Green extraction method of procyanidine in grape seeds

Abstract

The invention relates to the technical field of procyanidine extraction, and provides a green extraction method of procyanidine in grape pips, which comprises the following steps: carrying out physical wall breaking treatment on dry grape pips; mixing the pretreated grape seeds with the eutectic solvent precursor liquid, and carrying out solid-liquid separation to obtain a clear extracting solution; adding an azobenzene chitosan solution into the clarified extracting solution, mixing under a dark condition, irradiating by adopting light with a first specific wavelength to form a compound precipitate, and separating to obtain a precipitate; dispersing the compound precipitate in an aqueous medium, irradiating with light with a second specific wavelength to release the proanthocyanidins from the compound, separating to obtain a supernatant containing the proanthocyanidins, and recycling regenerated azobenzene chitosan; and concentrating and drying the supernate to obtain a procyanidine product. According to the technical scheme, the extraction efficiency and purity of the procyanidine are improved, and efficient selective precipitation separation and mild release are realized by utilizing the photoresponsive carrier.

Description

Technical Field

[0001] This invention relates to the field of proanthocyanidin extraction technology, specifically to a green extraction method for proanthocyanidins from grape seeds. Background Technology

[0002] Grape seeds are rich in proanthocyanidins, typically containing 5%-8%, making them an important raw material for proanthocyanidin extraction. The unique chemical structure of proanthocyanidins endows them with powerful antioxidant capabilities, enabling them to scavenge free radicals, inhibit lipid peroxidation, and protect cells from oxidative damage. This plays a vital role in preventing cardiovascular disease, delaying aging, and improving vision. Furthermore, proanthocyanidins possess various biological activities, including anti-inflammatory, antibacterial, and antitumor effects, giving them broad application prospects in the pharmaceutical field.

[0003] While existing extraction methods can achieve proanthocyanidin extraction to some extent, they still have shortcomings in terms of extraction efficiency and purity. Solvent extraction has low efficiency and requires long extraction times and large amounts of solvent; ultrasonic-assisted extraction and microwave-assisted extraction, although improving extraction efficiency, may damage the structure of proanthocyanidins, affecting their purity and bioactivity; supercritical fluid extraction, while obtaining high-purity proanthocyanidins, has high equipment costs, complex operation, and is difficult to implement on a large scale. Existing extraction methods also have many problems regarding process sustainability. The organic solvents used in solvent extraction are mostly volatile and toxic, causing environmental pollution, and solvent recovery and reuse are difficult. To address the problems of low extraction efficiency and purity, poor process sustainability, and insufficient selectivity in existing green extraction methods of proanthocyanidins from grape seeds, this invention proposes a green extraction method for proanthocyanidins from grape seeds. Summary of the Invention

[0004] This invention proposes a green extraction method for proanthocyanidins from grape seeds, which improves the extraction efficiency and purity of proanthocyanidins. It utilizes a photoresponsive carrier, azobenzyl chitosan, to achieve efficient and selective precipitation separation and gentle release. It also improves the sustainability of the process by reducing costs and waste through a closed-loop design of carrier regeneration and solvent reuse.

[0005] The technical solution of the present invention is as follows: This invention proposes a green extraction method for proanthocyanidins from grape seeds, comprising the following steps: S1. Physically break down the dried grape seeds to create microcracks on their surface; mix the pretreated grape seeds with a eutectic solvent precursor solution and extract by stirring at 40-70℃ for 60-120 min, then separate the solid and liquid to obtain a clear extract. S2. Add azobenzyl chitosan solution to the clarified extract, mix under light-protected conditions, and irradiate with light of a first specific wavelength to cause the azobenzyl chitosan to combine with the proanthocyanidins in the extract to form a complex precipitate, and separate to obtain the precipitate. S3. The complex is precipitated and dispersed in an aqueous medium and irradiated with a second specific wavelength of light to release proanthocyanidins from the complex. After separation, a supernatant containing proanthocyanidins is obtained, and the regenerated azobenzyl chitosan is recovered. S4. Concentrate and dry the supernatant containing proanthocyanidins to obtain the proanthocyanidin product.

[0006] In the proanthocyanidin extraction step, this invention employs a combination of physical cell disruption and eutectic solvent precursor liquid extraction to achieve efficient and green extraction. Physical cell disruption (such as rolling, pressing, or ball milling) creates microcracks on the surface of grape seeds. This process disrupts the cell structure of the grape seeds, increases the release channels of proanthocyanidins within the cells, and facilitates the subsequent extraction process.

[0007] As a further technical solution, the preparation method of the azobenzened chitosan solution includes: dissolving chitosan in an aqueous acetic acid solution with a concentration of 0.5%-2.0% to obtain a chitosan-acetic acid solution with a mass concentration of 1%-3%; activating 4-carboxyazobenzene in a solvent in the presence of a condensing agent and an activating agent to obtain an activated solution; reacting the activated solution with the chitosan-acetic acid solution at pH 4.5-6.0 under light-protected conditions for 12-36 hours; after the reaction is completed, purifying and drying the product to obtain azobenzened chitosan; dissolving the obtained azobenzened chitosan in an aqueous acetic acid solution with a concentration of 0.5%-2.0% to prepare a stock solution with a mass-volume concentration of 0.1%-1.0%.

[0008] As a further technical solution, the mass ratio of chitosan to 4-carboxyazobenzene is 10:3-5; the condensing agent is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dicyclohexylcarbodiimide; and the activator is N-hydroxysuccinimide or N-hydroxythiosuccinimide.

[0009] As a further technical solution, in step S1, the physical cell wall breaking treatment is roller pressing, rolling or ball milling; the crack width of the grape seeds after treatment is 0.5-2.0 mm.

[0010] As a further technical solution, in step S1, the eutectic solvent precursor liquid is prepared by dissolving a hydrogen bond acceptor and a hydrogen bond donor in water; the hydrogen bond acceptor is betaine, and the hydrogen bond donor is L-lactic acid; the molar ratio of betaine to L-lactic acid is 1:1.5-2.5; and the total mass concentration of the eutectic solvent precursor liquid is 5%-20%.

[0011] Hydrogen bonding in the eutectic solvent precursor solution enables specific interactions with proanthocyanidin molecules, enhancing the solubility of proanthocyanidins. Simultaneously, the release channels increased by physical cell disruption, combined with the excellent solubility of the eutectic solvent precursor solution, allow proanthocyanidins to dissolve more fully from grape seeds, improving extraction efficiency. Furthermore, this extraction system is environmentally friendly, avoiding the use of traditional organic solvents.

[0012] As a further technical solution, in step S1, the mass-to-volume ratio of the grape seeds to the eutectic solvent precursor liquid is 1 g: 10-14 mL; the extraction temperature is 45-55℃, and the extraction time is 70-90 min.

[0013] As a further technical solution, in step S2, after adding the azobenzyl chitosan solution, the final concentration of azobenzyl chitosan in the system is 0.01-0.2 g / L; the first specific wavelength light is blue light with a wavelength of 420-480 nm and a light intensity of 20-60 mW / cm². 2 The irradiation time is 10-60 minutes; stirring is performed during irradiation at a speed of 100-300 rpm.

[0014] As a further technical solution, in step S3, the second specific wavelength light is ultraviolet light with a wavelength of 340-380 nm and a light intensity of 20-50 mW / cm². 2 The irradiation time is 10-40 min; the aqueous medium is water or a buffer solution with pH 5.0-7.0.

[0015] As a further technical solution, in step S2, the supernatant obtained after separation of the precipitate is replenished with the consumed solute to the initial concentration and then reused in the extraction process of step S1; in step S3, the regenerated azobenzened chitosan recovered after separation is washed and dissolved and can be recycled for photocontrolled precipitation in step S2.

[0016] As a further technical solution, in step S4, the concentration is carried out under reduced pressure at 40-60°C; the drying is carried out by spray drying or freeze drying.

[0017] The working principle and beneficial effects of this invention are as follows: 1. In this invention, azobenzened chitosan is used as a photoresponsive carrier. Chitosan itself has good biocompatibility and modifiability. By chemically coupling 4-carboxylated azobenzene with chitosan, the photoresponsive group azobenzene is successfully introduced into the chitosan structure. The azobenzene group undergoes cis-isomerization under blue light (420-480 nm) irradiation, inducing a conformational change in the carrier. Through hydrophobic interactions, it forms a complex precipitation with proanthocyanidins, achieving highly efficient and selective precipitation and separation of proanthocyanidins. Ultraviolet light (340-380 nm) irradiation triggers retroisomerization, restoring the carrier's hydrophilicity and achieving a gentle release of proanthocyanidins. This photoresponsive mechanism avoids the damage to the target structure caused by traditional methods. Furthermore, through precise control of light intensity and irradiation time, high selectivity in the precipitation and release processes is achieved, thereby improving extraction efficiency and purity. 2. This invention significantly improves the sustainability of the process through a closed-loop design of photoresponsive carrier azobenzyl chitosan regeneration and solvent reuse. The supernatant after separation in step S2 can be directly reused in the extraction process after solute replenishment, avoiding solvent waste. The regenerated azobenzyl chitosan is recycled for photocontrolled precipitation after washing, and its cycle stability is far superior to that of traditional methods. This not only reduces raw material costs but also reduces waste generation. In addition, the mildness of the photocontrolled method further ensures the long-term performance of the carrier, providing a foundation for industrial applications. 3. This invention employs a synergistic extraction strategy combining physical cell wall disruption with a eutectic solvent precursor solution. First, mechanical forces, such as rolling, pressing, or ball milling, create microcracks on the surface of grape seeds, disrupting the cell wall structure and increasing the release channels for proanthocyanidins, thus improving their extractability. The eutectic solvent precursor solution is prepared by dissolving hydrogen bond acceptors (betaine) and hydrogen bond donors (L-lactic acid) in water. Its unique intermolecular hydrogen bond network structure can form various interactions with proanthocyanidin molecules, such as hydrogen bonds and van der Waals forces, thereby enhancing the solubility and extraction capacity of proanthocyanidins. Physical cell wall disruption provides favorable conditions for better contact and extraction of proanthocyanidins by the eutectic solvent precursor solution. The synergistic effect of both significantly improves the extraction efficiency of proanthocyanidins. Furthermore, the eutectic solvent precursor solution has advantages such as being green, environmentally friendly, and biodegradable, aligning with the concept of green extraction. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1 This embodiment provides a green extraction method for proanthocyanidins from grape seeds, the steps of which include: S1. Preparation of azobenzened chitosan solution: Weigh 10.0 g of chitosan powder and add 500 mL of 1% (w / w) acetic acid aqueous solution. Stir at 200 rpm at room temperature until completely dissolved to obtain a 2% (w / w) chitosan-acetic acid solution. Separately, take 3.96 g (0.016 mol) of 4-carboxyazobenzene and add 200 mL of a 1:1 (v / v) mixture of ethanol and water. While stirring at 250 rpm, add 3.82 g (0.020 mol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.30 g (0.020 mol) of N-hydroxysuccinimide. Activate at 250 rpm for 1 h at room temperature in the dark to obtain an activated 4-carboxyazobenzene solution. Slowly add this activated solution to the chitosan-acetic acid solution while gently stirring at 100 rpm, using 5% (w / w) water during the addition process. The pH of the system was adjusted to 5.0 to 5.5 with NaOH solution. After the addition was complete, the mixture was stirred at 120 rpm for 24 h at room temperature in the dark. After the reaction was completed, the reaction solution was poured into a large amount of anhydrous ethanol to precipitate the solid, which was then filtered and collected as a pale yellow flocculent solid. The solid was redissolved in 500 mL of 1% acetic acid aqueous solution and dialyzed in flowing deionized water for 72 h using a dialysis bag with a molecular weight cutoff of 3500 Da to completely remove unreacted 4-carboxylated azobenzene, carbodiimide, N-hydroxysuccinimide and byproducts. The dialyzed solution was freeze-dried to obtain azobenzened chitosan with a yield of approximately 11.2 g. 12.5 g of the azobenzened chitosan powder was dissolved in 2.5 L of 1% acetic acid aqueous solution and magnetically stirred overnight at 150 rpm to ensure complete dissolution, thus preparing a stock solution with a mass-volume concentration of 0.5%. S2. Raw material pretreatment: Take 2.0 kg of dried Cabernet Sauvignon grape seeds and use a roller press to perform a light compression treatment with a gap of 1.0 mm to obtain raw materials with uniform microcracks. S3. Preparation and extraction of extractant: 167 g (1.2 mol) of betaine and 216 g (2.4 mol) of 80% L-lactic acid were dissolved in 10 kg of deionized water to obtain a eutectic solvent precursor solution with a total concentration of approximately 12%. Grape seeds after gap treatment and 24 L of eutectic solvent precursor solution (solid-liquid ratio 1 g:12 mL) were added to an extraction tank and gently stirred at 180 rpm at 50 °C for 80 min. After extraction, the solution was filtered to obtain approximately 25 L of dark red extract, which was then clarified by filtration through a 0.45 μm membrane. S4. Photocontrolled precipitation and separation: 25 L of clarified extract was transferred to a custom photoreactor with a built-in 450 nm LED surface light source. Under light-protected conditions, 250 mL of azobenzened chitosan stock solution was added at a stirring rate of 200 rpm to bring the final concentration of azobenzened chitosan in the system to approximately 0.05 g / L. Under light-protected conditions, gentle stirring at 200 rpm was performed to ensure thorough mixing of the extract and polymer solution. Subsequently, 450 nm blue light (light intensity 40 mW / cm²) was turned on. 2 The light was used as the first specific wavelength light for irradiation, while gentle stirring was carried out continuously at 200 rpm. After the light irradiation and stirring were stopped, the mixture was allowed to stand for 30 min to allow the complex to precipitate completely. The supernatant was removed by siphon, and the bottom precipitate was collected by centrifugation at 4000 rpm for 10 min to obtain approximately 320 g of wet precipitate. S5. Photocontrolled release and recovery: Disperse the wet precipitate in 2 L of deionized water to form a suspension, and irradiate it under 365 nm ultraviolet light for 20 min (light intensity 30 mW / cm²). 2 During irradiation, the suspension was slowly stirred at 100 rpm. Under ultraviolet light, azobenzene reverted to its trans conformation, weakening the binding force between the polymer and proanthocyanidins. Proanthocyanidins were gradually released into the aqueous phase, and the solution turned clear and deep red. The released mixture was centrifuged at 8000 rpm for 15 minutes to separate the components. The supernatant was an aqueous solution containing proanthocyanidins. The precipitate was azobenzened chitosan restored to its initial state. After collection, it was washed with a small amount of water and redissolved in 1% acetic acid for recycling (the example verified that the precipitation efficiency remained above 90% after 10 cycles). S6. Product Preparation and Solvent Reuse: The supernatant containing proanthocyanidins was concentrated to a solid content of 25% by rotary evaporation at 45°C, and then spray-dried (inlet temperature 160°C, outlet temperature 85°C) to obtain 162 g of dark red powdered proanthocyanidins. The supernatant after the precipitation step, approximately 24.5 L, contained approximately 95% of the eutectic solvent precursor components. After adding a small amount of betaine and lactic acid to the initial concentration, it was directly reused for the next batch extraction. Tests showed that this precursor solution could be recycled at least 6 times without a significant decrease in extraction efficiency.

[0020] Example 2 This embodiment provides a green extraction method for proanthocyanidins from grape seeds, the steps of which include: S1. Preparation of azobenzened chitosan solution: Weigh 10.0 g of chitosan powder and add 1000 mL of 0.5% acetic acid aqueous solution. Stir at 200 rpm at room temperature until completely dissolved to obtain a 1% chitosan-acetic acid solution. Separately, take 5.0 g (0.020 mol) of 4-carboxyazobenzene and add 200 mL of a 1:1 mixture of ethanol and water. While stirring at 250 rpm, add 3.82 g (0.020 mol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.30 g (0.020 mol) of N-hydroxysuccinimide. Activate at 250 rpm for 1 h in the dark at room temperature to obtain an activated 4-carboxyazobenzene solution. Slowly add this activated solution to the chitosan-acetic acid solution while gently stirring at 100 rpm, using 5% [unclear - possibly a specific ingredient or ingredient] during the addition process. The pH of the system was adjusted to 4.5 with NaOH solution. After the addition was complete, the mixture was stirred at 120 rpm for 36 h at room temperature in the dark. After the reaction was completed, the reaction solution was poured into a large amount of anhydrous ethanol to precipitate the solid, which was then filtered and collected as a pale yellow flocculent solid. The solid was redissolved in 500 mL of 0.5% acetic acid aqueous solution and dialyzed in flowing deionized water for 72 h using a dialysis bag with a molecular weight cutoff of 3500 Da to completely remove unreacted 4-carboxylated azobenzene, carbodiimide, N-hydroxysuccinimide and byproducts. The dialyzed solution was freeze-dried to obtain azobenzened chitosan with a yield of approximately 11.8 g. 25.0 g of the azobenzened chitosan powder was dissolved in 2.5 L of 0.5% acetic acid aqueous solution and magnetically stirred overnight at 150 rpm to ensure complete dissolution, thus preparing a stock solution with a mass-volume concentration of 1.0%. S2. Raw material pretreatment: Take 2.0 kg of dried Cabernet Sauvignon grape seeds and crush them to obtain raw materials with uniform microcracks with a crack width of 0.5 mm. S3. Preparation and extraction of extractant: Dissolve 83.5 g (0.6 mol) of betaine and 108 g (1.2 mol) of 80% L-lactic acid in 10 kg of deionized water to obtain a eutectic solvent precursor solution with a total concentration of approximately 5%. Add the pressure-treated grape seeds and 20 L of the eutectic solvent precursor solution (solid-liquid ratio 1 g:10 mL) to the extraction tank and gently extract at 180 rpm for 120 min at 40 °C. After extraction, filter to obtain approximately 21 L of dark red extract, which is then clarified by filtration through a 0.45 μm membrane. S4. Photocontrolled precipitation and separation: 21 L of clarified extract was transferred to a custom photoreactor with a built-in 420 nm LED surface light source. 210 mL of azobenzyl chitosan stock solution was added under light-protected conditions at a stirring rate of 200 rpm, bringing the final concentration of azobenzyl chitosan in the system to approximately 0.10 g / L. Under light-protected conditions, the extract was gently stirred at 200 rpm to ensure thorough mixing with the polymer solution. Subsequently, 420 nm blue light (20 mW / cm²) was turned on as the first specific wavelength for irradiation, while gentle stirring was continued at 200 rpm for 10 min. After stopping the light and stirring, the mixture was allowed to stand for 30 min to allow complete precipitation of the complex. The supernatant was removed by siphon, and the bottom precipitate was collected by centrifugation at 4000 rpm for 10 min, yielding approximately 280 g of wet precipitate. S5. Photocontrolled Release and Recovery: The wet precipitate was dispersed in 2 L of deionized water to form a suspension, and irradiated under 340 nm ultraviolet light for 40 min (light intensity 20 mW / cm²). During irradiation, the suspension was slowly stirred at 100 rpm. Under ultraviolet light, azobenzene reverted to the trans conformation, the binding force between the polymer and proanthocyanidins weakened, and proanthocyanidins were gradually released into the aqueous phase, turning the solution into a clear deep red. The released mixture was centrifuged at 8000 rpm for 15 min to separate the components. The supernatant was an aqueous solution containing proanthocyanidins. The precipitate was azobenzened chitosan restored to its initial state. After collection, it was washed with a small amount of water and redissolved in 0.5% acetic acid for recycling. S6. Product Preparation and Solvent Reuse: The supernatant containing proanthocyanidins was concentrated to a solids content of 35% by rotary evaporation at 40°C, and then freeze-dried. Specific conditions were as follows: the concentrate was pre-frozen at -50°C for 6 hours, and then freeze-dried at -20°C and 20 Pa for 36 hours to obtain a deep red powdered proanthocyanidin product. The supernatant after the precipitation step, approximately 20.8 L, contained about 95% of the eutectic solvent precursor components. After adding a small amount of betaine and lactic acid to the initial concentration, it was directly reused for the next batch extraction.

[0021] Example 3 This embodiment provides a green extraction method for proanthocyanidins from grape seeds, the steps of which include: S1. Preparation of azobenzened chitosan solution: Weigh 10.0 g of chitosan powder and add 333 mL of 2.0% acetic acid aqueous solution. Stir at 200 rpm at room temperature until completely dissolved to obtain a 3% chitosan-acetic acid solution. Separately, take 3.0 g (0.012 mol) of 4-carboxyazobenzene and add 200 mL of a 1:1 mixture of ethanol and water. Under stirring at 250 rpm, add 4.12 g (0.020 mol) of dicyclohexylcarbodiimide and 4.60 g (0.020 mol) of N-hydroxythiosuccinimide. Activate at 250 rpm for 1 h in the dark at room temperature to obtain an activated 4-carboxyazobenzene solution. Slowly add this activated solution to the chitosan-acetic acid solution under gentle stirring at 100 rpm. Adjust the pH of the system to 6.0 with 5% NaOH solution during the addition. After the addition is complete, in the dark at room temperature, stir at 120°C. The reaction was stirred at rpm for 12 hours. After the reaction, the reaction solution was poured into a large amount of anhydrous ethanol to precipitate, and the pale yellow flocculent solid was collected by filtration. The solid was redissolved in 500 mL of 2.0% acetic acid aqueous solution and dialyzed in flowing deionized water for 72 hours using a dialysis bag with a molecular weight cutoff of 3500 Da to completely remove unreacted 4-carboxylated azobenzene, carbodiimide, N-hydroxythiosuccinimide and byproducts. The dialyzed solution was freeze-dried to obtain azobenzened chitosan with a yield of about 10.5 g. 2.5 g of the azobenzened chitosan powder was dissolved in 2.5 L of 2.0% acetic acid aqueous solution and magnetically stirred overnight at 150 rpm to ensure complete dissolution, and a stock solution with a mass-volume concentration of 0.1% was prepared. S2. Raw material pretreatment: Take 2.0 kg of dried Cabernet Sauvignon grape seeds and ball mill them to obtain raw materials with uniform microcracks with a crack width of 2.0 mm. S3. Preparation and extraction of extractant: 167 g (1.2 mol) of betaine and 270 g (3.0 mol) of 80% L-lactic acid were dissolved in 10 kg of deionized water to obtain a eutectic solvent precursor solution with a total concentration of approximately 20%. Grape seeds after gap treatment and 28 L of eutectic solvent precursor solution (solid-liquid ratio 1 g:14 mL) were added to an extraction tank and gently stirred at 180 rpm at 70 °C for 60 min. After extraction, the solution was filtered to obtain approximately 29 L of dark red extract, which was then clarified by filtration through a 0.45 μm membrane. S4. Photocontrolled precipitation and separation: 29 L of clarified extract was transferred to a custom photoreactor with a built-in 480 nm LED surface light source. Under light-protected conditions, 290 mL of azobenzened chitosan stock solution was added at a stirring rate of 300 rpm to bring the final concentration of azobenzened chitosan in the system to approximately 0.01 g / L. Under light-protected conditions, gentle stirring at 300 rpm was performed to ensure thorough mixing of the extract and polymer solution. Subsequently, 480 nm blue light (light intensity 60 mW / cm²) was turned on. 2 The light was used as the first specific wavelength light for irradiation, while the mixture was gently stirred at 300 rpm for 60 min. After the light irradiation and stirring were stopped, the mixture was allowed to stand for 30 min to allow the complex to precipitate completely. The supernatant was removed by siphon, and the bottom precipitate was collected by centrifugation at 4000 rpm for 10 min to obtain approximately 350 g of wet precipitate. S5. Photocontrolled release and recovery: The wet precipitate was dispersed in 2 L of pH 7.0 phosphate buffer solution to form a suspension, and then irradiated under 380 nm ultraviolet light for 10 min (light intensity 50 mW / cm²). 2 During irradiation, the suspension was slowly stirred at 100 rpm. Under ultraviolet light, azobenzene reverted to its trans conformation, the binding force between the polymer and proanthocyanidins weakened, and proanthocyanidins were gradually released into the aqueous phase, turning the solution into a clear deep red. The released mixture was centrifuged at 8000 rpm for 15 min to separate the components. The supernatant was an aqueous solution containing proanthocyanidins. The precipitate was azobenzened chitosan restored to its initial state. After collection, it was washed with a small amount of water and redissolved in 2.0% acetic acid for recycling. S6. Product Preparation and Solvent Reuse: The supernatant containing proanthocyanidins was concentrated to a solid content of 25% by rotary evaporation at 60°C, and then spray-dried (inlet temperature 180°C, outlet temperature 90°C) to obtain a dark red powdered proanthocyanidin product. The supernatant after the precipitation step, approximately 28.7 L, contained approximately 95% of the eutectic solvent precursor components. After adding a small amount of betaine and lactic acid to the initial concentration, it was directly reused for the next batch of extraction.

[0022] Comparative Example 1 Comparative Example 1 describes a synthetic polymer as a substitute for azobenzened chitosan. This comparative example provides a green extraction method for proanthocyanidins from grape seeds, comprising the following steps: S1. Preparation of the azobenzene polymer (PMAA-co-AAB) solution: Weigh 10.0 g of methacrylic acid (MAA) and 2.45 g of 4-acrylamidoazobenzene (AAB) monomer (molar ratio 20:1), dissolve in 200 mL of N,N-dimethylformamide (DMF), and add 0.12 g of azobisisobutyronitrile (AIBN) as an initiator. Under nitrogen protection, stir at 70 °C and 250 rpm for 12 h. After the reaction is complete, the reaction solution is added dropwise to a large amount of diethyl ether to precipitate, and the orange-red solid is collected by filtration. The solid is redissolved in 200 mL of 0.1 mol / L NaOH aqueous solution, and dialyzed in flowing deionized water for 72 h using a dialysis bag with a molecular weight cutoff of 3500 Da. The product is then lyophilized to obtain PMAA-co-AAB copolymer powder, with a yield of approximately 9.8 g. Take 12.5 g of the powder, dissolve it in 2.5 L of 0.1 mol / L NaOH aqueous solution, stir overnight, and prepare a stock solution with a mass-volume concentration of 0.5%. S2. Raw material pretreatment: Same as in Example 1; S3. Preparation of extractant and extraction: Same as in Example 1; S4. Photocontrolled precipitation and separation: Transfer 25 L of clarified extract to a photoreactor, add 250 mL of the above PMAA-co-AAB stock solution in the dark, to bring the final concentration of the system to approximately 0.05 g / L. Subsequent blue light irradiation (40 mW / cm²) is then performed. 2 The precipitation and centrifugation operations were the same as in Example 1, yielding approximately 300 g of wet precipitate; S5. Photocontrolled Release and Recovery: Disperse the wet precipitate in 2 L of deionized water and irradiate it under 365 nm ultraviolet light (30 mW / cm²) for 25 min with slow stirring. After centrifugation, the supernatant is an aqueous solution containing proanthocyanidins. After recovery, the precipitate (polymer) can only be partially dissolved in alkaline aqueous solution, and can be partially recycled after washing. S6. Product preparation and solvent reuse: Same as in Example 1.

[0023] Comparative Example 2 Comparative Example 2 uses a traditional organic solvent extraction method. This comparative example provides a green extraction method for proanthocyanidins from grape seeds, the steps of which include: S1. Preparation of azobenzyl chitosan solution: This step is not required in this comparative example; S2. Raw material pretreatment: Same as in Example 1; S3. Preparation and Extraction of Extractant: Weigh 24 L of 70% (v / v) ethanol aqueous solution and add it to the extraction vessel. Add the grape seeds after gap treatment (solid-liquid ratio 1g:12mL), and extract at 50℃ and 180 rpm for 80 min. After extraction, filter to obtain approximately 25 L of dark red extract, and then clarify by filtration through a 0.45 μm membrane. S4. Traditional Concentration and Precipitation Separation: 25 L of clarified extract was rotary evaporated at 45°C to concentrate it to 1 / 10 of its original volume (approximately 2.5 L). Then, an equal volume of chloroform was added to the concentrate for defatting extraction, and the upper aqueous phase was separated. Three times the volume of ethyl acetate was then added to the upper aqueous phase for extraction, repeated three times, and the ethyl acetate phases were combined. S5. Solvent recovery and product acquisition: The combined ethyl acetate phases were concentrated to dryness under reduced pressure at 40°C to obtain a dark red extract. S6. Product preparation: Dissolve the extract in a small amount of water and then spray dry (inlet temperature 160℃, outlet temperature 85℃) to obtain about 150 g of dark red powdered proanthocyanidin product.

[0024] Comparative Example 3 This comparative example provides a green extraction method for proanthocyanidins from grape seeds, the steps of which include: S1. Preparation of a low-grafting-rate azobenzene-modified chitosan solution: Weigh 1.24 g (0.005 mol) of 4-carboxyazobenzene. The activation and reaction with chitosan follow the same steps as in Example 1. Azobenzene-modified chitosan with a grafting rate of approximately 5% is obtained, yielding approximately 10.5 g. Take 12.5 g of this powder and dissolve it in 2.5 L of 1% acetic acid aqueous solution to prepare a stock solution with a mass-volume concentration of 0.5%. S2. Raw material pretreatment: Same as in Example 1; S3. Preparation of extractant and extraction: Same as in Example 1; S4. Photocontrolled precipitation and separation: The procedure is the same as in Example 1, but after adding the stock solution, the mixture is irradiated with blue light and stirred, and the standing time is extended to 60 minutes to ensure complete precipitation. The supernatant is siphoned off, and after centrifugation, approximately 260 g of wet precipitate is obtained. S5. Light-controlled release and recovery: Same as in Example 1; S6. Product preparation and solvent reuse: Same as in Example 1.

[0025] Comparative Example 4 This comparative example provides a green extraction method for proanthocyanidins from grape seeds, the steps of which include: S1. Preparation of a high-grafting-rate azobenzened chitosan solution: Weigh 6.20 g (0.025 mol) of 4-carboxyazobenzene. The activation and reaction with chitosan follow the same steps as in Example 1. Azobenzened chitosan with a grafting rate of approximately 20% is finally obtained, yielding approximately 12.8 g. Take 12.5 g of this powder and dissolve it in 2.5 L of 1% acetic acid aqueous solution to prepare a stock solution with a mass-volume concentration of 0.5%. S2. Raw material pretreatment: Same as in Example 1; S3. Preparation of extractant and extraction: Same as in Example 1; S4. Photocontrolled precipitation and separation: The operation is the same as in Example 1. After blue light irradiation, it only needs to stand for 15 minutes, siphon the supernatant, and centrifuge to obtain about 335 g of wet precipitate. S5. Photocontrolled release and recovery: The wet precipitate was dispersed in 2 L of deionized water and irradiated with ultraviolet light as in Example 1. After centrifugation, the precipitate was found to be viscous and the supernatant was light in color. The precipitate was dispersed again in 1 L of fresh deionized water and irradiated with ultraviolet light for 10 min. The supernatants were then combined. S6. Product preparation and solvent reuse: Same as in Example 1.

[0026] Comparative Example 5 This comparative example provides a green extraction method for proanthocyanidins from grape seeds, the steps of which include: S1. Preparation of chitosan solution: Weigh 12.5 g of ordinary chitosan powder, dissolve it directly in 2.5 L of 1% acetic acid aqueous solution, stir magnetically overnight, and prepare a stock solution with a mass-volume concentration of 0.5%. S2. Raw material pretreatment: Same as in Example 1; S3. Preparation of extractant and extraction: Same as in Example 1; S4. Precipitation and Separation: Transfer 25 L of clarified extract to the reactor, add 250 mL of the above chitosan stock solution in the dark, and stir at 200 rpm for 30 min; after stopping stirring, let stand directly for 30 min (without light exposure) to allow chitosan and proanthocyanidins and other components to co-precipitate through electrostatic interaction; remove the supernatant by siphon, and collect the bottom precipitate by centrifugation to obtain approximately 350 g of wet precipitate; S5. Acid Decomposition, Release, and Recovery: The wet precipitate was dispersed in 2 L of an aqueous solution containing 2% hydrochloric acid and stirred at 100 rpm for 60 min at 50 °C to dissolve the chitosan and release proanthocyanidins. Subsequently, the pH of the mixture was adjusted to 7.0 with 5% NaOH solution to allow the chitosan to redefine. The mixture was centrifuged, and the supernatant was an aqueous solution containing proanthocyanidins, while the precipitate was chitosan, which could be collected for redissolution and recycling. S6. Product preparation and solvent reuse: Same as in Example 1.

[0027] Comparative Example 6 This comparative example provides a green extraction method for proanthocyanidins from grape seeds, the steps of which include: S1. Preparation of azobenzyl chitosan solution: This step is not required in this comparative example; S2. Raw material pretreatment: Same as in Example 1; S3. Preparation of extractant and extraction: Same as in Example 1; S4. Salting-out precipitation and separation: Add 8.2 kg of ammonium sulfate solid to 25 L of clarified extract while stirring (to achieve 20% saturation), and continue stirring for 30 min until completely dissolved. Let stand overnight at 4°C. Siphon off the supernatant, and collect the bottom precipitate by centrifugation to obtain approximately 400 g of wet precipitate. S5. Resolution and Desalination: Disperse the wet precipitate in 2 L of deionized water and perform dialysis desalination using an ultrafiltration device with a molecular weight cutoff of 1000 Da until no sulfate ions are present in the effluent (detected by barium chloride). Obtain the desalted proanthocyanidin aqueous solution. S6. Product preparation and solvent reuse: Same as in Example 1.

[0028] Comparative Example 7 This comparative example provides a green extraction method for proanthocyanidins from grape seeds, the steps of which include: S1. Preparation of azobenzyl chitosan solution: Same as in Example 1; S2. Raw material pretreatment: Same as in Example 1; S3. Preparation and extraction of the extractant: Weigh 167.5 g (1.2 mol) of choline chloride and 252.3 g (1.2 mol) of citric acid monohydrate, dissolve them in 10 kg of deionized water to obtain a eutectic solvent precursor solution with a total concentration of approximately 12%. Subsequent extraction operations are the same as in Example 1; the viscosity of the extract increases significantly, and the filtration time is prolonged.

[0029] S4. Photocontrolled precipitation and separation: Same as in Example 1; S5. Light-controlled release and recovery: Same as in Example 1; S6. Product preparation and solvent reuse: Same as in Example 1.

[0030] Comparative Example 8 This comparative example provides a green extraction method for proanthocyanidins from grape seeds, the steps of which include: S1. Preparation of azobenzene-modified gelatin solution: Weigh 10.0 g of gelatin (Type A, 300 Bloom), add 500 mL of deionized water at 50°C, stir to dissolve, and obtain a 2% gelatin solution. The activation steps for 4-carboxyazobenzene are the same as in Example 1. Slowly add the activation solution to the gelatin solution under gentle stirring, adjust the pH to 7.0 with 5% NaOH, and react at 40°C in the dark for 12 hours. Subsequent precipitation, dialysis, and lyophilization steps are the same as in Example 1 to obtain azobenzene-modified gelatin. Take 12.5 g of this powder, dissolve it in 2.5 L of deionized water at 50°C, and prepare a 0.5% stock solution (keep warm in a 45°C water bath before use). S2. Raw material pretreatment: Same as in Example 1; S3. Preparation of extractant and extraction: Same as in Example 1; S4. Photocontrolled precipitation and separation: 25 L of clarified extract was transferred to a photoreactor and kept at 40°C. 250 mL of the above-mentioned warm azobenzened gelatin stock solution was added in the dark, to a final concentration of approximately 0.05 g / L. Subsequent blue light irradiation and settling were performed as in Example 1. However, the precipitate was flocculent and loose, easily agitated during siphoning. Instead, the precipitate was collected directly by centrifugation at 5000 rpm for 20 min, yielding approximately 180 g of wet precipitate. S5. Light-controlled release and recovery: Operation is the same as in Example 1; S6. Product preparation and solvent reuse: Same as in Example 1.

[0031] Comparative Example 9 This comparative example provides a green extraction method for proanthocyanidins from grape seeds, the steps of which include: S1. Preparation of azobenzened sodium alginate solution: Weigh 10.0 g of sodium alginate, add 500 mL of deionized water, stir to dissolve, and obtain a 2% sodium alginate solution; the activation steps for 4-carboxyazobenzene are the same as in Example 1. Slowly add the activation solution to the sodium alginate solution under gentle stirring, adjust the pH to 6.0 with 5% NaOH, and stir at room temperature in the dark for 24 h. Subsequent precipitation (using isopropanol as the precipitant), dialysis, and lyophilization steps are the same as in Example 1 to obtain azobenzened sodium alginate. Take 12.5 g of this powder, dissolve it in 2.5 L of deionized water, and prepare a 0.5% stock solution; S2. Raw material pretreatment: Same as in Example 1; S3. Preparation of extractant and extraction: Same as in Example 1; S4. Photocontrolled precipitation and separation: The procedure is the same as in Example 1, yielding approximately 280 g of wet precipitate; S5. Photocontrolled release and recovery: The wet precipitate was dispersed in 2 L of deionized water and irradiated with ultraviolet light as in Example 1. During the irradiation, the precipitate was observed to gradually dissolve and the solution became homogeneous. After the irradiation, solid-liquid separation could not be achieved by centrifugation. Instead, an ultrafiltration membrane with a molecular weight cutoff of 3500 Da was used to filter and separate the mixture. The permeate was an aqueous solution containing proanthocyanidins, and the retentate was a polymer solution. S6. Product preparation and solvent reuse: Same as in Example 1.

[0032] Experimental Example 1: The proanthocyanidins prepared in Examples 1-3 and Comparative Examples 1-9 were tested as follows: The purity of proanthocyanidins was specifically determined using the vanillin-hydrochloric acid method: 10 mg of extract was dissolved in 10 mL of methanol to obtain a 1 mg / mL test solution; 1.0 mL of sample solution + 1.5 mL of 1% vanillin methanol solution + 1.5 mL of concentrated hydrochloric acid (36%) was used as the reaction solution, and the reaction was carried out at 20℃ in the dark for 15 min, with the absorbance measured at 500 nm; methanol was used instead of vanillin solution as a blank group; a standard curve (5-500 μg / mL) was plotted using catechin as a standard. Proanthocyanidin purity (%) = Proanthocyanidin content / Total sample mass × 100%; Proanthocyanidin extraction rate (%) = (mass of dried proanthocyanidin product / dry weight of raw grape seeds) × 100%; Precipitation efficiency (%) = [1 - (concentration of proanthocyanidins in the supernatant after precipitation / concentration of proanthocyanidins in the extract before precipitation)] × 100%; Polymer cycle stability test: As described in Example 1, the recovered azobenzened chitosan was redissolved and prepared for precipitation in the next batch of extract. The precipitation efficiency of each cycle was recorded. When the precipitation efficiency dropped below 90%, the number of cycles was recorded. The results are shown in Table 1 below: Table 1

[0033] Based on the aforementioned data, although Comparative Example 1 showed acceptable extraction and precipitation efficiency upon initial use, its polymer cycling stability was extremely poor, and it could only "partially dissolve" after recovery, indicating poor solubility and structural stability under alkaline conditions. Compared to bio-based chitosan carriers, its biocompatibility may be lower, and the synthesis process involves DMF organic solvents, making it inferior to the methods in the examples in terms of cost and environmental friendliness.

[0034] Comparative Example 2 showed a similar extraction rate to the Example, but the product purity was low, confirming that the traditional extraction method has poor selectivity and contains many impurities. This method uses large amounts of flammable and explosive ethanol and involves cumbersome liquid-liquid extraction with toxic solvents such as chloroform and ethyl acetate, posing safety hazards, high solvent toxicity, and high energy consumption (concentrating large amounts of dilute solutions), which does not conform to the concept of green extraction.

[0035] The low grafting rate in Comparative Example 3 resulted in insufficient photoresponsive functional groups, leading to a significant decrease in precipitation efficiency to 82.3%, indicating a weakened ability to capture proanthocyanidins. Although the purity and extraction rate were acceptable, the precipitation rate was slow, requiring 60 minutes of settling, and the cycle stability was slightly lower than that of the Example, indicating a certain degree of performance degradation.

[0036] Comparative Example 4, with its high grafting ratio, exhibited extremely high initial precipitation efficiency and rapid precipitation rate, requiring only 15 minutes of settling. However, it suffered from low release efficiency, necessitating secondary UV irradiation, leading to decreased product purity, and reduced cycle stability due to excessive hydrophobicity. This indicates that an excessively high grafting ratio alters the polymer's hydrophilic-hydrophobic balance, hindering the gentle and complete release of the target compound and carrier cycling.

[0037] Comparative Example 5 achieved efficient co-precipitation through electrostatic interaction, but lacked photoselectivity, resulting in numerous impurities in the co-precipitate and low product purity. The release process requires strong acid and pH adjustment, which are harsh conditions that may damage the proanthocyanidin structure. Furthermore, the polymer recovery process is cumbersome and has poor recyclability, making it far less gentle and convenient than the photocontrolled method.

[0038] Comparative Example 6 showed acceptable precipitation efficiency via salting out, but it introduced a large amount of inorganic ammonium sulfate contamination, necessitating a complex desalination step, which increased process complexity and time costs. Furthermore, salting out exhibited poor selectivity, low product purity, and the inability to recycle key reagents.

[0039] In Comparative Example 7, changing the eutectic solvent resulted in a decrease in extraction efficiency and a significant increase in extract viscosity, leading to filtration difficulties and impacting the efficiency of subsequent steps. Although precipitation and circulation performance remained unaffected, this demonstrated that the betaine / lactic acid system offers advantages in extraction efficiency, solution properties, and process smoothness.

[0040] Comparative Example 8 uses gelatin as a carrier. Its heat sensitivity necessitates temperature control during operation, and the resulting precipitate exhibits poor physical properties—it is flocculent and loose, making simple siphon separation difficult and requiring high-speed centrifugation, thus increasing operational complexity. Its precipitation efficiency and cycle stability are both low, indicating that gelatin is poorly suited as a carrier in this system.

[0041] In Comparative Example 9, the sodium alginate carrier completely dissolved after ultraviolet irradiation, making solid-liquid separation impossible by centrifugation. Ultrafiltration membrane separation was necessary, significantly increasing the difficulty and cost of post-release separation. Although other indicators were acceptable, this deficiency limited the method's ease of operation and industrial feasibility.

[0042] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A green extraction method for proanthocyanidins from grape seeds, characterized in that the steps include... include: S1. Physically break down the dried grape seeds to create micro-cracks on their surface. The pretreated grape seeds were mixed with the eutectic solvent precursor liquid and extracted with stirring at 40-70℃ for 60-120 min. The solid-liquid mixture was then separated to obtain a clear extract. S2. Add azobenzyl chitosan solution to the clarified extract, mix under light-protected conditions, and irradiate with light of a first specific wavelength to cause the azobenzyl chitosan to combine with the proanthocyanidins in the extract to form a complex precipitate, and separate the precipitate. S3. The complex is precipitated and dispersed in an aqueous medium and irradiated with a second specific wavelength of light to release proanthocyanidins from the complex. After separation, a supernatant containing proanthocyanidins is obtained, and the regenerated azobenzyl chitosan is recovered. S4. Concentrate and dry the supernatant containing proanthocyanidins to obtain the proanthocyanidin product.

2. The green extraction method for proanthocyanidins from grape seeds according to claim 1, characterized in that, The preparation method of the azobenzened chitosan solution includes: dissolving chitosan in an aqueous acetic acid solution with a concentration of 0.5%-2.0% to obtain a chitosan-acetic acid solution with a mass concentration of 1%-3%; activating 4-carboxyazobenzene in a solvent in the presence of a condensing agent and an activating agent to obtain an activated solution; reacting the activated solution with the chitosan-acetic acid solution at pH 4.5-6.0 under light-protected conditions for 12-36 hours; after the reaction is completed, purifying and drying the product to obtain azobenzened chitosan; dissolving the obtained azobenzened chitosan in an aqueous acetic acid solution with a concentration of 0.5%-2.0% to prepare a stock solution with a mass volume concentration of 0.1%-1.0%.

3. The green extraction method for proanthocyanidins from grape seeds according to claim 2, characterized in that, The mass ratio of chitosan to 4-carboxyazobenzene is 10:3-5; the condensing agent is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dicyclohexylcarbodiimide; and the activator is N-hydroxysuccinimide or N-hydroxythiosuccinimide.

4. A green extraction method for proanthocyanidins from grape seeds according to claim 1, characterized in that, In step S1, the physical cell wall breaking treatment is roller pressing, rolling or ball milling; the crack width of the grape seeds after treatment is 0.5-2.0 mm.

5. The green extraction method for proanthocyanidins from grape seeds according to claim 1, characterized in that, In step S1, the eutectic solvent precursor solution is prepared by dissolving a hydrogen bond acceptor and a hydrogen bond donor in water; the hydrogen bond acceptor is betaine, and the hydrogen bond donor is L-lactic acid; the molar ratio of betaine to L-lactic acid is 1:1.5-2.5; and the total mass concentration of the eutectic solvent precursor solution is 5%-20%.

6. The green extraction method for proanthocyanidins from grape seeds according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of grape seeds to the eutectic solvent precursor liquid is 1g:10-14mL; the extraction temperature is 45-55℃, and the extraction time is 70-90min.

7. A green extraction method for proanthocyanidins from grape seeds according to claim 1, characterized in that, In step S2, after adding the azobenzyl chitosan solution, the final concentration of azobenzyl chitosan in the system is 0.01-0.2 g / L; the first specific wavelength light is blue light with a wavelength of 420-480 nm and a light intensity of 20-60 mW / cm². 2 The irradiation time is 10-60 minutes; stirring is performed during irradiation at a speed of 100-300 rpm.

8. A green extraction method for proanthocyanidins from grape seeds according to claim 1, characterized in that, In step S3, the second specific wavelength light is ultraviolet light with a wavelength of 340-380 nm and a light intensity of 20-50 mW / cm². 2 The irradiation time is 10-40 min; the aqueous medium is water or a buffer solution with pH 5.0-7.

0.

9. The green extraction method for proanthocyanidins from grape seeds according to claim 1, characterized in that, In step S2, the supernatant obtained after separation of the precipitate is replenished with the consumed solute to the initial concentration and then reused in the extraction process of step S1. In step S3, the regenerated azobenzened chitosan recovered after separation is washed and dissolved and can be recycled for photocontrolled precipitation in step S2.

10. The green extraction method for proanthocyanidins from grape seeds according to claim 1, characterized in that, In step S4, the concentration is carried out under reduced pressure at 40-60°C; the drying is carried out by spray drying or freeze drying.

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