Method for extracting resveratrol from peanut skin and application of resveratrol

Abstract

The invention relates to a method for extracting resveratrol from peanut skin and application of resveratrol, and belongs to the technical field of natural product extraction. The extraction method comprises the steps of peanut skin enzymolysis, impurity removal, high-speed counter-current chromatography extraction and separation, macroporous resin purification and the like. The resveratrol obtained by the method disclosed by the invention is high in extraction rate and high in purity.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product extraction, and relates to a method for extracting resveratrol from peanut skins and its application. Background Art

[0002] Peanuts, also known as groundnuts, longevity fruits, etc., are annual leguminous plants. Peanut skins are the seed coats of peanuts, named because the seed coats of the vast majority of peanut varieties are red. The raw product has a blood-tonifying effect. After peanuts are cooked or fried, the blood-tonifying effect of the red skins will be greatly weakened. Peanut skins can inhibit the dissolution of fibrin, increase the content of platelets, improve the quality of platelets, improve the defects of coagulation factors, strengthen the contraction function of capillaries, and promote the hematopoietic function of bone marrow.

[0003] Peanut skins contain rich polyphenolic substances, such as ferulic acid, procyanidins, catechins, etc., and also contain some alkaloids, and these substances all have strong antioxidant activities. Resveratrol is a non-flavonoid polyphenolic compound in peanut skins, which is a natural antioxidant and has biological activities such as reducing blood lipids, antiviral, and anti-tumor.

[0004] Although peanut skins have good physiological activities, in peanut product processing, peanut skins are usually removed. The comprehensive utilization level of peanut skins in China is relatively low. It is necessary to fully develop the active ingredients in peanut skins such as resveratrol and apply them to industries such as food, medicine, and health products.

[0005] The traditional solid-liquid extraction technology is mostly used for the extraction and application of resveratrol from peanut skins. It has high energy consumption, long time, and there is a risk of thermal degradation of substances. Summary of the Invention

[0006] The main purpose of the present invention is to provide a method for extracting resveratrol from peanut skins. The preparation process of this method is simple, and the obtained resveratrol has a relatively high purity and a relatively high bioavailability.

[0007] The present invention adopts the following technical solutions to achieve the above purpose:

[0008] A method for extracting resveratrol from peanut skins specifically includes the following steps:

[0009] Step A: Crush peanut skins and add water to prepare a homogenate, then add cellulase and xylanase thereto, enzymatically hydrolyze at 35 - 40 °C for 40 - 60 min. After enzymatic hydrolysis, adjust the alcohol content of the enzymatic hydrolysate to 30 - 35% with absolute ethanol, ultrasonicate for 20 - 30 min, and filter to obtain an extract;

[0010] Step B: Add an adsorbent to the extract obtained in Step A, centrifuge after 10 - 15 min, and take the supernatant;

[0011] Step C: Concentrate the supernatant obtained in Step B and separate it by high-speed counter-current chromatography, and collect the fraction at the target peak position.

[0012] Step D: Remove the lower phase from the fraction collected in Step C, concentrate it, load it onto macroporous resin, elute it with 75% ethanol solution as the elution solvent, collect the eluate, concentrate and dry it to obtain resveratrol.

[0013] Furthermore, in the above Step A, the material-liquid ratio of peanut skins to water is 1:(3 - 5) kg / L.

[0014] Furthermore, in the above Step A, the addition amount of cellulase is 0.3 - 0.35% of the mass of peanut skins, and the addition amount of xylanase is 0.1 - 0.2% of the mass of peanut skins.

[0015] Furthermore, in the above Step B, the addition amount of the adsorbent is 3 - 5 g / L.

[0016] Furthermore, in the above Step B, the adsorbent is a modified sodium alginate-bentonite composite adsorbent.

[0017] Even further, the preparation method of the modified sodium alginate-bentonite composite adsorbent is as follows:

[0018] Mix bentonite with molten sodium alginate, then cool and crush to obtain the composite adsorbent.

[0019] Even further, the mass ratio of bentonite to sodium alginate is 1:(0.4 - 0.6).

[0020] Even further, in the above Step C, the volume ratio of chloroform - n-butanol - methanol - water is 4:0.25:3:2.

[0021] Even further, the operating steps of the high-speed counter-current chromatography in the above Step C are as follows:

[0022] Concentrate the supernatant obtained in Step B as the sample; use chloroform:n-butanol:methanol:water = 4:0.25:3:2 (v / v) as the solvent system, add acetonitrile to the upper phase as the stationary phase, add acetic acid to the lower phase as the mobile phase, with a flow rate of 5 ml / min and a rotation speed of 500 revolutions per minute; the retention value of the stationary phase is 50 - 60%, the detection wavelength is 280 nm, and collect the fraction at the target peak position from 70 - 100 min.

[0023] Even further, in the above method, the addition amount of acetonitrile is 5 ml / L, and the addition amount of acetic acid is 0.8 ml / L.

[0024] The present invention has the following beneficial effects:

[0025] In the preparation method of resveratrol of the present invention, first, the tissue structure of peanut seed coats is destroyed by enzymatic hydrolysis, effectively promoting the dissolution of resveratrol and improving the extraction rate of resveratrol. However, when using this extraction method, there are more impurities in the obtained extract. For further purification, a modified sodium alginate-clay adsorbent is used to remove some impurities, which can further improve the purity of resveratrol. In the separation stage, high-speed counter-current chromatography is used to effectively separate resveratrol from impurities. In high-speed counter-current chromatography, the solvent system is optimized. The resveratrol obtained by this method has a higher purity and fewer impurities. After purification by macroporous resin, the purity of resveratrol can reach more than 95%. Description of the Drawings

[0026] Figure 1 : High-speed counter-current chromatography diagram in Example 1;

[0027] Figure 2 : High-speed counter-current chromatography diagram in Comparative Example 3;

[0028] Figure 3 : High-speed counter-current chromatography diagram in Comparative Example 4. Detailed Embodiments

[0029] The present invention will be further illustrated below with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art fall within the scope protected by the claims of this application. The cellulase and xylanase used in the following embodiments are all commercially available products, which can be obtained through online or offline channels. Among them, the enzyme activity of cellulase is 50 U / mg, and the enzyme activity of xylanase is 300000 U / g.

[0030] Example 1

[0031] First, prepare a modified sodium alginate-clay composite adsorbent:

[0032] Mix 1 kg of clay with 0.6 kg of molten sodium alginate, cool, crush, and sieve to obtain the composite adsorbent.

[0033] Secondly, prepare resveratrol:

[0034] Step A, crush 1 kg of peanut seed coats and add 5 L of water to prepare a homogenate, then add 3 g of cellulase and 2 g of xylanase thereto, enzymatically hydrolyze at 40 °C for 40 min. After enzymatic hydrolysis, adjust the alcohol content of the enzymatic hydrolysate to 35% with absolute ethanol, ultrasonicate for 20 min, and filter to obtain an extract;

[0035] Step B, add 5 g / L of the above composite adsorbent to the extract obtained in Step A, centrifuge after 10 min, and take the supernatant;

[0036] Step C: Concentrate the supernatant obtained in Step B and then separate and prepare resveratrol by high-speed counter-current chromatography:

[0037] Chromatographic conditions: rotation speed 500 r / min, column temperature 25 °C, UV detection wavelength 280 nm;

[0038] Use chloroform: n-butanol: methanol: water = 4: 0.25: 3: 2 (v / v / v / v) as the solvent system. Add the solvent system to a separatory funnel, shake well, let it stand for phase separation. Add 5 ml / L of acetonitrile to the upper phase as the stationary phase, and add 0.8 ml / L of acetic acid to the lower phase as the mobile phase. Place them in an ultrasonic container for ultrasonic degassing treatment respectively; After pumping the stationary phase into the chromatograph, adjust the forward rotation speed of the chromatograph to 500 r / min, set the flow rate of the mobile phase to 5 mL / min, start pumping the mobile phase until the two phases are in equilibrium; Dissolve the concentrated supernatant in the stationary phase solution - mobile phase solution (10 mL: 10 mL), inject it into the high-speed counter-current chromatography instrument through the injection valve for separation, monitor online with a UV detector, and collect the fractions at 70 - 100 min;

[0039] Step D: Remove the lower phase from the fractions collected in Step C, concentrate, load onto NKA-9 macroporous resin, elute with 75% ethanol solution as the elution solvent, collect the eluate, concentrate and dry to obtain resveratrol.

[0040] Example 2

[0041] First, prepare a modified sodium alginate - clay composite adsorbent:

[0042] Mix 1 kg of clay with 0.4 kg of molten sodium alginate, cool, crush, and sieve to obtain the composite adsorbent.

[0043] Secondly, prepare resveratrol:

[0044] Step A: Crush 1 kg of peanut skins and add 3 L of water to prepare a homogenate. Then add 3.5 g of cellulase and 1 g of xylanase thereto, enzymatically hydrolyze at 35 °C for 60 min. After enzymatic hydrolysis, adjust the alcohol content of the enzymatic hydrolysate to 30% with absolute ethanol, ultrasonicate for 30 min, and filter to obtain the extract;

[0045] Step B: Add 3 g / L of the above composite adsorbent to the extract obtained in Step A, centrifuge after 15 min, and take the supernatant;

[0046] Step C: Concentrate the supernatant obtained in Step B and then separate and prepare resveratrol by high-speed counter-current chromatography:

[0047] Chromatographic conditions: rotation speed 500 r / min, column temperature 25 °C, UV detection wavelength 280 nm;

[0048] Using chloroform: n-butanol: methanol: water = 4: 0.25: 3: 2 (v / v / v / v) as the solvent system, add the solvent system into a separatory funnel, shake well, let it stand for phase separation. Add 5 ml / L of acetonitrile to the upper phase as the stationary phase, and add 0.8 ml / L of acetic acid to the lower phase as the mobile phase. Place them in an ultrasonic container for ultrasonic degassing treatment respectively; after pumping the stationary phase into the chromatograph, adjust the forward rotation speed of the chromatograph to 500 r / min, set the flow rate of the mobile phase to 5 mL / min, and start pumping the mobile phase until the two phases are in equilibrium; dissolve the concentrated supernatant in the stationary phase solution - mobile phase solution (10 mL: 10 mL), inject it into a high-speed counter-current chromatography instrument through the injection valve for separation, monitor it online with a UV detector, and collect the fractions from 70 - 100 min;

[0049] Step D, remove the lower phase from the fractions collected in Step C, concentrate, load onto NKA-9 macroporous resin, elute with 75% ethanol solution as the elution solvent, collect the eluate, concentrate and dry it to obtain resveratrol.

[0050] Example 3

[0051] First, prepare a modified sodium alginate - clay composite adsorbent:

[0052] Mix 1 kg of clay with 0.5 kg of molten sodium alginate, cool, crush, and sieve to obtain the composite adsorbent.

[0053] Secondly, prepare resveratrol:

[0054] Step A, crush 1 kg of peanut skins and add 4 L of water to prepare a homogenate, then add 3.3 g of cellulase and 1.5 g of xylanase to it, enzymatically hydrolyze at 40 °C for 50 min. After enzymatic hydrolysis, adjust the alcohol content of the enzymatic hydrolysate to 30% with absolute ethanol, sonicate for 30 min, and filter to obtain the extract;

[0055] Step B, add 4 g / L of the above composite adsorbent to the extract obtained in Step A, centrifuge after 15 min, and take the supernatant;

[0056] Step C, concentrate the supernatant obtained in Step B and then separate and prepare resveratrol by high-speed counter-current chromatography:

[0057] Chromatographic conditions: rotation speed 500 r / min, column temperature 25 °C, UV detection wavelength 280 nm;

[0058] Using chloroform: n-butanol: methanol: water = 4:0.25:3:2 (v / v / v / v) as the solvent system, add the solvent system to a separatory funnel, shake well, let it stand for layering. Add 5 mL / L of acetonitrile to the upper layer as the stationary phase, and add 0.8 mL / L of acetic acid to the lower layer as the mobile phase. Place them in an ultrasonic container for ultrasonic degassing treatment respectively; after pumping the stationary phase into the chromatograph, adjust the forward rotation speed of the chromatograph to 500 r / min, set the flow rate of the mobile phase to 5 mL / min, start pumping the mobile phase until the two phases are in equilibrium; dissolve the concentrated supernatant in the stationary phase solution - mobile phase solution (10 mL:10 mL), inject it into the high-speed counter-current chromatography instrument through the injection valve for separation, monitor online with a UV detector, and collect the fractions at 70 - 100 min;

[0059] Step D, remove the lower layer from the fractions collected in Step C, concentrate, load onto NKA-9 macroporous resin, elute with 75% ethanol solution as the elution solvent, collect the eluate, concentrate and dry to obtain resveratrol.

[0060] Comparative Example 1

[0061] Crush 1 kg of peanut skins, add 4 L of water to prepare a homogenate, adjust the pH to 5.0, then add 3.3 g of cellulase and 1.5 g of xylanase, enzymatically hydrolyze at 40 °C for 50 min. After enzymatic hydrolysis, adjust the alcohol content of the enzymatic hydrolysate to 50% with anhydrous ethanol, ultrasonicate for 30 min, filter to obtain a resveratrol-containing extract, and dry to obtain a resveratrol extract.

[0062] Comparative Example 2

[0063] Step A, crush 1 kg of peanut skins, add 3 L of water to prepare a homogenate, then add 3.5 g of cellulase and 1 g of xylanase, enzymatically hydrolyze at 35 °C for 60 min. After enzymatic hydrolysis, adjust the alcohol content of the enzymatic hydrolysate to 30% with anhydrous ethanol, ultrasonicate for 30 min, filter to obtain an extract;

[0064] Step B, concentrate the extract obtained in Step A under reduced pressure, load onto NKA-9 macroporous resin, elute with 75% ethanol solution, collect the eluate to obtain a resveratrol extract.

[0065] Comparative Example 3

[0066] Step A, crush 1 kg of peanut skins, add 4 L of water to prepare a homogenate, then add 3.3 g of cellulase and 1.5 g of xylanase, enzymatically hydrolyze at 40 °C for 50 min. After enzymatic hydrolysis, adjust the alcohol content of the enzymatic hydrolysate to 30% with anhydrous ethanol, ultrasonicate for 30 min, filter to obtain an extract;

[0067] Step B, after concentrating the extract obtained in Step A, separate and prepare resveratrol by high-speed counter-current chromatography;

[0068] Chromatographic conditions: rotation speed 500 r / min, column temperature 25 °C, UV detection wavelength 280 nm;

[0069] Using chloroform: n-butanol: methanol: water = 4: 0.25: 3: 2 (v / v / v / v) as the solvent system, add the solvent system to a separatory funnel, shake well, let stand for phase separation. Add 5 ml / L of acetonitrile to the upper phase as the stationary phase, and add 0.8 ml / L of acetic acid to the lower phase as the mobile phase. Place them in an ultrasonic container for ultrasonic degassing treatment respectively; After pumping the stationary phase into the chromatograph, adjust the forward rotation speed of the chromatograph to 500 r / min, set the flow rate of the mobile phase to 5 mL / min, start pumping the mobile phase until the two phases are in equilibrium; Dissolve the concentrated supernatant in the stationary phase solution - mobile phase solution (10 mL: 10 mL), inject it into a high-speed countercurrent chromatography instrument through the injection valve for separation, monitor online with a UV detector, and collect the fractions at 70 - 85 min;

[0070] Step C: Remove the lower phase from the fractions collected in Step B, concentrate, load onto NKA-9 macroporous resin, elute with 75% ethanol solution as the elution solvent, collect the eluate, concentrate and dry to obtain resveratrol.

[0071] Comparative Example 4

[0072] Step A: Crush 1 kg of peanut skins and add 4 L of water to prepare a homogenate, then add 3.3 g of cellulase and 1.5 g of xylanase thereto, enzymatically hydrolyze at 40 °C for 50 min. After enzymatic hydrolysis, adjust the alcohol content of the enzymatic hydrolysate to 30% with absolute ethanol, ultrasonicate for 30 min, and filter to obtain an extract;

[0073] Step B: Add 4 g / L of clay to the extract obtained in Step A, centrifuge after 15 min, and take the supernatant;

[0074] Step C: Concentrate the supernatant obtained in Step B and then separate and prepare resveratrol by high-speed countercurrent chromatography:

[0075] Chromatographic conditions: rotation speed 500 r / min, column temperature 25 °C, UV detection wavelength 280 nm;

[0076] Using chloroform: n-butanol: methanol: water = 4:0.25:3:2 (v / v / v / v) as the solvent system, add the solvent system to a separatory funnel, shake well, let it stand for layer separation. The upper phase is used as the stationary phase and the lower phase is used as the mobile phase. Place them in ultrasonic containers respectively for ultrasonic degassing treatment. After pumping the stationary phase into the chromatograph, adjust the forward rotation speed of the chromatograph to 500 r / min, set the flow rate of the mobile phase to 5 mL / min, and start pumping the mobile phase until the two phases are in equilibrium. Dissolve the concentrated supernatant in the stationary phase solution - mobile phase solution (10 mL:10 mL), inject it into a high-speed countercurrent chromatography instrument through the injection valve for separation, monitor online with a UV detector, and collect the fractions at 65 - 75 min.

[0077] Step D: Remove the lower phase from the fractions collected in Step C, concentrate, load onto NKA-9 macroporous resin, elute with 75% ethanol solution as the elution solvent, collect the eluate, concentrate and dry to obtain resveratrol.

[0078] Performance test

[0079] Weigh the resveratrol or resveratrol extract obtained in Examples 1 - 3 and Comparative Examples 1 - 3, calculate the product yield; prepare a standard reference solution with a standard product, dilute it to different concentrations, measure the absorbance at a detection wavelength of 306 nm to prepare a standard curve; prepare sample solutions with the resveratrol or resveratrol extract obtained in Examples 1 - 3 and Comparative Examples 1 - 3, measure the absorbance, and calculate the purity of resveratrol in the sample solution according to the standard curve. The results are shown in Table 1 below.

[0080] Table 1 Product yield and resveratrol content in different embodiments

[0081] Embodiment Product yield (mg / kg) Resveratrol purity (%) Example 1 13.52 96.8 Example 2 14.85 97.3 Example 3 15.79 95.9 Comparative Example 1 26.84 56.6 Comparative Example 2 22.58 63.1 Comparative Example 3 18.41 75.4 Comparative Example 4 17.26 81.2

[0082] As can be seen from the results in Table 1, in Comparative Examples 1 and 2 using traditional extraction methods, more products are obtained, but the purity of resveratrol is relatively low and there are more impurities. In Comparative Example 3 without an adsorbent and Comparative Example 4 with an adsorbent different from that of the present invention, the product yield is relatively high, but the purity of resveratrol is relatively low.

[0083] Self-attached Figures 1-3 It can be seen from the attached that the extract components in Comparative Examples 3 and 4 are different, and the peak emergence in high-speed countercurrent chromatography is different, and complete separation is not achieved, resulting in a relatively low purity of resveratrol.

Claims

1. A method for extracting resveratrol from peanut skins, characterized in that, It includes the following steps: Step A: Crush peanut skins and add water to prepare a homogenate. Then add cellulase and xylanase thereto, and carry out enzymatic hydrolysis at 35 - 40 °C for 40 - 60 min. After enzymatic hydrolysis, adjust the alcohol content of the enzymatic hydrolysis solution to 30 - 35% with absolute ethanol, carry out ultrasonic treatment for 20 - 30 min, and filter to obtain an extract; Step B: Add an adsorbent to the extract obtained in Step A, centrifuge after 10 - 15 min, and take the supernatant; Step C: Concentrate the supernatant obtained in Step B and separate it by high-speed counter-current chromatography, and collect the fraction at the target peak position; Step D: Remove the lower phase from the fraction collected in Step C, concentrate it, load it onto a macroporous resin, elute with a 75% ethanol solution as an elution solvent, collect the eluate, concentrate and dry it to obtain resveratrol.

2. The extraction method according to claim 1, wherein In the said Step A, the material-liquid ratio of peanut skins to water is 1:(3 - 5) kg / L.

3. The extraction method according to claim 1, wherein In the said Step A, the addition amount of cellulase is 0.3 - 0.35% of the mass of peanut skins, and the addition amount of xylanase is 0.1 - 0.2% of the mass of peanut skins.

4. The extraction method according to claim 1, characterized in that In the said Step B, the addition amount of the adsorbent is 3 - 5 g / L.

5. The extraction method according to claim 1, wherein In the said Step B, the adsorbent is a modified sodium alginate-bentonite composite adsorbent.

6. The extraction method according to claim 5, characterized in that, The preparation method of the said modified sodium alginate-bentonite composite adsorbent is: Mix bentonite with molten sodium alginate, then cool and crush to obtain the composite adsorbent.

7. The extraction method according to claim 1, characterized in that, The mass ratio of the said bentonite to sodium alginate is 1:(0.4 - 0.6).

8. The extraction method according to claim 1, characterized in that The operation steps of the high-speed counter-current chromatography in the said Step C are: Concentrate the supernatant obtained in Step B as a sample; use chloroform:n-butanol:methanol:water = 4:0.25:3:2 (v / v) as a solvent system, add acetonitrile to the upper phase as a stationary phase, add acetic acid to the lower phase as a mobile phase, with a flow rate of 5 ml / min and a rotation speed of 500 revolutions per minute; the retention value of the stationary phase is 50 - 60%, the detection wavelength is 280 nm, and collect the fraction at the target peak position from 70 - 100 min.

9. The extraction method according to claim 8, wherein The addition amount of the said acetonitrile is 5 ml / L, and the addition amount of acetic acid is 0.8 ml / L.

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