggregate into nuclei, which can enrich hydrophobic substances; the hydrophilic parts face outward, thereby interacting with polar and non-polar components and improving the extraction rate. In addition, the extraction reagent used in the present invention can also prevent the degradation of proanthocyanidins during the extraction process, and the obtained proanthocyanidins have better antioxidant activity and are more stable.

[0019] In summary, the extraction conditions of the present invention are mild, the extraction rate is higher, the obtained product has high purity and is more stable, and it solves the problems of low extraction rate of proanthocyanidins, large consumption of organic reagents, long extraction time, and easy destruction during the extraction process in the traditional solution extraction method. Description of the Drawings

[0020] Figure 1 is the test result 1 of DPPH free radical scavenging ability.

[0021] Figure 2 is the test result 2 of DPPH free radical scavenging ability. Detailed Embodiments

[0022] The present invention provides a method for extracting proanthocyanidins from peanut skin. The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0023] Examples 1 to 5

[0024] (1) Drying: 10 g of peanut skins were dried at a certain temperature according to Table 1.

[0025] (2) Crushing: The peanut skins were crushed and sieved according to Table 1.

[0026] (3) Defatting: The peanut skin powder was soaked in an organic reagent for defatting for 24 h, and then the organic solvent was removed by suction filtration. The peanut skin powder was placed in a fume hood to dry, and defatted peanut skin powder was obtained.

[0027] (4) Extraction: 5 g of defatted peanut skin powder was placed in a beaker, and an aqueous benzethonium solution with a certain concentration was added according to Table 1. After mixing evenly, ultrasonic extraction was carried out.

[0028] (5) Centrifugation: The peanut skin extract was centrifuged at 5000 rpm / min - 8000 rpm / min for 10 min - 15 min according to Table 1, and the supernatant was taken.

[0029] (6) Primary concentration: The supernatant was concentrated by a rotary evaporator to a certain concentration to obtain a primary concentrated solution containing procyanidins from peanut skins;

[0030] (7) Purification: The macroporous resin was soaked in absolute ethanol for 24 h, then washed with deionized water until there was no ethanol, and then soaked in 5% HCl (w / w) and 5% NaOH (w / w) solutions for 6 h respectively, and washed with deionized water until neutral, and reserved. 100 g of pretreated resin was loaded into a glass chromatography column, the sample loading flow rate was 1.0 mL / min, the sample loading concentration was 3 mg / mL, and the sample loading volume was 100 mL. After adsorption saturation, impurities were first washed away with deionized water, and eluted with a 50% ethanol solution, the elution flow rate was 1.5 mL / min, and the elution volume was 160 mL.

[0031] (8) Secondary concentration and freeze-drying: The eluate was concentrated by a rotary evaporator and then freeze-dried to obtain procyanidin powder from peanut skins, and the sample numbers were Sample 1 to 5 respectively.

[0032] Table 1: Parameter table of experimental examples

[0033]

[0034] Continued Table 1: Parameter table of experimental examples

[0035]

[0036] Control Examples 1 to 4

[0037] According to the parameters of Experimental Example 2 in Table 1, the difference lies in the types and dosages of reagents used in the extraction step. For details, see Table 2.

[0038] Table 2: List of extraction reagents used in Control Examples 1-4

[0039]

[0040] Control Example 5

[0041] According to the parameters of Experimental Example 2 in Table 1, the difference lies in the parameters of the extraction step. For details, see Table 3.

[0042] Table 3: List of extraction process parameters used in Control Examples 5-8

[0043]

[0044] Test method:

[0045] Test 1: Extraction rate and purity test

[0046] Test the proanthocyanidin extraction rate of each group of examples and control examples, and determine the proanthocyanidin content by the hydrochloric acid-vanillin method. The results are shown in Table 4. The specific formula is as follows:

[0047] Extraction rate % = (mass of proanthocyanidins obtained in each group of tests / mass of peanut red skins) × 100%

[0048] Purity % = (mass of proanthocyanidins measured / mass of proanthocyanidins obtained in each group of tests) × 100%

[0049] Table 4: Statistical table of extraction rate and purity results

[0050] Sample number Mass of obtained proanthocyanidins (mg) Extraction rate (%) Purity (%) Sample 1 174.1 3.5％ 99.5 Sample 2 172.8 3.5％ 99.8 Sample 3 178.9 3.6％ 99.5 Sample 4 175.3 3.5％ 99.3 Sample 5 176.5 3.5％ 99.4 Control 1 119.3 2.4％ 83.4 Control 2 121.1 2.4％ 89.5 Control 3 102.6 2.1％ 75.6 Control 4 157.2 3.1％ 90.1 Control 5 85.4 1.7％ 91.3 Control 6 155.2 3.1％ 79.4 Control 7 149.3 3.0％ 75.6 Control 8 90.7 1.8％ 86.3

[0051] As can be seen from Table 4 above, the average extraction rate of the 5 samples of the present invention reached 3.5%, and the average purity could reach 99.5%, far higher than that of the reference substance. When changing the type of extraction solvent, extraction temperature, ultrasonic power, extraction time and extraction pH, it has an adverse effect on the extraction rate or purity. For example, in Comparative Examples 6-8, when changing the extraction time, extraction pH and extraction temperature respectively, the purity decreased very significantly. It can be seen that the proanthocyanidins are very sensitive to these three factors and are prone to degradation during the extraction process. In Comparative Example 5, although benzethonium aqueous solution was also selected as the extraction solvent, ultrasonic treatment was not used. This is because ultrasonic assistance destroys the cell membrane, enabling the substances contained in the cell sap to be fully dissolved, providing convenient conditions for extraction. It can be seen that benzethonium reagents play an amphoteric role, not only acting on the cell membrane, but also directly acting on proanthocyanidins. When equal to or greater than the critical micelle concentration, benzethonium reagents will form spherical micelles in aqueous solution. The hydrophobic part aggregates into a nucleus, which can enrich hydrophobic substances; the hydrophilic part faces outward, enriching hydrophilic proanthocyanidins, facilitating the subsequent elution of proanthocyanidins, thus separating them from proanthocyanidins and other non-polar components, and improving the extraction rate. At the same time, it can also prevent the degradation of proanthocyanidins during the extraction process. The obtained proanthocyanidins have better antioxidant activity, are more stable, and further improve the purity.

[0052] Test Two: DPPH Free Radical Scavenging Ability Test

[0053] Take 0.05 g of 2,2-diphenyl-1-picrylhydrazyl (containing 10%-20% benzene) and dissolve it, and make up the volume to 50 mL in a volumetric flask to obtain a DPPH· solution with a mass concentration of 1.0 mg / mL. Take 1.0 mL of the DPPH· solution in a test tube, add 1.0 mL of absolute ethanol, then add 3.0 mL of water, shake well and react in the dark for 30 min, and measure the absorbance of the above solution at 517 nm and record it as +A0.

[0054] Prepare test solutions with different concentrations from the test samples and vitamin C, with concentrations of 10, 20, 30, 40, 50 μg / mL. Take 1 mL of each test solution with different concentrations and vitamin C solution and add them to a test tube, then add 1 mL of the DPPH· solution, add 3.0 mL of water, react in the dark for 30 min, and measure the absorbance at 517 nm and record it as A1.

[0055] Take 1 mL of each test solution with different concentrations and vitamin C solution and add them to a test tube, then add 1 mL of absolute ethanol, add 3.0 mL of water, react in the dark for 30 min, and measure the absorbance at 517 nm and record it as A2.

[0056] The calculation of the DPPH free radical scavenging ability is as follows: Scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%

[0057] Wherein: A0, A1, and A2 are the absorbance values measured at 517 nm for the DPPH· solution and absolute ethanol, the sample solution and the DPPH· solution, and the mixture of the sample solution and absolute ethanol, respectively.

[0058] Taking the concentration of the test solution as the x-axis and the scavenging rate as the y-axis to plot a curve, and the results are shown in Appendix Figure 1 and Appendix Figure 2 , the sample prepared by the present invention has a stronger DPPH free radical scavenging ability, indicating that the proanthocyanidins therein have better antioxidant activity, and the extraction solvent used in the present invention is more conducive to protecting proanthocyanidins from being destroyed.

Claims

1. A method for extracting proanthocyanidins from peanut skins, which comprises drying, pulverizing, and degreasing peanut skins, characterized in that, The defatted peanut skin powder is extracted with an aqueous solution of a Brij reagent. After centrifugation, primary concentration, purification, re-concentration, and freeze-drying, proanthocyanidin dry powder is obtained. For the extraction, the defatted peanut skin powder is mixed with the aqueous solution of the Brij reagent at a mass-to-volume ratio of 1 g: 10 ml - 60 ml to prepare a peanut skin solution, which is then subjected to ultrasonic extraction at a temperature of 30°C - 50°C for 30 min - 70 min with an ultrasonic power of 300 W - 500 W. The concentration of the aqueous solution of the Brij reagent is such that the weight-to-volume ratio of the Brij reagent to the aqueous solution is 0.5% - 3%, and the pH is 4.0 - 5.

0. The Brij reagent is any one of Brij78, Brij76, Brij35, Brij56, or Brij58. For the purification, the primary concentrate obtained after primary concentration is separated and eluted by macroporous resin to obtain an eluate. The macroporous resin is one of S-8, LX-8, AB-8, or D-101.

2. The method for extracting proanthocyanidins from peanut skins according to claim 1, wherein For the drying, the peanut skin is dried at 45°C - 55°C for 4 h - 5 h; the pulverization means pulverizing the dried peanut skin to a particle size not higher than 60 mesh.

3. A method for extracting proanthocyanidins from peanut skins according to claim 1, characterized in that, For the defatting, the pulverized peanut skin powder is placed in an organic solvent and soaked for 24 h. The organic solvent used is n-hexane or petroleum ether.

4. A method for extracting proanthocyanidins from peanut skins according to claim 1, characterized in that, The centrifugation means centrifuging the peanut skin solution at 5000 rpm / min - 8000 rpm / min for 10 min - 15 min, and taking the supernatant for subsequent operations.

5. A method for extracting proanthocyanidins from peanut skins according to claim 1, characterized in that, The primary concentration means concentrating the supernatant after centrifugation by a rotary evaporator to obtain a primary concentrate containing peanut skin proanthocyanidin.

6. A method for extracting proanthocyanidins from peanut skins according to claim 1, characterized in that, For the re-concentration and freeze-drying, the eluate obtained after purification is re-concentrated by a rotary evaporator and then freeze-dried at -75°C - 85°C for 45 h - 50 h to obtain proanthocyanidin dry powder of peanut skin.

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