Method for ultrasonic-assisted extraction of resveratrol from peanut sprouts and application of resveratrol

Abstract

The invention provides an ultrasonic-assisted peanut sprout resveratrol extraction method and application thereof, and belongs to the technical field of natural active ingredient extraction. The invention provides an application of at least one peanut variety selected from Zhongju flower 100, Yuhua flower 23 and Yuhua flower 99 in extraction of resveratrol. According to the method provided by the invention, the peanut sprouts are used as raw materials, and the resveratrol is extracted in an ultrasonic-assisted manner by adopting optimized parameters, so that the extraction rate of the resveratrol can be greatly improved, and the content of the resveratrol in the screened Zhongjuhua 100 peanut sprouts is obviously higher than that of other varieties. Meanwhile, the extract obtained by the method has good antioxidant activity, the antioxidant ability of the Yuhua 93 peanut sprouts is the strongest during 3-5 days of sprouting, and the Yuhua 93 peanut sprouts are suitable for being used for preparing products with high antioxidant activity.

Description

A method for ultrasonic-assisted extraction of resveratrol from peanut sprouts and its application Technical Field

[0001] This application belongs to the field of natural active ingredient extraction technology, specifically relating to a method for ultrasonic-assisted extraction of resveratrol from peanut sprouts and its application. Background Technology

[0002] Resveratrol (Res), also known as stilbene triol, is a non-flavonoid polyphenol compound produced by plants in response to pathogen infection or other inducing factors. Modern medical research has shown that resveratrol is effective in preventing and treating tumors, cardiovascular diseases, nervous system disorders, as an antioxidant, and in treating diabetes, and is widely used in pharmaceuticals and health foods. Currently, resveratrol has been found in various plants, with peanuts and other plants attracting significant attention due to their relatively high content. After peanuts sprout, their internal macromolecules degrade, forming smaller molecules with higher nutritional value and stronger bioactivity, and the resveratrol content increases significantly. Therefore, peanut sprouts are an excellent material for extracting resveratrol.

[0003] However, although peanut sprouts have a clear advantage over unsprouted peanuts in terms of resveratrol content, the extraction rate still needs to be improved. Summary of the Invention

[0004] This invention provides a method for ultrasonic-assisted extraction of resveratrol from peanut sprouts, achieving a high resveratrol extraction rate by optimizing peanut varieties.

[0005] This invention provides the application of peanuts in the extraction of resveratrol, wherein the peanuts include at least one of the following varieties: Zhongyuhua 100, Yuhua 23 and Yuhua 99.

[0006] Preferably, resveratrol is extracted from peanuts in the form of peanut sprouts; the peanut sprouts include peanut sprouts that have germinated for 4 to 6 days.

[0007] This invention provides a method for ultrasonic-assisted extraction of resveratrol from peanut sprouts, comprising the following steps: preparing peanut sprouts; mixing the peanut sprouts with an ethanol aqueous solution and then performing ultrasonic extraction to obtain an extract; wherein the peanuts include at least one of the following varieties: Zhongyuhua 100, Yuhua 23, and Yuhua 99.

[0008] Preferably, the volume concentration of the ethanol aqueous solution is 70% to 90%.

[0009] Preferably, the ratio of peanut sprouts to ethanol aqueous solution is 1g:25~30mL.

[0010] Preferably, the ultrasonic treatment time is 35-75 minutes; the ultrasonic treatment temperature is 30-70°C.

[0011] Preferably, the germination time of the peanut sprouts is 4 to 6 days.

[0012] Preferably, the germination time of the peanut sprouts is 5 days; the volume concentration of the ethanol aqueous solution is 81%; the material-to-liquid ratio of the peanut sprouts to the ethanol aqueous solution is 1g:28mL; the ultrasonic treatment time is 64min; and the ultrasonic treatment temperature is 60℃.

[0013] This invention provides the application of the method in the development of antioxidant functional products, wherein the peanut includes Yuhua 93.

[0014] Preferably, the antioxidant includes the ability to scavenge ABTS free radicals.

[0015] This invention provides the application of peanuts in the extraction of resveratrol, wherein the peanuts include at least one of the following varieties: Zhongyuhua 100, Yuhua 23, and Yuhua 99. This invention uses seeds of multiple peanut varieties as initial raw materials for resveratrol extraction, and uses the extraction yield of resveratrol as the screening standard. Experiments show that different peanut varieties can significantly affect the resveratrol content during germination. Among peanuts germinated for 3-5 days, Zhongyuhua 100 showed the best extraction rate. Other peanut varieties showed advantages at different germination stages. For example, at 3 days of germination, Yuhua 23, Yuhua 99, and Yuanza 9307 were more advantageous than Yuhua 93 and Yuhua 138; at 4 days of germination, Yuhua 99 had a significant advantage over the other four peanut varieties; at 5 days of germination, Yuhua 23 reached the same level as Zhongyuhua 100, while Yuhua 99 and Yuanza 9307 showed a significant advantage over Yuhua 93 and Yuhua 138. Therefore, considering various peanut varieties, Zhongyuhua 100, Yuhua 23, and Yuhua 99 express a large amount of resveratrol during germination, which can serve as advantageous materials for resveratrol extraction. At the same time, it provides a foundation for developing peanut varieties rich in resveratrol or for breeding research.

[0016] This invention provides a method for ultrasonic-assisted extraction of resveratrol from peanut sprouts. Based on the application of this method in the development of antioxidant functional products, the peanut variety includes Yuhua 93. Experiments have shown that the ultrasonic-assisted extraction method for resveratrol from peanut sprouts yields a crude extract rich in antioxidant components, and Yuhua 93 exhibits a significant advantage over other peanut varieties in scavenging free radicals. This provides a new approach for the development of antioxidant products. Attached Figure Description

[0017] Figure 1 shows the high-performance liquid chromatography (HPLC) chromatogram of resveratrol standard; Figure 2 shows the standard curve of resveratrol; Figure 3 shows the effect of solid-liquid ratio on the resveratrol extraction yield from peanut sprouts (Note: different letters indicate different groups, P < 0.05); Figure 4 shows the effect of ethanol concentration on the resveratrol extraction yield from peanut sprouts (Note: different letters indicate different groups, P < 0.05); Figure 5 shows the effect of ultrasonic time on the resveratrol extraction yield from peanut sprouts (Note: different letters indicate different groups, P < 0.05); Figure 6 shows the effect of ultrasonic temperature on the resveratrol extraction yield from peanut sprouts. Figure 7 shows the response surface plot and contour plot of the interaction between the material-liquid ratio and ethanol concentration; Figure 8 shows the response surface plot and contour plot of the interaction between the material-liquid ratio and ultrasonic time; Figure 9 shows the response surface plot and contour plot of the interaction between ethanol concentration and ultrasonic time; Figure 10 shows the bar chart of the change in resveratrol content during the germination process of different peanut varieties; Note: Different letters indicate different groups, P < 0.05; Figure 11 shows the bar chart of the comparison of ABTS antioxidant activity of peanut sprouts of different varieties; Note: Different letters indicate different groups, P < 0.05. Detailed Implementation

[0018] This invention provides the application of peanuts in the extraction of resveratrol, wherein the peanuts include at least one of the following varieties: Zhongyuhua 100, Yuhua 23 and Yuhua 99.

[0019] In this invention, resveratrol is preferably extracted from peanuts in the form of peanut sprouts. The peanut sprouts preferably include those that have germinated for 4-6 days, and can be 5 days old. When the germination time of the peanut sprouts is 5 days, the peanut variety is preferably Zhongyuhua 100 and / or Yuhua 23. When the germination time of the peanut sprouts is 4 days, the peanut variety is preferably Zhongyuhua 100 and / or Yuhua 99. Germination preferably includes germination in the dark. Experiments show that different germination days can affect the resveratrol extraction rate, which may be due to the regulation of specific functional gene expression levels during germination in different peanut varieties.

[0020] This invention provides a method for ultrasonic-assisted extraction of resveratrol from peanut sprouts, comprising the following steps: preparing peanut sprouts; mixing the peanut sprouts with an ethanol aqueous solution and then performing ultrasonic extraction to obtain an extract; wherein the peanuts include at least one of the following varieties: Zhongyuhua 100, Yuhua 23, and Yuhua 99.

[0021] This invention produces peanut sprouts from peanuts.

[0022] In this invention, the method for producing peanut sprouts preferably involves removing impurities from the surface of peanut seeds, soaking them in water, and then germinating them in the dark. The impurity removal method preferably involves washing the seed surface with deionized water. The soaking time is preferably 7-10 hours. The soaking temperature is preferably 28-32°C, and can be 30°C. The soaking is preferably carried out in an artificial climate chamber. The germination time in the dark is preferably 4-6 days, and can be 5 days. The germination temperature in the dark is preferably 28-30°C, and can be 29°C. During the germination in the dark, the deionized water is preferably changed daily.

[0023] After obtaining peanut sprouts, the present invention mixes the peanut sprouts with an ethanol aqueous solution and then performs ultrasonic extraction to obtain an extract.

[0024] In this invention, to achieve full extraction, the peanut sprouts preferably undergo a pulverizing process. The peanut sprouts are preferably pulverized to a powder of 20 mesh or less, and can be 40-100 mesh.

[0025] In this invention, the volume concentration of the ethanol-water solution is preferably 70%~90%, can be 75%~85%, or can be 81%. The material-to-liquid ratio of the peanut sprouts to the ethanol-water solution is preferably 1g:25~30mL, can be 1g:27~29mL, or can be 1g:28mL. The ethanol-water solution, as the extraction solution, can effectively and fully extract resveratrol from the peanut sprouts.

[0026] In this invention, the ultrasonic treatment time is preferably 35-75 min, but can be 40-70 min, 45-65 min, or 50-64 min. The ultrasonic treatment temperature is preferably 30-70℃, but can be 40-65℃ or 50-60℃. The ultrasonic treatment utilizes the cavitation effect of ultrasound to fully extract resveratrol from peanut sprout powder, which can greatly improve the extraction rate of the active ingredient.

[0027] After ultrasonic extraction, the crude extract is collected and purified. The preferred purification method involves centrifuging the crude extract, collecting the supernatant, removing water, reconstituted with methanol, centrifuging a second time, collecting the supernatant, filtering through a filter membrane, and collecting the filtrate to obtain purified resveratrol. The centrifugation speed is preferably 8000-12000 rpm, and can be 10000 rpm. The centrifugation time is preferably 12-17 min, and can be 15 min. The water removal method is preferably nitrogen drying. The second centrifugation speed is preferably 6000-10000 rpm, and can be 8000 rpm. The second centrifugation time is preferably 4-6 min, and can be 5 min. The pore size of the filter membrane is preferably 0.45 μm.

[0028] This invention provides the application of the extract prepared by the method in the development of antioxidant functional products, wherein the peanut includes Yuhua 93.

[0029] In this invention, the antioxidant preferably includes the ability to scavenge ABTS free radicals.

[0030] In this invention, the extract prepared by the method includes resveratrol and other antioxidant active ingredients. In embodiments of this invention, experiments were conducted using different peanut varieties as raw materials to extract resveratrol. The results showed that compared with Zhongyuhua 100, Gonghua 23, Fan Gonghua 138, Yuhua 99, and Yuanza 9307, Yuhua 93 exhibited the strongest antioxidant capacity during the 3-5 day germination period. This indicates that, during germination, the peanut sprouts of Yuhua 93, in addition to synthesizing resveratrol, also produce other types of antioxidant active ingredients, giving its extract a significant advantage in scavenging ABTS free radicals. Therefore, Yuhua 93 can be used for the cultivation of peanut varieties rich in antioxidant active ingredients and for the study of their internal mechanisms.

[0031] The following detailed description, in conjunction with embodiments, illustrates a method for ultrasonic-assisted extraction of resveratrol from peanut sprouts and its application, but these descriptions should not be construed as limiting the scope of protection of this invention.

[0032] Example 1: A method for extracting resveratrol from peanut sprouts. 1. Raw material preparation: Peanuts were soaked in the dark at 30°C for 8 hours, and then sprouted in the dark at 29°C for 5 days. The obtained peanut sprouts were freeze-dried for 96 hours and then pulverized through a 20-mesh sieve.

[0033] 2. Extraction process: Weigh 1.000 g of peanut sprout powder and place it in a 100 mL Erlenmeyer flask. Add ethanol solution to dissolve and extract resveratrol from the peanut sprout powder. Then, sonicate for a certain period of time and collect the crude extract.

[0034] 3. Separation and purification: Take 10 mL of the crude extract, centrifuge at 10000 r / min for 15 min, take 4 mL of the supernatant and blow it dry with nitrogen, add 1 mL of methanol to reconstitute, shake thoroughly to mix, centrifuge at 8000 r / min for 5 min, and use a disposable sterile syringe to draw an appropriate amount of the supernatant solution and filter it through a 0.45 μm microporous filter to obtain the test solution. Keep the entire operation as dark as possible, and set up three replicates for each experiment.

[0035] 4. HPLC detection conditions were as follows: column: MicroPulite Platinum C18 (4.6 × 250 mm / 5 μm); mobile phase: acetonitrile:water (30:70, V / V); flow rate: 1 mL / min; column temperature: 28℃; detection wavelength: 306 nm; injection volume: 20 μL. The HPLC chromatogram of resveratrol standards is shown in Figure 1. The resveratrol standard curve is shown in Figure 2.

[0036] The extraction rate of resveratrol was calculated according to Formula I.

[0037] X = (C × V) × n / M × 1000 Formula I.

[0038] In the formula: X is the resveratrol extraction rate, mg / g; M is the mass of crushed and sieved peanut sprout powder, g; V is the volume of the extract after dilution, mL; n is the dilution factor; C is the mass concentration of resveratrol, g / mL.

[0039] Example 2: Process Parameter Optimization Experiment (Response Surface Methodology) Based on the extraction method in Example 1, single-factor optimization was performed. The single-factor variables were the ratio of peanut sprout powder to ethanol solution (X1, 1:15, 1:20, 1:25, 1:30, and 1:35), the ethanol solution concentration (X2, 70%, 75%, 80%, 85%, and 90%), the ultrasonic time (X3, 35 min, 45 min, 55 min, 65 min, and 75 min), and the ultrasonic temperature (X4, 30℃, 40℃, 50℃, 60℃, and 70℃). The resveratrol extraction yield was used as the evaluation index. The results of the single-factor variable optimization are shown in Figures 3-6. Based on the single-factor optimization, a Box-Behnken design was used with Design Expert 13 software, employing the ratio of peanut sprout powder to ethanol solution, the ethanol solution concentration, and the ultrasonic time as independent variables, and the resveratrol extraction yield as the response value for further optimization.

[0040] The results are shown in Figures 7-9. The experimental results indicate that the order of influence of each factor on the extraction yield is: solid-liquid ratio > ultrasonic time > ethanol concentration > ultrasonic temperature. Through model prediction and validation, the optimal process parameters were determined to be: solid-liquid ratio 28.45 mL / g (corrected to 1:28), ethanol concentration 81.19% (corrected to 81%), ultrasonic time 63.97 min (corrected to 64 min), and ultrasonic temperature 60℃.

[0041] Three parallel validation experiments were conducted under these optimal conditions, and the average resveratrol extraction yield was measured to be 2.49 μg / g, which is close to the theoretical prediction of 2.42 μg / g, indicating that the model is reliable.

[0042] Example 3: Comparison of Resveratrol Content in Peanut Sprouts of Different Varieties. Peanut sprouts were prepared using the following six peanut varieties: Yuhua 93, Gonghua 23, Fan Gonghua 138, Zhongyuhua 100, Yuhua 99, and Yuanza 9307. Peanut seeds were germinated for 3, 4, and 5 days according to the method in Example 1. Resveratrol was extracted using the optimized process in Example 2 to obtain a crude extract.

[0043] As shown in Figure 10, the resveratrol content of all varieties increased significantly with the increase of germination days. On the 5th day of germination, Zhongyuhua 100 had the highest resveratrol content, reaching 3.15 μg / g, which was significantly higher than other varieties, while varieties such as Yuhua 23 were second highest. Zhongyuhua 100, Yuhua 23, Yuhua 99, and Yunza 9307 are suitable for processing peanut sprouts enriched with resveratrol.

[0044] Example 4: Comparison of Antioxidant Activity of Peanut Sprouts from Different Varieties. The ABTS free radical scavenging ability of the peanut sprout extracts from Example 3 was determined. Free radical generation was achieved using an ABTS and potassium persulfate reaction system. The specific steps were as follows: 5 mL of a 7 mM ABTS solution and 88 μL of a 140 mM potassium persulfate solution were mixed and placed in the dark for 12 h to obtain the ABTS stock solution. This solution was prepared one day in advance and used on the same day. A certain amount of the ABTS stock solution was diluted 65 times with anhydrous ethanol until its absorbance at 732 nm was 0.70 ± 0.02 (OD). 734 The solution was prepared fresh for each use. The crude resveratrol extracts from different peanut sprout varieties in Example 3 were diluted 5 times. 0.1 mL of the diluted solution was added to 3.9 mL of the ABTS working solution and reacted in the dark for 8 min. The absorbance was measured at 732 nm. Three replicates were performed, and anhydrous ethanol was used for zeroing.

[0045] The results are shown in Figure 11. During the 3-5 day germination period, Yuhua 93 exhibited the strongest ABTS scavenging ability, with concentrations of 5.19 mg / g, 5.63 mg / g, and 6.49 mg / g, respectively, all significantly higher than other varieties. Yuhua 93 is suitable for developing antioxidant functional products.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of peanuts in the extraction of resveratrol, characterized in that, The peanuts include at least one of the following varieties: Zhongyuhua 100, Yuhua 23 and Yuhua 99.

2. The application according to claim 1, characterized in that, Resveratrol is extracted from peanuts in the form of peanut sprouts; the peanut sprouts include peanut sprouts that have germinated for 4 to 6 days.

3. A method for ultrasonic-assisted extraction of resveratrol from peanut sprouts, characterized in that, Includes the following steps: Peanuts are processed into peanut sprouts; the peanut sprouts are mixed with an ethanol aqueous solution and then subjected to ultrasonic extraction to obtain an extract; the peanuts include at least one of the following varieties: Zhongyuhua 100, Yuhua 23 and Yuhua 99.

4. The method according to claim 3, characterized in that, The volume concentration of the ethanol aqueous solution is 70%~90%.

5. The method according to claim 3, characterized in that, The ratio of peanut sprouts to ethanol aqueous solution is 1g:25~30mL.

6. The method according to claim 3, characterized in that, The ultrasonic treatment time is 35~75 min; the ultrasonic treatment temperature is 30~70℃.

7. The method according to claim 3, characterized in that, The germination time of the peanut sprouts is 4 to 6 days.

8. The method according to any one of claims 3 to 7, characterized in that, The germination time of the peanut sprouts is 5 days; the volume concentration of the ethanol aqueous solution is 81%; the material-to-liquid ratio of the peanut sprouts to the ethanol aqueous solution is 1g:28mL; the ultrasonic treatment time is 64min; and the ultrasonic treatment temperature is 60℃.

9. The use of the extract prepared by the method according to any one of claims 3 to 8 in the development of antioxidant functional products, wherein the peanut includes Yuhua 93.

10. The application according to claim 9, characterized in that, The antioxidant properties include the ability to scavenge ABTS free radicals.

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