Preparation method and application of oligomeric grape seed procyanidine

Abstract

The invention discloses a preparation method and application of oligomeric grape seed procyanidine, and belongs to the field of wine making byproduct processing. The preparation method comprises the following steps: pretreating grape pomace to obtain grape seed powder; degreasing with n-hexane, and carrying out ethanol ultrasonic-dark extraction to obtain a procyanidine crude product; and purifying with AB-8 macroporous resin to obtain a pure product. And depolymerizing in the high-pressure reaction kettle to obtain a product. The average polymerization degree of the product is less than 5, the monomer content is greater than or equal to 47%, the recovery rate is greater than or equal to 88%, the bioavailability is high, and the activity is strong. The equipment is conventional, the solvent is green, the problems of heavy pollution and difficulty in industrialization in the prior art are solved, the product can be used for preparing health care products, medicines or cosmetics, the added value of wine brewing byproducts is increased, and the industrial value is remarkable.

Description

Technical Field

[0001] This invention belongs to the field of processing and utilization technology of winemaking by-products, specifically relating to a method for preparing and applying oligomeric grape seed proanthocyanidins. Background Technology

[0002] Statistics show that in the international winemaking industry, winemaking byproducts can account for up to 30% of the total weight of wine grapes, with grape seeds making up 38% to 52% of the grape pomace. Grape seeds are rich in proanthocyanidins. Rational utilization of these byproducts can protect the environment, maintain sustainable development, and increase the added value of the wine industry. Proanthocyanidins (PC), also known as condensed tannins, are oligomers or polymers formed by the polymerization of flavan-3-ol monomers. They are mainly linked by C4-C8 bonds, but C4-C6 bonds also exist. They are found in various plants, such as grapes, blueberries, dragon fruit, and goji berries, and are the second most abundant natural phenolic compound after lignin. Proanthocyanidins are classified into oligomeric proanthocyanidins (DP < 5) and polymeric proanthocyanidins (DP ≥ 5) based on their degree of polymerization (DP).

[0003] Proanthocyanidins are functional bioactive substances beneficial to health, possessing effects such as antioxidation, anti-aging, anti-inflammation, antibacterial properties, and reducing cardiovascular disease risk. However, the bioavailability of proanthocyanidins is closely related to their degree of polymerization; only oligomeric proanthocyanidins with a degree of polymerization less than 5 and soluble in water can be absorbed and utilized by the human body. However, over 65% of the proanthocyanidins extractable from grape seeds are polymeric proanthocyanidins. Therefore, an efficient and environmentally friendly method to reduce the degree of polymerization of proanthocyanidins is of significant importance and value to industrial development.

[0004] Existing research has shown that one publicly disclosed method for preparing oligomeric proanthocyanidins involves soaking grape seeds in water followed by explosion (2.5-3.5 MPa) and defatting. The defatted sample is then mixed with an extract to obtain a crude extract of high-polymer proanthocyanidins. This crude extract is then mixed with a lyase and reacted under anaerobic conditions to obtain oligomeric proanthocyanidins. This method uses an explosion method in the extraction step, which requires sophisticated equipment and is highly dangerous, resulting in samples with extremely low purity. Furthermore, another publicly disclosed method for preparing oligomeric proanthocyanidins involves adding an aqueous solution of threonine, followed by aspartic acid and glutamic acid, then sonicating, dialysis, and / or nanofiltration to obtain the oligomeric proanthocyanidins. This method uses multiple amino acids, requires a large amount of membrane with low flux, and is costly, significantly limiting production and making industrial-scale production difficult. A publicly disclosed continuous production method for oligomeric proanthocyanidins with uniform degree of polymerization involves mixing a raw material containing high-polymer proanthocyanidins with an alkaline catalytic degradation solution, stirring until homogeneous, and then subjecting it to catalytic degradation. The resulting product is then obtained through membrane separation, enrichment, recycling, concentration, and drying. This method utilizes a strongly alkaline catalytic solution and a weak acid solution for neutralization, which can easily pollute the environment and is complex. Another publicly disclosed method for the efficient extraction and conversion of oligomeric proanthocyanidins involves mixing high-polymer proanthocyanidins with a eutectic solvent, followed by resin adsorption and elution, and drying to obtain the oligomeric proanthocyanidins. However, the eutectic solvent used in this method has drawbacks: high viscosity affecting mass transfer efficiency, hygroscopicity altering the physicochemical properties of the sample, difficulty in recovery, and high cost.

[0005] Therefore, developing an efficient, environmentally friendly, and industrially scalable method for preparing oligomeric proanthocyanidins can effectively increase yield and reduce costs, which has significant industrial importance and value. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing and applying oligomeric grape seed proanthocyanidins, which addresses the shortcomings of the prior art. This method solves the technical problems of existing grape seed proanthocyanidin extraction methods, such as high equipment requirements, high risk factor, low yield, difficulty in achieving industrial production, serious environmental pollution, and poor product stability.

[0007] The present invention adopts the following technical solution: A method for preparing oligomeric grape seed proanthocyanidins includes the following steps: Grape pomace is pretreated to obtain grape seed powder; Grape seed powder was extracted using an extraction solvent to obtain crude grape seed proanthocyanidins. The crude product was purified using macroporous resin to obtain pure grape seed proanthocyanidins. The pure product was dissolved in water and subjected to a depolymerization reaction in a high-pressure reactor to obtain oligomeric grape seed proanthocyanidins.

[0008] Preferably, the preprocessing includes: Wash the grape skins and pomace, dry them at 30-60℃ for 12-36 hours, separate the grape seeds, crush them and pass them through a 20-80 mesh sieve.

[0009] Preferably, the extraction solvent is n-hexane, the ratio of grape seed powder to n-hexane is 1:1~5 (g / mL), the extraction time is 12~48h, and the precipitate is collected by centrifugation; The precipitate was mixed with 30-95% ethanol at a ratio of 1:3-7 (g / mL), and ultrasonically extracted at 20-80℃ for 5-60 min. The mixture was then extracted in the dark for 6-48 h, with stirring every 4 h during the extraction. After ultrasonication, extraction, and stirring, the mixture was centrifuged to collect the supernatant, concentrated under reduced pressure, and then freeze-dried to obtain crude grape seed proanthocyanidins.

[0010] Preferably, the macroporous resin is activated by sequentially soaking in 60-80% ethanol, 2-6% HCl, and 2-6% NaOH solutions, rinsed with distilled water until neutral, and then stored in 30-95% ethanol for later use.

[0011] Preferably, the macroporous resin is type AB-8 with a particle size of 16-80 mesh.

[0012] Preferably, the purification process includes: The crude product was prepared into an aqueous solution of 10-150 mg / mL and loaded onto the sample. It was then rinsed with distilled water to remove impurities, followed by elution with 40-70% acetone-water solution. The eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain pure grape seed proanthocyanidins.

[0013] Preferably, the conditions for the depolymerization reaction are: The pure proanthocyanidin was prepared into an aqueous solution of 0.5~2.0 mg / mL, placed in a high-pressure reactor, and nitrogen was introduced to purge the air. The reaction was carried out at 80~150℃ with stirring for 45~180 min. After cooling, the sample was taken, concentrated under reduced pressure and freeze-dried to obtain oligomeric grape seed proanthocyanidins.

[0014] Another technical solution of the present invention is an oligomeric grape seed proanthocyanidin with an average degree of polymerization of less than 5, a monomer content of greater than or equal to 47%, and a recovery rate of greater than or equal to 88%.

[0015] Preferably, the average degree of polymerization is 1.26 to 4.20, and the monomer content is 47.36% to 89.09%.

[0016] Another technical solution of the present invention is the application of oligomeric grape seed proanthocyanidins in the preparation of health products, pharmaceuticals or cosmetics.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: A method for preparing oligomeric grape seed proanthocyanidins is disclosed, with "pretreatment → extraction → macroporous resin purification → high-pressure reactor depolymerization" as its core process, thus constructing a complete preparation chain for oligomeric grape seed proanthocyanidins in principle. The pretreatment stage achieves preliminary purification of the raw materials, laying the foundation for subsequent extraction; extraction selectively dissolves and separates proanthocyanidins from impurities using solvents; macroporous resin purification utilizes adsorption-elution characteristics to improve product purity; and high-pressure reactor depolymerization breaks the polymerization bonds of high-polymer proanthocyanidins under high temperature and pressure, reducing the degree of polymerization. From an application perspective, this solution avoids the problems of high equipment requirements (such as steam explosion) and high pollution (such as alkaline catalyst liquid) in existing technologies, achieving industrial compatibility with conventional equipment. Furthermore, the logical connections between each step are tight, and the product of each step serves as a qualified raw material for subsequent steps, ensuring a stable and controllable preparation process. It also covers all the necessary technical features to solve the technical problem, providing a basis for the detailed definition of subsequent dependent claims, maximizing the scope of protection while ensuring the feasibility of the technical solution.

[0018] Furthermore, the technical solution of "washing grape pomace → drying at 30~60℃ for 12~36h → sorting grape seeds → pulverizing through a 20~80 mesh sieve" effectively removes dust, pesticide residues, and other impurities from the surface of the pomace, preventing contamination during subsequent extraction. The low-temperature drying at 30~60℃ ensures the pomace is dry enough for subsequent sorting while preventing high temperatures from damaging the heat-sensitive proanthocyanidins in the grape seeds. The 12~36h drying time ensures sufficient moisture removal without over-drying the raw material. Sorting the grape seeds separates the target raw material from the pomace, improving raw material utilization. Pulverizing through a 20~80 mesh sieve controls the particle size, increasing the contact area between the grape seeds and the subsequent extraction solvent, improving extraction efficiency, and preventing difficulties in subsequent centrifugation due to excessively small particle size. This solution, by refining pretreatment parameters, ensures the purity of the raw material and extraction efficiency from the source, providing a prerequisite for the subsequent preparation of high-quality crude proanthocyanidins and solving the problem of high impurities in subsequent products due to incomplete pretreatment.

[0019] Furthermore, a two-step extraction method of "hexane defatting → ethanol ultrasonic extraction-light-protected extraction" is adopted. Hexane, as a non-polar solvent, can selectively dissolve oily impurities in grape seed powder. A ratio of 1:1~5 (g / mL) and an extraction time of 12~48h ensure thorough defatting. Centrifugation to collect the precipitate achieves the separation of raw material and solvent after defatting. The precipitate is mixed with 30~95% ethanol at a ratio of 1:1~7 (g / mL). Ethanol, as a polar solvent, can effectively dissolve proanthocyanidins. An ultrasonic temperature of 20~80℃ and an ultrasonic time of 5~60min utilize the ultrasonic cavitation effect to accelerate the dissolution of proanthocyanidins. Light-protected extraction for 6~48h avoids the oxidation and degradation of proanthocyanidins caused by light. Stirring once every 4h and two ultrasonic-extraction-stirring cycles further improve the extraction efficiency. Centrifugation to collect the supernatant, vacuum concentration and freeze drying achieve the separation and drying of crude proanthocyanidins. The solution balances defatting effect and proanthocyanidin extraction rate through solvent selection and parameter optimization, avoiding problems such as high impurities or insufficient extraction caused by single solvent extraction. At the same time, vacuum concentration freeze drying can achieve drying at low temperature, preserving the activity of proanthocyanidins.

[0020] Furthermore, the technical solution involves sequentially activating the macroporous resin with 60-80% ethanol, 2-6% HCl, and 2-6% NaOH solutions, followed by rinsing with distilled water until neutral, and then storing it in 30-95% ethanol. The 60-100% ethanol solution removes residual monomers, pore-forming agents, and other organic impurities from the resin synthesis process, ensuring unobstructed resin pores. The 2-6% HCl solution removes inorganic impurities such as metal cations from the resin surface, and the 2-6% NaOH solution removes acidic impurities. Alternating acid and alkali immersion ensures thorough impurity removal. Rinsing with distilled water until neutral prevents residual acids and alkalis from damaging proanthocyanidins or affecting the resin's adsorption performance during subsequent purification. Storage in 30-95% ethanol maintains the resin's swollen state, preventing pore collapse and ensuring subsequent adsorption efficiency. This multi-step activation process significantly improves the adsorption capacity and selectivity of the macroporous resin, solving the problem of poor purification results caused by incomplete resin activation. It provides a guarantee for efficient purification of crude proanthocyanidins, and the activation parameters are within a reasonable range to suit the processing needs of different batches of resin.

[0021] Furthermore, the AB-8 type macroporous resin, with a particle size of 16-80 mesh, is a weakly polar resin. Its pore structure and surface chemistry allow for the specific adsorption of proanthocyanidins. Compared to other resin types (such as D101), it exhibits higher selectivity and lower desorption rate for proanthocyanidins, effectively improving the purity of the purified product. The 16-80 mesh particle size range ensures that the resin particles are of appropriate size; excessively large particles result in a small specific surface area and low adsorption efficiency, while excessively small particles lead to excessively high column pressure, potentially causing unstable eluent flow rates and affecting the continuity of the purification process. This approach, by specifying the resin type and particle size, avoids problems such as low purification efficiency and insufficient product purity caused by improper resin selection, making the purification process parameters easier to control. Simultaneously, the AB-8 type resin is low-cost and readily available, suitable for industrial production needs, and the 16-80 mesh particle size range is compatible with conventional chromatography column operations, further enhancing the practicality of the technical solution.

[0022] Furthermore, the technical solution involves preparing a crude product into a 10-150 mg / mL aqueous solution for loading, rinsing with distilled water to remove impurities, eluting with a 40-70% acetone-water solution, collecting the eluent, and concentrating and lyophilizing to obtain the pure product. The 10-150 mg / mL concentration of the crude product aqueous solution ensures uniform dispersion of proanthocyanidins in the solution, avoiding excessive concentration leading to resin saturation or insufficient concentration wasting elution solvent. Rinsing with distilled water utilizes differences in water solubility to remove residual soluble sugars, pigments, and other impurities from the crude product without affecting the resin's adsorption of proanthocyanidins. The 40-70% acetone-water solution, used as the eluent, can disrupt the adsorption between proanthocyanidins and the resin by adjusting its polarity, achieving efficient elution while avoiding the use of highly toxic solvents, thus meeting environmental protection requirements. Concentration and lyophilization at low temperatures prevents proanthocyanidin degradation due to high temperatures. This solution effectively improves product purity by refining the parameters of each purification step, solving the problems of incomplete impurity removal and low elution efficiency in existing purification methods. Moreover, the operation steps are simple and easily scalable for large-scale production.

[0023] Furthermore, the product is depolymerized by preparing a 0.5~2.0 mg / mL aqueous solution from the pure product, followed by a high-pressure reactor, cooling and sampling, and then concentration and freeze-drying. The 0.5~2.0 mg / mL aqueous solution concentration ensures uniform dispersion of proanthocyanidins, providing sufficient contact conditions for the depolymerization reaction. Nitrogen purging removes air and prevents oxygen in the air from causing oxidative degradation of proanthocyanidins, ensuring product quality. The reaction temperature of 80~150℃ and the reaction time of 45~180 min, by controlling the heat input, break the C4-C8 or C4-C6 polymerization bonds of the high-polymer proanthocyanidins, reducing the degree of polymerization to below 5. The temperature and time ranges are reasonable and can be flexibly adjusted according to the target degree of polymerization. Stirring ensures uniform temperature of the reaction system and avoids local overheating that could lead to product carbonization. This solution avoids the problems of existing depolymerization methods, such as the use of amino acids, lyases (high cost), alkaline catalysts (high pollution), and dialysis membranes (large usage, high cost, and limited output). It uses water as a solvent and nitrogen as a protective gas, making it green and environmentally friendly. At the same time, the high-pressure reactor equipment is conventional and easy to industrialize. The depolymerization parameters are highly controllable, ensuring stable product quality.

[0024] Furthermore, an average degree of polymerization <5 conforms to the definition of oligomeric proanthocyanidins, ensuring that the product can be effectively absorbed and utilized by the human body, thus solving the core problem of low bioavailability of polymeric proanthocyanidins. A monomer content ≥47% indicates a high proportion of active ingredients (such as catechins and epicatechins) in the product, enhancing its functional activity (such as antioxidant and anti-aging effects). A recovery rate ≥88% indicates high raw material utilization during preparation, reducing resource waste and lowering production costs. From an application perspective, this parameter combination gives the product significant advantages in the fields of health products, pharmaceuticals, and cosmetics. Compared to existing products, it has higher bioavailability, stronger activity, and better production economics. Simultaneously, the parameter range is fully verified through example data, ensuring stable and controllable product performance and providing a solid product quality foundation for subsequent application of the claims.

[0025] Based on the product's characteristics of "low average degree of polymerization and high monomer content," it can exert antioxidant and immune-boosting effects in the health supplement field, assist in anti-inflammation and reduce the risk of cardiovascular disease in the pharmaceutical field, and play an anti-aging and whitening role in the cosmetic field, solving the problem that existing high-polymer proanthocyanidins are difficult to effectively apply in these fields due to low bioavailability. From an application perspective, this solution opens up a clear market direction for the product. Compared with the vague application scenarios of existing technologies, it directly relates to three high-demand fields, enhancing the industrialization value of the technology. At the same time, combined with the product's green preparation process, there are no harmful substance residues during application, meeting the safety standards of health supplements, pharmaceuticals, and cosmetics, further enhancing the market competitiveness of the technical solution.

[0026] In summary, this invention utilizes a four-step process of pretreatment, extraction, purification, and depolymerization to prepare oligomeric grape seed proanthocyanidins using conventional equipment and green solvents. This avoids the problems of high equipment requirements, significant pollution, and difficulty in industrialization associated with existing technologies, while balancing product quality, environmental friendliness, and economic efficiency. It can be widely applied in the fields of health products, pharmaceuticals, and cosmetics.

[0027] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention, Figures 1 to 6, which are intended to be used in the following description of the embodiments, will be briefly introduced. Obviously, the figures described below are only some embodiments of the present invention. For those skilled in the art, other figures can be obtained based on these figures without creative effort.

[0029] Figure 1 shows the C and EC response peak diagrams for the comparative example and each embodiment, where (a) is the comparative example, (b) is embodiment 1, (c) is embodiment 2, (d) is embodiment 3, and (e) is embodiment 4. Figure 2 shows the ECG response peak diagrams of the comparative example and each embodiment, where (a) is the comparative example, (b) is embodiment 1, (c) is embodiment 2, (d) is embodiment 3, and (e) is embodiment 4. Figure 3 shows the EGC response peak diagrams of the comparative example and each embodiment, where (a) is the comparative example, (b) is embodiment 1, (c) is embodiment 2, (d) is embodiment 3, and (e) is embodiment 4. Figure 4 shows the C-PHL and EC-PHL response peak diagrams for the comparative example and each embodiment, where (a) is the comparative example, (b) is Example 1, (c) is Example 2, (d) is Example 3, and (e) is Example 4. Figure 5 shows the ECG-PHL response peak diagrams of the comparative example and each embodiment, where (a) is the comparative example, (b) is Example 1, (c) is Example 2, (d) is Example 3, and (e) is Example 4. Figure 6 shows the EGC-PHL response peak diagrams of the comparative example and each embodiment, where (a) is the comparative example, (b) is embodiment 1, (c) is embodiment 2, (d) is embodiment 3, and (e) is embodiment 4. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0031] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0032] Unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0033] In this invention, unless otherwise specified, percentage (%) or parts refer to weight percentage or parts relative to the composition.

[0034] Unless otherwise specified, the components or preferred components involved in this invention can be combined with each other to form new technical solutions.

[0035] In this invention, unless otherwise specified, the numerical range "a~b" represents an abbreviation of any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "6~22" means that all real numbers between "6~22" have been listed in this document, and "6~22" is simply an abbreviation of these numerical combinations.

[0036] The "scope" disclosed in this invention can be in the form of a lower limit and an upper limit, and can be one or more lower limits and one or more upper limits, respectively.

[0037] In this invention, the term "and / or" as used herein refers to any combination of one or more of the associated listed items, as well as all possible combinations, and includes such combinations.

[0038] In this invention, unless otherwise stated, the various reactions or operation steps may be performed sequentially or not. Preferably, the reaction methods described herein are performed sequentially.

[0039] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be used in this invention.

[0040] This invention provides a method for preparing and applying oligomeric grape seed proanthocyanidins. By setting the temperature and pressure of a high-pressure reactor and using water as a solvent, the proanthocyanidins extracted from grape seeds are depolymerized, reducing the average degree of polymerization of grape seed proanthocyanidins. This method is environmentally friendly, with a proanthocyanidin degradation product recovery rate greater than 88%, and the average degree of polymerization of samples obtained under different treatment conditions is reduced to varying degrees.

[0041] This invention discloses a method for preparing oligomeric grape seed proanthocyanidins, comprising the following steps: S1. Grape pomace pretreatment yields grape seeds, which are then ground and sieved to obtain grape seed powder; The preprocessing is as follows: Wash the grape skins and pomace produced from winemaking, dry them at 30-60℃ for 12-36 hours, sort out the grape seeds, crush them, and pass them through a 20-80 mesh sieve.

[0042] S2. Extract anthocyanins from grape seed powder by adding an extraction solvent, concentrate under reduced pressure, and freeze dry to obtain crude grape seed proanthocyanidins. The ratio of grape seed powder to n-hexane is 1:1~5 (g / mL). Mix well and extract for 12-48 hours. Centrifuge at 3850 r / min for 15 min and collect the precipitate.

[0043] The specific extraction process of crude grape seed proanthocyanidins is as follows: The precipitated sample was mixed with 30%~95% ethanol at a ratio of 1:3~7 (g / mL), and ultrasonically extracted at 40% power and 20~80℃ for 5~60 min. The sample was then extracted in the dark for 6~48 h, with stirring every 4 h. Under the same conditions, the ultrasonic extraction and stirring were repeated once. The sample was centrifuged at 3850 r / min for 15 min, and the supernatant was collected. The supernatant was then concentrated under reduced pressure at 38℃ and 90 r / min to 5%~50% of the original volume, and then freeze-dried to obtain crude grape seed proanthocyanidins.

[0044] S3. The crude grape seed proanthocyanidin was purified by adsorption and elution using activated macroporous resin. The eluent was collected, concentrated under reduced pressure, and freeze-dried to obtain pure grape seed proanthocyanidin. The specific method for activating chromatography column packing is as follows: Soak an appropriate amount of 16-80 mesh AB-8 macroporous resin in 60-80% ethanol for 24 hours, then rinse with distilled water until no ethanol odor remains. Soak in 2-6% HCl solution for 24 hours, then rinse with distilled water until neutral. Soak in 2-6% NaOH solution for 24 hours each time, then rinse with distilled water until neutral. Store the resin in 30-95% ethanol solution for later use. The order of soaking in 2-6% HCl and 2-6% NaOH solutions is not required, but the soaking time must be the same.

[0045] The specific method for packing a chromatography column is as follows: Take an appropriate amount of activated AB-8 macroporous resin and pack it into a column using the wet packing method. Select a glass chromatography column with an inner diameter of 5 cm and a column length of 30 cm without a frit core. Use the wet packing method and wash with 5 column volumes of distilled water until the eluent is clear and has no alcohol odor.

[0046] The specific purification method for crude grape seed proanthocyanidins is as follows: Prepare a crude grape seed proanthocyanidin solution at a concentration of 10-150 mg / mL. The loading volume should be equal to the column volume divided by 5-40. For example, with a column volume of 400 mL, the loading volume should be 400 / (5-40) g of crude proanthocyanidin. Wash rapidly with 10 column volumes of distilled water at 10-50 BV / h to remove soluble sugars and pigments. Then elute the adsorbed sample with 1-20 column volumes of 40%-70% acetone-water solution at 2-50 BV / h and collect the eluent. Simultaneously, concentrate the eluent under reduced pressure at 38℃ and 90 rpm to 5%-60% of its original volume. Freeze-dry the concentrate to obtain pure grape seed proanthocyanidin. Throughout the column packing and elution process, ensure the resin remains moist to prevent cracking, which could affect the purification effect.

[0047] S4. After further processing in a high-pressure reactor, low-polymerization grape seed proanthocyanidins are obtained.

[0048] The specific method for depolymerizing pure grape seed proanthocyanidins is as follows: Prepare a 0.5-2.0 mg / mL aqueous solution of pure grape seed proanthocyanidins. Mix the solution using a high-speed disperser at 10000 rpm for 1 min to ensure complete dissolution. Add the sample solution to the lining of a high-pressure reactor, tighten the lid, and purge with nitrogen until all air is expelled from the sample solution. Tighten the vent knob, connect the cooling water, and incubate at 80-150℃ for 45-180 min. Readjust the temperature to room temperature and maintain an appropriate time, ensuring the sample remains stirred to prevent uneven heating. After the equipment cools to room temperature, open the lid, take a sample, concentrate under reduced pressure, and freeze-dry to obtain oligomeric grape seed proanthocyanidins.

[0049] An oligomeric grape seed proanthocyanidin, prepared by the above method, has an average degree of polymerization of less than 5, a monomer content of greater than or equal to 47%, and a recovery rate of greater than or equal to 88%.

[0050] Preferably, the average degree of polymerization is 1.26 to 4.20, and the monomer content is 47.36% to 89.09%.

[0051] The raw material selected in this invention is grape seeds, a byproduct of wine processing. The effective use of grape seeds improves the comprehensive utilization rate of wine byproducts, reduces environmental pollution and resource waste, and increases the economic value of wine processing byproducts.

[0052] The degradation solvent selected in this invention is distilled water, which is readily available, low in cost, and environmentally friendly.

[0053] The one-pot method effectively reduces the average degree of polymerization of proanthocyanidins in grape seeds, with a recovery rate of over 88%, high degradation efficiency, simple method, and easy industrial production.

[0054] The present invention discloses a method for preparing oligomeric grape seed proanthocyanidins and its application, which can be used in health products, pharmaceuticals and cosmetics.

[0055] A system for preparing oligomeric grape seed proanthocyanidins includes a pretreatment unit, a crude extraction unit, a purification unit, and a depolymerization unit; The pretreatment unit includes a washing device, a drying device, a sorting device, a crushing device, and a screening device. The output end of the washing device is connected to the input end of the drying device to transport the washed grape pomace to the drying device. The output end of the drying device is connected to the input end of the sorting device to transport the dried grape pomace to the sorting device. The output end of the sorting device is connected to the input end of the crushing device to transport the sorted grape seeds to the crushing device. The output end of the crushing device is connected to the input end of the screening device to transport the crushed grape seeds to the screening device, and the output end of the screening device outputs grape seed powder. The crude extraction unit includes a first mixing tank, a first centrifuge, a second mixing tank, an ultrasonic device, an extraction tank, a second centrifuge, a first vacuum concentration device, and a first freeze-drying device. The input end of the first mixing tank is connected to the output end of a sieving device for receiving grape seed powder and mixing it with n-hexane. The output end of the first mixing tank is connected to the input end of the first centrifuge for conveying the mixture of grape seed powder and n-hexane to the first centrifuge. The output end of the first centrifuge is connected to the input end of the second mixing tank for conveying the precipitate obtained from centrifugation to the second mixing tank. The output end of the second mixing tank is connected to the input end of the ultrasonic device for removing the precipitate from the precipitate. A mixture of starch and ethanol is fed to an ultrasonic device; the output of the ultrasonic device is connected to the input of an extraction tank to feed the ultrasonically treated mixture to the extraction tank; the output of the extraction tank is connected to the input of a second centrifuge device to feed the extracted mixture to the second centrifuge device; the output of the second centrifuge device is connected to the input of a first vacuum concentration device to feed the supernatant obtained by centrifugation to the first vacuum concentration device; the output of the first vacuum concentration device is connected to the input of a first freeze-drying device to feed the concentrated supernatant to the first freeze-drying device; the output of the first freeze-drying device outputs crude grape seed proanthocyanidins. The purification unit includes a resin activation device, a chromatography column, a sample loading device, a rinsing device, an elution device, a second vacuum concentration device, and a second lyophilization device. The output of the resin activation device is connected to the input of the chromatography column, used to deliver the activated AB-8 macroporous resin to the column. The input of the sample loading device is connected to the output of the first lyophilization device and a solvent supply device, used to mix crude grape seed proanthocyanidins with a solvent to form an aqueous solution, which is then delivered to the chromatography column. The output of the rinsing device is connected to the input of the chromatography column, used to deliver distilled water to the column. The output of the elution device is connected to the input of the chromatography column, used to deliver an acetone-water solution to the column. The output of the chromatography column is connected to the input of the second vacuum concentration device, used to deliver the eluent to the second vacuum concentration device. The output of the second vacuum concentration device is connected to the input of the second lyophilization device, used to deliver the concentrated eluent to the second lyophilization device, and the output of the second lyophilization device outputs pure grape seed proanthocyanidins. The depolymerization unit includes a third mixing tank, a high-speed disperser, a high-pressure reactor, a third vacuum concentration device, and a third freeze-drying device. The input end of the third mixing tank is connected to the output end of the second freeze-drying device and a water supply device, respectively, for mixing pure grape seed proanthocyanidins with water to form an aqueous solution. The output end of the third mixing tank is connected to the input end of the high-speed disperser, for conveying the aqueous solution to the high-speed disperser. The output end of the high-speed disperser is connected to the input end of the high-pressure reactor, for conveying the mixed aqueous solution to the high-pressure reactor. The output end of the high-pressure reactor is connected to the input end of the third vacuum concentration device, for conveying the processed sample to the third vacuum concentration device. The output end of the third vacuum concentration device is connected to the input end of the third freeze-drying device, for conveying the concentrated sample to the third freeze-drying device. The output end of the third freeze-drying device outputs oligomeric grape seed proanthocyanidins.

[0056] The high-pressure reactor includes a reactor body, a liner, a nitrogen inlet device, a condensate device, and a stirring device. The liner is located inside the reactor body and is used to contain the sample liquid. The nitrogen inlet device is connected to the reactor body and is used to introduce nitrogen into the reactor body. The condensate device is connected to the reactor body and is used to cool the reactor body. The stirring device is located inside the reactor body and below the liner and is used to stir the sample liquid.

[0057] The liner prevents the sample liquid from directly contacting the vessel body, thus preventing the vessel material from contaminating the sample liquid; the nitrogen inlet device can expel air from the vessel, preventing the oxidation of proanthocyanidins; the condensate device can quickly cool the vessel body to room temperature after the reaction, improving operational efficiency; the stirring device ensures that the sample liquid is heated evenly during the reaction, ensuring that the depolymerization reaction proceeds fully and improving the quality stability of oligomeric grape seed proanthocyanidins.

[0058] The purification unit also includes a resin storage device, which is connected to the output end of the resin activation device and is used to store the activated AB-8 macroporous resin; the resin storage device contains a 30%-95% ethanol solution.

[0059] The resin storage device can temporarily store the activated AB-8 macroporous resin, making it convenient to use it at any time according to production needs; the 30%-95% ethanol solution can maintain the resin in a swollen state, prevent the resin from drying and cracking, and ensure that the adsorption performance of the resin is not affected by storage.

[0060] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, not all, of the embodiments of the present invention. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0061] Example 1 1. Pretreatment: Clean the grape skins and pomace produced during winemaking, air dry or dry at 40℃, sort out the grape seeds, crush them, and pass them through a 40-60 mesh sieve to obtain grape seed powder.

[0062] 2. Crude Extraction: Grape seed powder and n-hexane were mixed at a ratio of 1:3 (g / mL) and soaked for 24 hours. The precipitate was centrifuged at 3850 r / min for 15 minutes. The precipitate was mixed with 70% ethanol solution at a ratio of 1:5 (g / mL), and then sonicated at 40% power and below 38℃ for 20 minutes. Extraction was carried out in the dark for 12 hours, with stirring every 4 hours. The supernatant was collected by centrifugation and concentrated under reduced pressure (38℃, 90 r / min) to less than 30% of the original volume. The sample solution was freeze-dried to obtain crude grape seed proanthocyanidins.

[0063] 3. Activation of chromatography column packing: Take an appropriate amount of 16-80 mesh AB-8 macroporous resin and soak it in 70% ethanol solution for 24 hours, then rinse it with distilled water until there is no alcohol odor; then soak it in 4% HCl solution for 24 hours, and rinse the resin with distilled water until neutral; soak it in 4% NaOH solution for 24 hours, rinse the resin with distilled water until neutral, and then soak it in 70% ethanol solution for later use.

[0064] 4. Packing the chromatography column: Take an appropriate amount of macroporous resin, pack the column using the wet method, and then rinse the chromatography column with 5 column volumes (BV) of distilled water until the eluent is clear and has no alcohol odor.

[0065] 5. Purification of crude proanthocyanidins: Load 10 mg / mL of crude proanthocyanidin aqueous solution onto the column and rapidly wash the column with 10 column volumes of distilled water to remove soluble sugars, pigments, etc. from the crude proanthocyanidins; elute with 4 column volumes of 40% acetone solution, collect the eluent, concentrate under reduced pressure (38°C, 90 r / min) to below 40% of the original volume, freeze-dry the sample solution to obtain pure proanthocyanidins.

[0066] 6. Depolymerization of proanthocyanidins: Take a fully dissolved 1.0 mg / mL proanthocyanidin aqueous solution, add it to the lining of the high-pressure reactor, tighten the lid, purge with nitrogen for 1 min, close the venting knob, turn on the condenser, and run the reactor at 80℃ and 500 r / min for 60 min. Then set the temperature to room temperature and wait for the equipment to cool down. Open the lid and freeze-dry the sample to obtain oligomeric grape seed proanthocyanidin powder.

[0067] The extract prepared in this embodiment has a proanthocyanidin mass fraction of ≥47.71%, an average degree of polymerization of 4.20, a monomer content of 47.36%, and an average recovery rate of 95.40%.

[0068] Example 2 1. Pretreatment: Clean the grape skins and pomace produced during winemaking, air dry or dry at 40℃, sort out the grape seeds, crush them, and pass them through a 40-60 mesh sieve to obtain grape seed powder.

[0069] 2. Crude Extraction: Grape seed powder and n-hexane were mixed at a ratio of 1:1 (g / mL) and soaked for 24 hours. The precipitate was centrifuged at 3850 r / min for 15 minutes. The precipitate was mixed with 60% ethanol solution at a ratio of 1:3 (g / mL), and then sonicated at 40% power and below 38℃ for 20 minutes. Extraction was carried out in the dark for 12 hours, with stirring every 4 hours. The supernatant was collected by centrifugation and concentrated under reduced pressure (38℃, 90 r / min) to less than 30% of the original volume. The sample solution was freeze-dried to obtain crude grape seed proanthocyanidins.

[0070] 3. Activation of chromatography column packing: Take an appropriate amount of 16-80 mesh AB-8 macroporous resin and soak it in 60% ethanol solution for 24 hours, then rinse it with distilled water until there is no alcohol odor; then soak it in 2% HCl solution for 24 hours, and rinse the resin with distilled water until neutral; soak it in 2% NaOH solution for 24 hours, rinse the resin with distilled water until neutral, and then soak it in 60% ethanol solution for later use.

[0071] 4. Packing the chromatography column: Take an appropriate amount of macroporous resin, pack the column using the wet method, and then rinse the chromatography column with 5 column volumes (BV) of distilled water until the eluent is clear and has no alcohol odor.

[0072] 5. Purification of crude proanthocyanidins: Load 100 mg / mL of crude proanthocyanidin aqueous solution onto the column and rapidly wash the column with 10 column volumes of distilled water to remove soluble sugars, pigments, etc. from the crude proanthocyanidins; elute with 4 column volumes of 70% acetone solution, collect the eluent, concentrate under reduced pressure (38°C, 90 r / min) to below 40% of the original volume, freeze-dry the sample solution to obtain pure proanthocyanidins.

[0073] 6. Depolymerization of proanthocyanidins: Take a fully dissolved 0.5 mg / mL proanthocyanidin aqueous solution, add it to the lining of the high-pressure reactor, tighten the lid, purge with nitrogen for 1 min, close the venting knob, turn on the condenser, and run the reactor at 100℃ and 500 r / min for 60 min. Then set the temperature to room temperature and wait for the equipment to cool down. Open the lid and freeze-dry the sample to obtain oligomeric grape seed proanthocyanidin powder.

[0074] The extract prepared in this embodiment has a proanthocyanidin mass fraction of ≥46.64%, an average degree of polymerization of 3.97, a monomer content of 48.06%, and an average recovery rate of 91.29%.

[0075] Example 3 1. Pretreatment: Clean the grape skins and pomace produced during winemaking, air dry or dry at 40℃, sort out the grape seeds, crush them, and pass them through a 40-60 mesh sieve to obtain grape seed powder.

[0076] 2. Crude Extraction: Grape seed powder and n-hexane were mixed at a ratio of 1:5 (g / mL) and soaked for 24 hours. The precipitate was centrifuged at 3850 r / min for 15 minutes. The precipitate was mixed with 80% ethanol solution at a ratio of 1:7 (g / mL), and then sonicated at 40% power and below 38℃ for 20 minutes. Extraction was carried out in the dark for 12 hours, with stirring every 4 hours. The supernatant was collected by centrifugation and concentrated under reduced pressure (38℃, 90 r / min) to less than 30% of the original volume. The sample solution was freeze-dried to obtain crude grape seed proanthocyanidins.

[0077] 3. Activation of chromatography column packing: Take an appropriate amount of 16-80 mesh AB-8 macroporous resin and soak it in 80% ethanol solution for 24 hours, then rinse it with distilled water until there is no alcohol odor; then soak it in 6% HCl solution for 24 hours, and rinse the resin with distilled water until neutral; soak it in 6% NaOH solution for 24 hours, rinse the resin with distilled water until neutral, and then soak it in 95% ethanol solution for later use.

[0078] 4. Packing the chromatography column: Take an appropriate amount of macroporous resin, pack the column using the wet method, and then rinse the chromatography column with 5 column volumes (BV) of distilled water until the eluent is clear and has no alcohol odor.

[0079] 5. Purification of crude proanthocyanidins: Load 60 mg / mL of crude proanthocyanidin aqueous solution onto the column and rapidly wash the column with 10 column volumes of distilled water to remove soluble sugars, pigments, etc. from the crude proanthocyanidins; elute with 4 column volumes of 60% acetone solution, collect the eluent, concentrate under reduced pressure (38°C, 90 r / min) to below 40% of the original volume, freeze-dry the sample solution to obtain pure proanthocyanidins.

[0080] 6. Depolymerization of proanthocyanidins: Take a fully dissolved 1 mg / mL proanthocyanidin aqueous solution, add it to the lining of the high-pressure reactor, tighten the lid, purge with nitrogen for 1 min, close the venting knob, turn on the condenser, and run the reactor at 120℃ and 500 r / min for 45 min. Then set the temperature to room temperature and wait for the equipment to cool down. Open the lid and freeze-dry the sample to obtain oligomeric grape seed proanthocyanidin powder.

[0081] The extract prepared in this embodiment has a proanthocyanidin mass fraction of ≥48.21%, an average degree of polymerization of 3.39, a monomer content of 48.61%, and an average recovery rate of 90.56%.

[0082] Example 4 1. Pretreatment: Clean the grape skins and pomace produced during winemaking, air dry or dry at 40℃, sort out the grape seeds, crush them, and pass them through a 40-60 mesh sieve to obtain grape seed powder.

[0083] 2. Crude Extraction: Grape seed powder and n-hexane were mixed at a ratio of 1:3 (g / mL) and soaked for 24 hours. The precipitate was centrifuged at 3850 r / min for 15 minutes. The precipitate was mixed with 30% ethanol solution at a ratio of 1:5 (g / mL), and then sonicated at 40% power and below 38℃ for 20 minutes. Extraction was carried out in the dark for 12 hours, with stirring every 4 hours. The supernatant was collected by centrifugation and concentrated under reduced pressure (38℃, 90 r / min) to less than 30% of the original volume. The sample solution was freeze-dried to obtain crude grape seed proanthocyanidins.

[0084] 3. Activation of chromatography column packing: Take an appropriate amount of 16-80 mesh AB-8 macroporous resin and soak it in 70% ethanol solution for 24 hours, then rinse it with distilled water until there is no alcohol odor; then soak it in 4% HCl solution for 24 hours, and rinse the resin with distilled water until neutral; soak it in 4% NaOH solution for 24 hours, rinse the resin with distilled water until neutral, and then soak it in 70% ethanol solution for later use.

[0085] 4. Packing the chromatography column: Take an appropriate amount of macroporous resin, pack the column using the wet method, and then rinse the chromatography column with 5 column volumes (BV) of distilled water until the eluent is clear and has no alcohol odor.

[0086] 5. Purification of crude proanthocyanidins: Load 150 mg / mL of crude proanthocyanidin aqueous solution onto the column and rapidly wash the column with 10 column volumes of distilled water to remove soluble sugars, pigments, etc. from the crude proanthocyanidins; elute with 4 column volumes of 60% acetone solution, collect the eluent, concentrate under reduced pressure (38°C, 90 r / min) to below 40% of the original volume, freeze-dry the sample solution to obtain pure proanthocyanidins.

[0087] 6. Depolymerization of proanthocyanidins: Take a fully dissolved 2.0 mg / mL proanthocyanidin aqueous solution, add it to the lining of the high-pressure reactor, tighten the lid, purge with nitrogen for 1 min, close the venting knob, turn on the condenser, and run the reactor at 150℃ and 500 r / min for 180 min. Then set the temperature to room temperature and wait for the equipment to cool down. Open the lid and freeze-dry the sample to obtain oligomeric grape seed proanthocyanidin powder.

[0088] The extract prepared in this embodiment has a proanthocyanidin mass fraction of ≥47.83%, an average degree of polymerization of 1.26, a monomer content of 89.09%, and an average recovery rate of 88.33%.

[0089] Comparative Example This comparative example provides a method for extracting seed proanthocyanidins from grape pomace, including the following steps: 1) Pretreatment: Wash the waste grape skins and pomace produced during winemaking, air dry or dry at a low temperature below 40℃, sort out the grape seeds, crush them and pass them through a 40-60 mesh sieve to obtain grape seed powder; 2) Crude extraction: Using hexane as solvent, the grape seed powder was soaked for 24 hours at a solid-liquid ratio of 1:3 (g / mL). After soaking, the precipitate was collected and centrifuged at 3850 r / min for 15 minutes using a 500 mL centrifuge bottle. The precipitate was then collected. 70% ethanol was added to the precipitate as the extraction solvent at a solid-liquid ratio of 1:5 (g / mL). The mixture was stirred evenly and ultrasonicated at 38°C with 40% power for 20 minutes. The mixture was then statically extracted in the dark for 12 hours, with stirring or vortexing approximately every 4 hours. After static extraction, the mixture was centrifuged at 3850 r / min for 15 minutes using a 500 mL centrifuge bottle. The supernatant was collected. The supernatant was then distilled under reduced pressure using a rotary evaporator at 38°C and 90 r / min to concentrate the liquid to less than 30% of its original volume. The liquid was then removed and freeze-dried to obtain crude grape seed proanthocyanidins.

[0090] 3) Purification: Purification was performed using AB-8 macroporous resin. First, the AB-8 macroporous resin was pretreated: An appropriate amount of resin was soaked in 95% (v / v) ethanol solution for 24 hours until fully swollen. Then, it was filtered through a gauze bag and rinsed with distilled water until no alcohol odor remained. Next, it was soaked in 4% HCl solution for 24 hours, removed, and rinsed with distilled water until neutral. Then, it was soaked in 4% NaOH solution for 24 hours, removed, and rinsed with distilled water until neutral. Finally, the resin was placed in 70% ethanol solution for later use. An appropriate amount of AB-8 macroporous resin was wet-packed into a glass chromatography column. After packing, 5 times the volume of ethanol was used for further processing. Wash the chromatography column with distilled water by column volume (BV) until the eluent is clear and free of alcohol odor; dissolve the crude product extracted in step 2) in distilled water to a concentration of approximately 0.1 g / mL and load it onto a glass chromatography column; quickly wash the chromatography column with 10 column volumes of distilled water to remove soluble sugars, pigments, etc. from the crude proanthocyanidin product; elute the proanthocyanidins with 4 column volumes of 60% (volume fraction) acetone solution and collect the clear eluent; distill the collected clear eluent under reduced pressure using a rotary evaporator with parameters set to 38°C and 90 r / min to concentrate the liquid to less than 40% of its original volume, remove it, freeze-dry it, and obtain pure grape seed proanthocyanidins.

[0091] The extract prepared in this example has a proanthocyanidin content of ≥76.21%, an average degree of polymerization of 5.01, a monomer content of 43.41%, a crude extraction yield of 9.72%, a purified yield after crude extraction of 36.40%, and a total extraction rate of 3.54%.

[0092] Please refer to Figures 1 to 6, which are comparative LC-MS chromatograms of the comparative examples and various embodiments. C-catechin, EC-epicatechin, ECG-epicatechin gallate, EGC-epicalgalcatechin, and -PHL represent the phloroglucinol adducts of the corresponding substances.

[0093] The following figures illustrate the C and EC response peaks of a particular sample, using Figure 1 as an example (comparative example and various embodiments): Each figure below shows the response peaks of catechin (C) and epicatechin (EC) of a particular sample. The retention time of C is 6.27 min, and the retention time of EC is 7.09 min, meaning that the peaks appearing at these times represent the corresponding substances.

[0094] Table 1 Product Quality Inspection Indicators of Examples

[0095] Note: Recovery rate is the mass of the degraded proanthocyanidin extract / the amount of sample injected.

[0096] As shown in Table 1, the method of the present invention can effectively improve the recovery rate of proanthocyanidins after degradation, effectively avoid raw material loss, and significantly reduce the average degree of polymerization of proanthocyanidins, while increasing the content of monomers to varying degrees.

[0097] The methods for measuring each parameter in Table 1 are as follows: 1. Method for determining the purity of proanthocyanidins 1) Reagent preparation and standard curve determination Preparation of the colorimetric solution: Weigh 0.15g vanillin and dilute to 10ml in a brown volumetric flask with methanol; measure 3ml of concentrated hydrochloric acid and dilute to 10ml in a volumetric flask with methanol. Take 2.5ml of each and mix them in equal volumes to make 5ml of colorimetric solution for later use. Preparation of the standard curve: Weigh 17mg of catechin standard and dilute to 25ml in a brown bottle with methanol. Quantitatively dilute to prepare standard solutions of 0.34, 0.17, 0.085, 0.0425, 0.0215, and 0.0106mg / ml. Construct a standard curve with absorbance on the ordinate and catechin concentration on the abscissa.

[0098] 2) Sample determination Dissolve the sample in methanol and dilute to a suitable concentration. Take 1 ml of sample and add 5 ml of colorimetric solution. React in a 25℃ water bath in the dark for 15 min. Measure the absorbance at 500 nm and substitute it into the standard curve to calculate the proanthocyanidin mass content.

[0099] 3) Purity calculation In the methanol system, the C8 or C position on the A ring of the proanthocyanidin molecule ６ The active site cross-links with vanillin molecules to generate specific chromophores, which is a characteristic reaction of proanthocyanidins. Since each unit of proanthocyanidins can participate in the reaction in the methanol system, the total proanthocyanidin mass content in the system can be measured by establishing a standard curve using catechin monomer standards. The absorbance value obtained by substituting it into the standard curve, dividing it by the dilution factor, and then dividing it by the average degree of polymerization of the sample, yields the proanthocyanidin purity of the sample.

[0100] 2. Method for determining the average degree of polymerization of the extract 1) Determination Method: The content of phenolic compounds in the extract was detected by ultra-high performance liquid chromatography-mass spectrometry-quadrupole time-of-flight (UPLC-MS / MSQTOF). Appropriate amounts of catechin, epicatechin, epigallocatechin, and epicatechin gallate reference standards, dried to constant weight at 80℃, were accurately weighed and prepared into standard solutions with chromatographic grade methanol at concentrations between 10 and 100 μg / mL. 10 μL of each solution was accurately injected into the chromatograph.

[0101] The chromatographic conditions are as follows: Column: Kinetex C18 (100 mm × 2.1 mm, 1.7 μm); Mobile phases: Phase A - formic acid solution (0.1%), Phase B - acetonitrile; Flow rate: 0.4 mL / min; Injection volume: 2 μL; Column temperature: 35°C; Gradient elution: 0-3 min, 5% B; 3-6 min, 20% B; 6-12 min, 45% B; 12-18 min, 100% B; 18-20 min, 5% B.

[0102] Mass spectrometry conditions: negative ion mode; Spray voltage: 4.5kV; Ion source temperature: 550°C; Gas pressure: 50 psi.

[0103] The standard curve was obtained by regressing the peak area integral value against the monomer injection concentration.

[0104] 2) Sample Determination: Tannins in the sample were cleaved using the phloroglucinol method. First, the phloroglucinol reaction solution and sodium acetate stop solution were prepared: phloroglucinol (100 g / L) and ascorbic acid (20 g / L) were added to a chromatographic grade methanol solution containing 0.2 mol / L HCl to prepare the reaction solution; a 40 mM sodium acetate aqueous solution was prepared to prepare the stop solution. The sample was dissolved in chromatographic grade methanol to a concentration of 10 mg / mL. 100 μL of the sample solution and 100 μL of the phloroglucinol cleavage solution were mixed and reacted in a light-protected water bath at 50°C for 20 minutes. Then, 1 mL of sodium acetate stop solution was added to terminate the reaction. After thorough mixing, 1 mL of the solution was filtered through a 0.22 μm organic filter membrane and placed in a vial for liquid chromatography-mass spectrometry (LC-MS) analysis. The injection volume was 10 μL. Detection was performed under the above chromatographic conditions, and the peak area integral values ​​of each monomer were calculated. The content of each monomer after sample cleavage was obtained using the above standard curve. The average degree of polymerization of each sample was obtained by calculating the extended and terminal units.

[0105] 3. Methods for determining the monomer content in extracts 1) Determination method: The content of phenolic compounds in the extract was detected by ultra-high performance liquid chromatography-mass spectrometry-quadrupole time-of-flight (UPLC-MS / MSQTOF). Appropriate amounts of catechin, epicatechin, gallocatechin, and catechin gallate reference standards, dried to constant weight at 80℃, were accurately weighed and prepared into standard solutions with chromatographic grade methanol at concentrations between 10 and 100 μg / mL. 10 μL of each solution was accurately injected into the chromatograph.

[0106] The chromatographic conditions are as follows: Column: Kinetex C18 (100 mm × 2.1 mm, 1.7 μm); Mobile phases: Phase A - formic acid solution (0.1%), Phase B - acetonitrile; Flow rate: 0.4 mL / min; Injection volume: 2 μL; Column temperature: 35°C.

[0107] Gradient elution: 0-3 min, 5% B; 3-6 min, 20% B; 6-12 min, 45% B; 12-18 min, 100% B; 18-20 min, 5% B.

[0108] Mass spectrometry conditions: negative ion mode; Spray voltage: 4.5kV; Ion source temperature: 550°C; Gas pressure: 50 psi.

[0109] The standard curve was obtained by regressing the peak area integral value against the monomer injection concentration.

[0110] 2) Sample determination: Accurately weigh the proanthocyanidin product, dissolve it in chromatographic grade methanol, and prepare a 1 mg / mL proanthocyanidin solution. Before injection, filter the solution through a 0.22 μm organic filter membrane. The injection volume is 10 μL. Detect the sample according to the above chromatographic conditions and calculate the peak area integral value of each monomer. Use the above standard curve to obtain the content of each monomer. The sum of the contents of each monomer is the monomer content of the proanthocyanidin product.

[0111] In summary, the present invention provides a method for preparing and applying oligomeric grape seed proanthocyanidins, which effectively reduces the average degree of polymerization of grape seed proanthocyanidins in a one-pot process, with a recovery rate of over 90%, high degradation efficiency, simple method, and easy industrial production.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing oligomeric grape seed proanthocyanidins, characterized in that, Includes the following steps: Grape pomace is pretreated to obtain grape seed powder; Grape seed powder was extracted using an extraction solvent to obtain crude grape seed proanthocyanidins. The crude product was purified using macroporous resin to obtain pure grape seed proanthocyanidins. The pure product was dissolved in water and subjected to a depolymerization reaction in a high-pressure reactor to obtain oligomeric grape seed proanthocyanidins.

2. The method for preparing oligomeric grape seed proanthocyanidins according to claim 1, characterized in that, The preprocessing includes: Wash the grape skins and pomace, dry them at 30-60℃ for 12-36 hours, separate the grape seeds, crush them and pass them through a 20-80 mesh sieve.

3. The method for preparing oligomeric grape seed proanthocyanidins according to claim 1, characterized in that, The extraction solvent is n-hexane, and the ratio of grape seed powder to n-hexane is 1:1~5 (g / mL). The extraction time is 12~48h, and the precipitate is collected by centrifugation. The precipitate was mixed with 30-95% ethanol at a ratio of 1:3-7 (g / mL), and ultrasonically extracted at 20-80℃ for 5-60 min. The mixture was then extracted in the dark for 6-48 h, with stirring every 4 h during the extraction. After ultrasonication, extraction, and stirring, the mixture was centrifuged to collect the supernatant, concentrated under reduced pressure, and then freeze-dried to obtain crude grape seed proanthocyanidins.

4. The method for preparing oligomeric grape seed proanthocyanidins according to claim 1, characterized in that, The macroporous resin was activated by sequentially soaking in 60-80% ethanol, 2-6% HCl, and 2-6% NaOH solutions, rinsed with distilled water until neutral, and then stored in 30-95% ethanol for later use.

5. The method for preparing oligomeric grape seed proanthocyanidins according to claim 4, characterized in that, The macroporous resin is type AB-8, with a particle size of 16~80 mesh.

6. The method for preparing oligomeric grape seed proanthocyanidins according to claim 1, characterized in that, The purification process includes: The crude product was prepared into an aqueous solution of 10-150 mg / mL and loaded onto the sample. It was then rinsed with distilled water to remove impurities, followed by elution with 40-70% acetone-water solution. The eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain pure grape seed proanthocyanidins.

7. The method for preparing oligomeric grape seed proanthocyanidins according to claim 1, characterized in that, The conditions for the depolymerization reaction are: The pure proanthocyanidin was prepared into an aqueous solution of 0.5~2.0 mg / mL, placed in a high-pressure reactor, and nitrogen was introduced to purge the air. The reaction was carried out at 80~150℃ with stirring for 45~180 min. After cooling, the sample was taken, concentrated under reduced pressure and freeze-dried to obtain oligomeric grape seed proanthocyanidins.

8. The oligomeric grape seed proanthocyanidins prepared by the method according to any one of claims 1 to 7 have an average degree of polymerization of less than 5, a monomer content of greater than or equal to 47%, and a recovery rate of greater than or equal to 88%.

9. The oligomeric grape seed proanthocyanidins according to claim 8, characterized in that, The average degree of polymerization is 1.26 to 4.20, and the monomer content is 47.36% to 89.09%.

10. The application of the oligomeric grape seed proanthocyanidins according to claim 9 in the preparation of health products, pharmaceuticals or cosmetics.

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