A method for efficiently extracting and purifying polyphenols from raisins

Abstract

The application discloses a method for efficiently extracting and purifying polyphenols from raisins, and relates to the technical field of purification. The application provides specific methods and applications. The application solves the problems of low recovery efficiency of raisin polyphenols, complex process, repeated extraction of various organic reagents, environmental pollution and difficult recovery in the traditional extraction and purification process of raisin polyphenols in the prior art. The application uses an ethanol aqueous solution as a solvent, and the solvent can be easily recovered and recycled by rotary evaporation, thereby significantly reducing environmental pollution. The application also obtains suitable process parameters, conditions and material ratios, and clearly defines the selection of materials.

Description

Technical Field

[0001] This invention relates to the field of purification technology, and more specifically to a method for efficiently extracting and purifying polyphenols from raisins. Background Technology

[0002] The Xinjiang Uyghur Autonomous Region, with its unique climate, produces nutritious and high-quality fruits, making it a major fruit production base in China. In particular, Xinjiang ranks first in the country in both planting area and yield of grapes. Furthermore, raisins made from certain high-quality grape varieties produced in the region enjoy a high reputation in both international and domestic markets.

[0003] Raisins are rich in various bioactive components, such as flavonoids, polysaccharides, and polyphenols, which endow them with high biological activity and nutritional and health benefits, making them an indispensable health food in the daily diet. Due to their high polyphenol content, raisins exhibit powerful active functions, providing adjunctive therapeutic effects such as anti-inflammatory and anti-diabetic benefits.

[0004] Macroporous resins (MRs) have attracted much attention in the field of polyphenol purification due to their advantages such as high reusability, low solvent consumption, strong selectivity and low cost.

[0005] However, in traditional processes, the adsorption / desorption process requires a long time to reach equilibrium, and the recovery efficiency of polyphenolic compounds is relatively low.

[0006] Therefore, whether a method can be provided for the efficient extraction and purification of polyphenols from raisins to overcome the above-mentioned technical deficiencies is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a method for efficiently extracting and purifying polyphenols from raisins.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for efficiently extracting and purifying polyphenols from raisins includes the following steps: (1) Grape intervention treatment: The raisins were crushed into raisin powder; (2) Ultrasonic-assisted extraction: Add raisin powder to an ethanol aqueous solution and extract with ultrasonic assistance to obtain crude raisin polyphenol extract; (3) Preparation of crude raisin polyphenol extract: Centrifuge the crude raisin polyphenol extract, take the supernatant, remove the solvent, dry it into powder, and obtain crude raisin polyphenol extract; (4) Ultrasonic-assisted macroporous resin separation and purification of raisin polyphenols: The crude extract of raisin polyphenols was reconstituted and the pH was adjusted. The extract was added to macroporous resin and ultrasonically adsorbed. The resin was then packed into a column for column chromatography separation. The resin surface was washed with distilled water to remove residual liquid. The resin was then repeatedly eluted with ethanol aqueous solution until the eluent was clear and transparent. The raisin polyphenol eluent was collected. (5) Preparation of purified raisin polyphenols: The solvent was removed from the raisin polyphenol effluent and the raisin polyphenols were freeze-dried to obtain purified raisin polyphenols.

[0010] Preferred: Step (1) Grape intervention treatment specifically involves washing, pitting, and cutting raisins into pieces, vacuum drying at 60°C to constant weight, and then pulverizing to obtain raisin powder.

[0011] Preferred step (2): Add raisin powder to an ethanol aqueous solution with a volume fraction of 50-60% at a material-to-liquid ratio of 1:10-30 mL / g, and extract with ultrasonic assistance at 50-70℃ for 60-65 min to obtain crude raisin polyphenol extract.

[0012] Preferred step (3) is as follows: after centrifuging the crude extract of raisin polyphenols at 10,000 rpm for 15 min, take the supernatant, remove the solvent, and freeze-dry to obtain the crude extract of raisin polyphenols.

[0013] Preferred: Step (4) specifically involves: redissolving the crude extract of raisin polyphenols in distilled water to a concentration of 2.5-5 mg / mL, adjusting the pH to 1-7, adding macroporous resin at a resin mass to solution volume ratio of 1 g : 5 mL, performing ultrasonic adsorption at 25°C for 30 min, adding the macroporous resin to the chromatography column, and then washing the saturated macroporous resin 5 times with distilled water to remove unadsorbed residue on the resin surface. The macroporous resin is then repeatedly eluted with 40 mL of 90% ethanol aqueous solution until the effluent is clear and transparent, and the raisin polyphenol effluent is collected.

[0014] Preferred: Macroporous resin: D101 type macroporous resin.

[0015] Preferred step (5): The solvent is removed by rotary evaporation of the raisin polyphenol effluent at 40°C, and then freeze-dried into powder to obtain purified raisin polyphenol.

[0016] The present invention also provides purified raisin polyphenols prepared by any of the above methods.

[0017] The present invention also provides the application of any of the above methods or the above-described raisin polyphenol purifications in food, cosmetics and health supplements.

[0018] As can be seen from the above technical solution, compared with the prior art, this invention discloses a method for efficiently extracting and purifying polyphenols from raisins. The technical effects achieved are as follows: This invention solves the problems of low recovery efficiency of raisin polyphenol compounds, complex processes, and environmental pollution and difficulty in recycling caused by repeated extraction with multiple organic reagents in traditional purification processes. This invention uses an ethanol-water solution as a solvent, and the solvent is easily recovered and recycled through rotary evaporation, significantly reducing environmental pollution. This invention also obtains suitable process parameters, conditions, and material ratios, and clarifies the selection of materials. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 The attached figure is a flowchart of the extraction and purification process of raisin polyphenols provided by the present invention.

[0021] Figure 2 The attached figure shows the effect of different extraction conditions provided by the present invention on the extraction yield of raisin polyphenols, wherein (a): the effect of liquid-to-solid ratio on the extraction yield of total raisin polyphenols; (b): the effect of ethanol concentration on the extraction yield of total raisin polyphenols; (c): the effect of extraction time on the extraction yield of total raisin polyphenols; and (d): the effect of extraction temperature on the extraction yield of total raisin polyphenols.

[0022] Figure 3 The attached figure shows the factors affecting the adsorption and desorption performance of macroporous resin for raisin polyphenols provided by the present invention, wherein: (a): the effect of resin type on adsorption and desorption capacity; (b): the effect of sample concentration on resin adsorption capacity; (c): the effect of ethanol concentration on desorption rate of D101 resin; (d): the effect of sample pH value on adsorption capacity of D101 resin.

[0023] Figure 4 The attached figure is a dynamic desorption curve of the D101 macroporous resin provided by the present invention.

[0024] Figure 5 The attached figure shows a comparison of the purity of raisin polyphenols before and after purification provided by this invention.

[0025] Figure 6 The attached figure is a comparison chart of polyphenol content extracted from raisins using different solvents provided by the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] This invention discloses a method for efficiently extracting and purifying polyphenols from raisins. See the process flow diagram below. Figure 1 .

[0028] In the examples, all raw materials not mentioned are commercially available, and all experimental methods not mentioned are conventional experimental methods, which will not be described in detail here.

[0029] Example 1 A method for efficiently extracting and purifying polyphenols from raisins includes the following steps: (1) Grape intervention treatment: Wash the raisins, remove the pits, cut them into pieces, vacuum dry them at 60℃ to constant weight, pulverize them to obtain raisin powder, and seal them for storage at 4℃ for later use. (2) Ultrasonic-assisted extraction: Raisin powder was added to a beaker containing 50% ethanol aqueous solution at a material-to-liquid ratio of 1:10 mL / g, and ultrasonic-assisted extraction was performed at 50℃ for 55 min to obtain crude raisin polyphenol extract. (3) Preparation of crude extract of raisin polyphenols: After centrifuging the crude extract of raisin polyphenols at 10,000 rpm for 15 min, the supernatant was collected, the solvent was removed, and the crude extract of raisin polyphenols was freeze-dried and stored in a -20℃ refrigerator for later use. (4) Ultrasonic-assisted separation and purification of raisin polyphenols using macroporous resin The crude extract of raisin polyphenols was reconstituted with distilled water to a concentration of 2.5 mg / mL, and the pH was adjusted to 1. D101 macroporous resin was added at a resin mass to solution volume ratio of 1 g : 5 mL. The adsorption was performed at 25℃ with ultrasonic assistance for 30 min. The macroporous resin was then added to a glass resin chromatography column, and the saturated macroporous resin was washed 5 times with distilled water to remove the unadsorbed residue on the resin surface. The macroporous resin was repeatedly eluted with 40 mL of 90% ethanol aqueous solution until the eluent was clear and transparent. The raisin polyphenol eluent was collected. (5) Preparation of purified raisin polyphenols The solvent was removed by rotary evaporation of the raisin polyphenols at 40°C, and the product was freeze-dried into powder. The purified raisin polyphenols were then stored at -20°C for later use.

[0030] Example 2 A method for efficiently extracting and purifying polyphenols from raisins includes the following steps: (1) Grape intervention treatment: Wash the raisins, remove the pits, cut them into pieces, vacuum dry them at 60℃ to constant weight, pulverize them to obtain raisin powder, seal them and store them at 4℃ for later use. (2) Ultrasonic-assisted extraction: Raisin powder was added to a 50% ethanol solution at a material-to-liquid ratio of 1:20 mL / g, and extracted with ultrasound at 60℃ for 60 min to obtain crude raisin polyphenol extract. (3) Preparation of crude extract of raisin polyphenols: After centrifuging the crude raisin polyphenol extract at 10,000 rpm for 15 min, the supernatant was collected, the solvent was removed, and the crude raisin polyphenol extract was freeze-dried and stored at -20℃ for later use. (4) Ultrasonic-assisted separation and purification of raisin polyphenols using macroporous resin The crude extract of raisin polyphenols was reconstituted with distilled water to a concentration of 3.5 mg / mL, and the pH was adjusted to 5. D101 macroporous resin was added at a resin mass to solution volume ratio of 1 g: 5 mL. The adsorption was performed at 25℃ with ultrasonic assistance for 30 min. The macroporous resin was then added to a chromatography column, and the D101 macroporous resin was washed 5 times with distilled water to remove unadsorbed residue on the resin surface. The macroporous resin was then repeatedly eluted with 40 mL of 90% ethanol solution until the eluent was clear and transparent. The raisin polyphenol eluent was collected. (5) Preparation of purified raisin polyphenols The solvent was removed by rotary evaporation of the raisin polyphenols at 40°C, and the product was freeze-dried into powder. The purified raisin polyphenols were then stored at -20°C for later use.

[0031] Example 3 A method for efficiently extracting and purifying polyphenols from raisins includes the following steps: (1) Grape intervention treatment: Wash, pit, and cut the raisins into pieces. Vacuum dry them at 60°C to constant weight, then pulverize them and pack them into sample bags and seal them to obtain raisin powder. Store them at 4°C for later use. (2) Ultrasonic-assisted extraction: Raisin powder was added to a 60% ethanol aqueous solution at a material-to-liquid ratio of 1:30 mL / g, and ultrasonic extraction was performed at 70℃ for 65 min to obtain crude raisin polyphenol extract. (3) Preparation of crude extract of raisin polyphenols: After centrifuging the crude raisin polyphenol extract at 10,000 rpm for 15 min, the supernatant was collected, the solvent was removed, and the crude raisin polyphenol extract was freeze-dried and stored at -20℃ for later use. (4) Ultrasonic-assisted separation and purification of raisin polyphenols using macroporous resin The crude extract of raisin polyphenols was reconstituted with distilled water to a concentration of 5 mg / mL, and the pH was adjusted to 7. D101 macroporous resin was added at a resin mass to solution volume ratio of 1 g : 5 mL. The adsorption was performed at 25℃ with ultrasonic assistance for 30 min. The macroporous resin was then added to a chromatography column, and the saturated macroporous resin was washed 5 times with distilled water to remove the unadsorbed residue on the resin surface. The macroporous resin was repeatedly eluted with 40 mL of 90% ethanol solution until the eluent was clear and transparent. The raisin polyphenol eluent was collected. (5) Preparation of purified raisin polyphenols The solvent was removed by rotary evaporation of the raisin polyphenols at 40°C, and the product was freeze-dried into powder. The purified raisin polyphenols were then stored at -20°C for later use.

[0032] Comparative test Comparative Experiment 1 Effects of different extraction conditions on the yield of raisin polyphenols The experimental variable was the step of "adding raisin powder to an ethanol aqueous solution, extracting with ultrasonic assistance, and obtaining crude raisin polyphenol extract (after a certain time)".

[0033] Experimental results: see Figure 2 : Effect of solid-liquid ratio on the total polyphenol extraction yield of raisins: From Figure 2 As can be seen in (a), the extraction yield of total polyphenols from raisins reaches its maximum when the liquid-to-solid ratio is 20 mL / g. As the liquid-to-solid ratio continues to increase, the extraction yield of total polyphenols from raisins shows a decreasing trend. This is because the increase in the liquid-to-solid ratio can expand the concentration gradient inside and outside the cells, and the mass transfer rate of polyphenol compounds also increases accordingly, thus increasing the extraction yield. However, when the liquid-to-solid ratio is too high, the mass transfer process is mainly limited by the absolute amount of raw materials, which leads to the gradual weakening of the promoting effect of the concentration gradient on the mass transfer rate, resulting in a decreasing trend in the extraction yield.

[0034] Effect of ethanol concentration on the total polyphenol extraction yield from raisins: From Figure 2 As can be seen in (b), the total polyphenol extraction yield of raisins reaches its maximum when the ethanol concentration is 50%. When the ethanol concentration is further increased, the total polyphenol extraction yield of raisins shows a downward trend. This is because excessively high ethanol concentrations can cause plant cells to dehydrate, thereby inhibiting the diffusion efficiency of polyphenol compounds from the plant matrix to the solvent, resulting in a decrease in the extraction yield.

[0035] Effect of extraction time on the total polyphenol yield from raisins: From Figure 2 As can be seen in (c), the total polyphenol content of raisins reached its maximum when the extraction time was 60 min. As the extraction time continued to increase, the total polyphenol content of raisins showed a decreasing trend. The main reason for this may be that the increased extraction time led to an increase in solution temperature, which triggered the oxidative polymerization of polyphenol compounds and the degradation of their structure, resulting in a decrease in polyphenol content.

[0036] Effect of extraction temperature on the total polyphenol extraction yield from raisins: From Figure 2 As shown in Figure (d), the extraction yield of total polyphenols from raisins reaches its maximum at an extraction temperature of 60℃. Subsequently, as the extraction temperature continues to increase, the extraction yield of total polyphenols from raisins decreases. This is because heating may cause damage to the cell structure, leading to the fragmentation of the cell wall and cell membrane, which promotes the release of polyphenol compounds and increases the extraction rate. However, as the temperature continues to increase, the viscosity and surface tension of the solvent decrease significantly, and the high temperature weakens the cavitation intensity, which in turn leads to a decrease in extraction efficiency.

[0037] The optimal extraction conditions for raisin polyphenols were determined to be: a liquid-to-solid ratio of 20 mL / g, an ethanol concentration of 50%, an extraction time of 60 min, and an extraction temperature of 60℃.

[0038] Comparative Experiment 2 The experimental variable was the following steps: "Redissolve crude raisin polyphenol extract in distilled water, adjust the pH value, add macroporous resin, perform ultrasonic adsorption at 25°C for 30 min, add macroporous resin to a chromatography column, wash the saturated macroporous resin five times with distilled water to remove unadsorbed residue on the resin surface, and then repeatedly elute the macroporous resin with 40 ml of ethanol solution until the eluent is clear and transparent, and collect the raisin polyphenol eluent".

[0039] The experimental results are shown in Figure 3 : like Figure 3 As shown in (a), XAD-7 macroporous resin has the strongest adsorption capacity, but its adsorption capacity is the lowest among the five macroporous resins. D101 macroporous resin, on the other hand, has strong adsorption capacity and excellent desorption capacity; therefore, D101 macroporous resin was selected. Figure 3 As shown in (b), the resin adsorption capacity gradually increases with increasing polyphenol concentration in the solution. This is because the adsorption equilibrium shifts to a state more favorable for adsorption, thereby increasing the amount of polyphenol adsorbed on the resin and thus more effectively promoting the adsorption reaction. However, the adsorption rate decreases again after reaching 3.5 mg / mL. Figure 3In (c), when the ethanol concentration of the eluent is below 90%, the desorption capacity of polyphenols increases significantly with increasing ethanol concentration. However, when the concentration exceeds 90%, the desorption capacity decreases significantly. This decrease is because the hydrogen bonds between polyphenols and resins are relatively strong and not easily broken by low-concentration eluents, but at excessively high concentrations, other adsorbed impurities (such as proteins and pigments) will also be eluted. Figure 3 Figure (d) shows that the adsorption capacity of D101 macroporous resin initially increases and then gradually decreases with increasing pH of the sample solution, reaching its maximum at pH 5. This is because lower pH favors the presence of hydroxyl groups in the polyphenol molecules as hydrogen bond donors, which readily form hydrogen bonds with acceptors on the resin surface, thereby enhancing adsorption. Conversely, as pH increases, these hydrogen bonds may weaken, thus reducing adsorption capacity. Based on these observations, D101 macroporous resin, a sample solution pH of 5, a concentration of 3.5 mg / mL, and an eluent ethanol volume fraction of 90% were selected for subsequent experiments.

[0040] Comparative Experiment 3 Effect of different volumes of 90% ethanol solution on the concentration of raisin polyphenols The experimental variable was "repeatedly eluting the macroporous resin with different volumes of 90% ethanol aqueous solution until the effluent was clear and transparent".

[0041] The experimental results are shown in Figure 4 : Under dynamic conditions, most of the raisin polyphenols adsorbed on the D-101 macroporous resin can be eluted from the resin using approximately 40 mL of 90% ethanol solution, with a relatively concentrated elution peak.

[0042] Technical effect verification Parallel experiments were conducted to verify the method in Example 2. After purification, the purity of raisin polyphenols increased from 21.09±1.9% to 80.52±0.95%, which is 3.8 times that before purification. (See...) Figure 5 .

[0043] Comparative Test 4 The difference from the examples is that ethanol (extraction solvent) is the experimental variable.

[0044] The experimental results are shown in Figure 6 : Ethanol, as one of the solvents for extracting polyphenols from raisins, yielded a polyphenol content of 3.79 mg / g. Although this is lower than that of methanol (4.09 mg / g, approximately 11.98%) and acetone (4.30 mg / g, approximately 7.33%), its toxicity is far lower than methanol and it is safer than acetone, making it suitable for both laboratory and industrial production. Ethanol is widely used in the food, pharmaceutical, and health product industries. It exhibits good biocompatibility, ensuring no residue in the extract; it also demonstrates strong solubility and high selectivity for polyphenolic compounds. As a renewable resource, ethanol production is environmentally friendly and cost-effective, and its moderate volatility facilitates handling and solvent recovery. Considering safety, biocompatibility, solubility, environmental friendliness, and cost-effectiveness, ethanol is an ideal choice for polyphenol extraction.

[0045] In summary, this invention focuses on obtaining polyphenol components with higher activity, higher extraction rate, and environmental friendliness. Based on this, this invention selects ultrasound-assisted extraction as the extraction method, aiming to rapidly and efficiently obtain green and pollution-free natural active polyphenols from raisins (this invention can also be used for dried apricots and walnuts).

[0046] The present invention also obtained suitable process parameters, conditions, and material ratios, and clarified the selection of materials.

[0047] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0048] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for efficiently extracting and purifying polyphenols from raisins, characterized in that, Includes the following steps: (1) Grape intervention treatment: The raisins were crushed into raisin powder; (2) Ultrasonic-assisted extraction: Add raisin powder to an ethanol aqueous solution and extract with ultrasonic assistance to obtain crude raisin polyphenol extract; (3) Preparation of crude raisin polyphenol extract: Centrifuge the crude raisin polyphenol extract, take the supernatant, remove the solvent, dry it into powder, and obtain crude raisin polyphenol extract; (4) Ultrasonic-assisted macroporous resin separation and purification of raisin polyphenols: The crude extract of raisin polyphenols was reconstituted and the pH was adjusted. The extract was added to macroporous resin and ultrasonically adsorbed. The resin was then packed into a column for column chromatography separation. The resin surface was washed with distilled water to remove residual liquid. The resin was then repeatedly eluted with ethanol aqueous solution until the eluent was clear and transparent. The raisin polyphenol eluent was collected. (5) Preparation of purified raisin polyphenols: The solvent was removed from the raisin polyphenol effluent and the raisin polyphenols were freeze-dried to obtain purified raisin polyphenols.

2. The method as described in claim 1, characterized in that, The grape intervention process in step (1) specifically involves washing, pitting, and cutting the raisins into pieces, vacuum drying at 60°C to constant weight, and then pulverizing them to obtain raisin powder.

3. The method as described in claim 2, characterized in that, Step (2) Add raisin powder to an ethanol aqueous solution with a volume fraction of 50-60% at a material-to-liquid ratio of 1:10-30 mL / g, and extract with ultrasonic assistance at 50-70℃ for 60-65 min to obtain crude raisin polyphenol extract.

4. The method as described in claim 3, characterized in that, Step (3) is as follows: After centrifuging the crude extract of raisin polyphenols at 10,000 rpm for 15 min, take the supernatant, remove the solvent, and freeze-dry to obtain the crude extract of raisin polyphenols.

5. The method as described in claim 4, characterized in that, Step (4) is as follows: the crude extract of raisin polyphenols is reconstituted with distilled water to a concentration of 2.5-5 mg / mL, and the pH value is adjusted to 1-7. The macroporous resin is added at a resin mass to solution volume ratio of 1 g : 5 mL. After ultrasonic adsorption at 25℃ for 30 min, the macroporous resin is added to the chromatography column. Then, the macroporous resin after adsorption saturation is washed 5 times with distilled water to remove the unadsorbed residue on the resin surface. The macroporous resin is repeatedly eluted with 40 mL of 90% ethanol aqueous solution until the effluent is clear and transparent. The raisin polyphenol effluent is collected.

6. The method as described in claim 5, characterized in that, The macroporous resin is D101 type macroporous resin.

7. The method as described in claim 6, characterized in that, Step (5) The solvent in the raisin polyphenol effluent is removed by rotary evaporation at 40°C, and then freeze-dried into powder to obtain purified raisin polyphenol.

8. The purified raisin polyphenols prepared by any one of the methods described in claims 1 to 7.

9. The use of the method according to any one of claims 1 to 7 or the purified raisin polyphenols according to claim 8 in food, cosmetics and health supplements.

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