Efficient extraction method of grape seed procyanidine

Abstract

The invention relates to the field of plant active ingredient extraction, and particularly discloses an efficient extraction method of grape seed procyanidine. The preparation method comprises the following steps: taking grape seed powder, and carrying out supercritical fluid extraction on the grape seed powder by taking ethylene as a supercritical fluid and acetone as an entrainer to obtain degreased grape seed residues; mixing the degreased grape pip residues with an acidic alcohol solution containing poloxamer, and carrying out ultrasonic extraction; and purifying the extracting solution by using resin functionalized by beta-cyclodextrin, carrying out gradient elution by using ethanol as an eluent, adding a protective agent into the eluent, and carrying out low-temperature vacuum drying. By adopting the extraction method, the procyanidine substance in the grape seeds can be efficiently extracted, and as the poloxamer can selectively solubilize the oligomer and the beta-cyclodextrin functional resin can realize specific adsorption of the oligomer, the content of the oligomer in the product is increased.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant active ingredient extraction, more particularly, it relates to a high-efficiency extraction method of grape seed proanthocyanidin. BACKGROUND

[0002] Grape seed proanthocyanidin, as a natural antioxidant, has shown a wide application prospect in the fields of food, cosmetics and medicine due to its unique chemical properties and biological activities. Grape seed proanthocyanidin has a C6-C3-C6 structure, which is a polyphenol compound formed by C4-C8 or C4-C6 condensation with flavan-3-ol as the basic constituent unit. The oligomeric proanthocyanidin has n = 2-5, and the high polymer proanthocyanidin has n > 5. The antioxidant activity of proanthocyanidin is affected by the degree of polymerization. The oligomer has high activity, the higher the degree of polymerization, the lower the antioxidant activity, and the bioavailability of the oligomer is also higher than that of the high polymer. With the in-depth understanding of proanthocyanidin and the discovery of the biological activity of proanthocyanidin with different degrees of polymerization, the market demand for high-quality grape seed proanthocyanidin, especially the product rich in oligomers, continues to rise, which also promotes the research and development of related production technologies.

[0003] At present, the main production methods of grape seed proanthocyanidin include solvent extraction, enzymatic hydrolysis and ultrasonic assistance, etc. For example, a method for extracting and separating water-soluble proanthocyanidin from grape seeds is provided in Chinese patent CN 114890973B. The method uses CO2 supercritical extraction for degreasing, then uses ethanol as the extraction liquid for ultrasonic extraction, and then uses macroporous resin adsorption resin for purification and spray drying after refrigeration. The method has the following disadvantages: the CO2 supercritical degreasing equipment has high pressure, and only degreasing is not gelatinous and waxy, and residual impurities will affect the subsequent purification efficiency; the use of conventional macroporous resin has low selectivity for proanthocyanidin oligomers; the inlet air temperature of spray drying may cause oxidation and polymerization of proanthocyanidin, resulting in degradation of heat-sensitive components. At the same time, more importantly, the extraction method ignores the extraction of proanthocyanidin oligomers, so that the content of oligomers in the product is low, which cannot fully meet the market demand for high-quality proanthocyanidin products rich in oligomers.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] In order to solve the above problems, the present application provides a high-efficiency extraction method of grape seed proanthocyanidin.

[0006] The present application adopts the following technical solution: In a first aspect, the present application provides a high-efficiency extraction method of grape seed proanthocyanidin, which comprises: (1) taking grape seed powder, using ethylene as a supercritical fluid, and using acetone as a entrainer to extract the grape seed powder by supercritical fluid extraction to obtain defatted grape seed residue; (2) mixing the defatted grape seed dregs with an acidic alcohol solution containing 1-3 wt% poloxamer, performing ultrasonic extraction at 45-55℃, and obtaining an extraction liquid rich in procyanidin oligomers after filtration; (3) performing grafting treatment on the macroporous resin with β-cyclodextrin to obtain a functionalized resin, performing membrane filtration on the extraction liquid, and performing functionalized resin chromatography on the filtered extraction liquid, and performing gradient elution on the chromatography with ethanol as the eluent to obtain an eluate; (4) adding an ascorbic acid protective agent to the eluate after filtration, and performing vacuum drying at 50-60℃ to obtain a grape seed procyanidin extract.

[0007] Further, the process parameters of the supercritical fluid extraction are as follows: extraction temperature is 20-23℃, extraction pressure is 11-15 Mpa, extraction time is 25-30 min, extraction agent flow rate is 12-14 kg / h, and the addition amount of the entraining agent is 3-6%.

[0008] By using the above technical solution, compared with the existing CO2 supercritical extraction defatting process, the pressure of the ethylene supercritical fluid equipment is greatly reduced, and the gum and lipid can be removed synchronously by using acetone as the entraining agent, thereby improving the subsequent purification efficiency.

[0009] Further, in step (2), when the defatted grape seed dregs are subjected to ultrasonic extraction, the acidic alcohol solution is a citric acid-ethanol solution with a volume concentration of 40-50% and a pH of 3.5-4.5.

[0010] By using the above technical solution, on the one hand, low temperature and acidic environment can inhibit the oxidation of anthocyanins during the extraction process; on the other hand, the poloxamer in the acidic alcohol solution is a non-ionic triblock copolymer surfactant with a unique "hydrophilic-hydrophobic-hydrophilic" structure, the hydrophobic core captures oligomers, and the hydrophilic shell enhances dispersion; and because it can form micelles in the solution, the micelle size is about 8 nm, which naturally repels high polymers, thereby selectively solubilizing oligomers, increasing the content of oligomers in the extraction liquid, and ultimately obtaining an extraction liquid rich in procyanidin oligomers. Preferably, poloxamer 407 is selected as the solubilizing agent.

[0011] Further, in step (2), when the defatted grape seed dregs are subjected to ultrasonic extraction, the ultrasonic power is 400-500 W, and the total extraction time is 10-20 min.

[0012] Further, in step (3), the step of performing grafting treatment on the macroporous resin with β-cyclodextrin includes: selecting a styrene-based macroporous resin and performing activation treatment; The activated resin is mixed with epichlorohydrin in an alkaline solution, and a catalytic amount of tetrabutylammonium bromide is added to perform a shaking reaction, to obtain an epoxy resin; The beta-cyclodextrin is dissolved in an alkaline solution, and the epoxy resin is added, and polyethylene glycol 400 is added as a phase transfer catalyst, and a grafting reaction is performed under a nitrogen atmosphere, to obtain a functionalized resin grafted with beta-cyclodextrin.

[0013] By adopting the technical scheme, the styrene macroporous resin is activated first to have better reactivity; then the resin is reacted with epichlorohydrin to have an epoxy group, and the beta-CD is stably grafted through the epoxy group "bridging" chemical bond, so that the falling defect of the physical embedding method is broken through. Since the beta-cyclodextrin has a specific cavity structure, the size and shape of the oligomeric procyanidin molecule can match the cavity of the beta-cyclodextrin, so that the oligomeric procyanidin molecule can selectively enter the cavity to form an inclusion compound. The beta-cyclodextrin functionalized resin can specifically adsorb the oligomer, thereby increasing the content of the oligomer in the product and improving the yield of the oligomer in the purification of the grape seed procyanidin.

[0014] Further, the molar ratio of the beta-cyclodextrin to the epoxy resin is 1.1-1.3:1.

[0015] Further, the temperature of the grafting reaction is 60-70 DEG C, and the reaction time is 18-24 h.

[0016] Further, the volume ratio of the activated resin to epichlorohydrin is 2.5-3.5:1.

[0017] Further, in the preparation of the epoxy resin, the reaction temperature is 53-57 DEG C, and the reaction time is 4-8 h.

[0018] Further, in step (4), the mass concentration of the ascorbic acid protective agent added is 0.05-0.07 wt%, and the temperature of vacuum drying is 55-60 DEG C.

[0019] By adopting the above technical scheme, the ascorbic acid protective agent with a mass concentration of 0.05-0.07 wt% is added during vacuum drying, and the vacuum drying temperature is controlled at 55-60 DEG C, so that the ascorbic acid can better play an antioxidant role, and a more stable antioxidant microenvironment is formed in cooperation with vacuum drying, so that the degradation of the heat-sensitive grape seed procyanidin due to temperature and oxidation factors during the drying process is effectively avoided, thereby ensuring the quality and active ingredient content of the extract and improving the product quality.

[0020] In summary, the present application has the following beneficial effects: The application provides a high-efficiency extraction method of grape seed proanthocyanidins, which is supercritical fluid extraction with ethylene as a supercritical fluid and acetone as an entrainer. Compared with a traditional supercritical defatting method, the pressure requirement is reduced, the dependence on high-pressure equipment is reduced, and the energy consumption is reduced. Meanwhile, the gum and lipids in grape seeds can be removed synchronously, which can avoid the pollution of the resins used subsequently, and then the subsequent purification efficiency is improved. In the ultrasonic extraction process, low temperature of 45-55 DEG C and acidic alcohol solution are used, so that the oxidation polymerization and C ring cracking of proanthocyanidins with heat sensitivity and pH instability are inhibited. 1-3wt% of poloxamer is added to selectively solubilize proanthocyanidin oligomers and improve the content of the oligomers in the extraction solution. The macroporous resin is treated by grafting with β-cyclodextrin to obtain a functional resin, and the functional resin can realize specific adsorption of proanthocyanidin oligomers, so that the content of proanthocyanidin oligomers in the product is improved. The ascorbic acid protective agent is added in the vacuum drying stage of the eluent, which can form an antioxidant microenvironment in cooperation with vacuum drying, so that the degradation of heat-sensitive proanthocyanidins and other components is avoided in the vacuum drying process at 50-60 DEG C, the quality of the extract is ensured, and finally the high-efficiency extraction of grape seed proanthocyanidins is realized. DETAILED DESCRIPTION

[0021] The embodiments of the application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only used to illustrate the application, and should not be regarded as limiting the scope of the application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are conventional products that can be purchased on the market.

[0022] The specific embodiments of the application are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the application, and are not used to limit the application.

[0023] Preparation example of raw materials and / or intermediates Preparation example 1 The preparation example provides a β-cyclodextrin functional resin, and a preparation method thereof comprises the following steps: 1. Resin activation: D101 macroporous resin is soaked in ethanol overnight, washed with deionized water until neutral, then washed with 5% NaOH solution for dynamic circulation for 4 column volumes, washed with water until neutral, then washed with 5% HCl solution for dynamic circulation for 4 column volumes, washed with water until neutral, and then vacuum dried to constant weight.

[0024] 2. Epoxy functionalization: Epoxy chloropropane was mixed with the activated resin in a volume ratio of 3:1, then 40% NaOH solution and catalytic amount of tetrabutylammonium bromide were added, the resulting reaction system was placed in a constant temperature shaker at 55°C for 6h, then washed with acetone and deionized water in turn until no chloride ion was obtained, and the epoxy resin was obtained.

[0025] 3. β-cyclodextrin grafting: β-cyclodextrin was dispersed in 10% NaOH solution, and the epoxy resin of step (2) was added in an amount of β-CD:epoxy resin molar ratio of 1.1:1, then 3wt% polyethylene glycol 400 was added, and the reaction was stirred at 65°C under nitrogen protection for 24h, then washed with hot water, anhydrous ethanol and acetone in turn, and washed with ethanol to remove physically adsorbed β-cyclodextrin, and the β-cyclodextrin functionalized resin was obtained.

[0026] Preparation Example 2 The difference between this preparation example and Preparation Example 1 is that in the β-cyclodextrin grafting step, the molar ratio of β-CD to epoxy resin is 1.2:1.

[0027] Preparation Example 3 The difference between this preparation example and Preparation Example 1 is that in the epoxy functionalization step, the reaction temperature is 50°C.

[0028] Preparation Example 4 The difference between this preparation example and Preparation Example 1 is that in the epoxy functionalization step, the reaction temperature is 65°C.

[0029] Preparation Example 5 The difference between this preparation example and Preparation Example 1 is that in the β-cyclodextrin grafting step, the molar ratio of β-CD to epoxy resin is 0.8:1.

[0030] Preparation Example 6 The difference between this preparation example and Preparation Example 1 is that in the β-cyclodextrin grafting step, the amount of polyethylene glycol 400 added is 1wt%.

[0031] Using the adsorption performance test, the ordinary conventional D101 type macroporous resin was used as a control, and the specific adsorption capacity of the β-cyclodextrin functionalized resin provided in the above Preparation Examples 1-6 for anthocyanin oligomers was investigated: The above resins were respectively packed into columns, washed with water and ethanol, then an ethanol solution containing 100mg / L of procyanidin B2 (dimer) was used for dynamic loading, the flow rate was 2BV / h, and the effluent was collected. Then the concentration of procyanidin B2 in the effluent was detected by HPLC, and the adsorption rate was calculated according to the following formula: Adsorption rate (%) = (1-C / C0)*100% Wherein, C is the concentration of the effluent; C0 is the initial concentration.

[0032] The results are shown in Table 1: Table 1. Adsorption rate of proanthocyanidin oligomers on resins Resin Adsorption rate (%) Conventional D101 type macroporous resin 38 Preparation Example 1 94 Preparation Example 2 95 Preparation Example 3 91 Preparation Example 4 64 Preparation Example 5 47 Preparation Example 6 53 As shown in Table 1, compared with conventional resins, the adsorption rate of proanthocyanidin oligomers on the resins functionalized with β-cyclodextrin is significantly improved, and the preparation examples 1 and 2 are optimal. In contrast, the adsorption rates of the preparation examples 4-6 are lower, which is mainly because in the step of epoxy group functionalization, the crosslinking degree of the resin is reduced when the reaction temperature exceeds 60℃, thereby affecting the grafting rate of β-cyclodextrin (preparation example 4); and when the molar ratio of β-CD to epoxy resin is less than 1, the grafting rate of β-cyclodextrin is significantly reduced (preparation example 5); at the same time, in the step of β-cyclodextrin grafting, if the amount of polyethylene glycol 400 as the phase transfer catalyst is too low, the grafting efficiency will be affected. Example

[0033] Example 1 The present embodiment provides a high-efficiency extraction method of grape seed proanthocyanidin, which comprises the following steps: 1. Take grape seed powder, use ethylene as a supercritical fluid, and use acetone as a entrainer to perform supercritical fluid extraction on the grape seed powder, the extraction temperature is 22℃, the extraction pressure is 12Mpa, the extraction time is 25min, the extraction agent flow rate is 13kg / h, and the addition amount of the entrainer is 4%, to obtain defatted grape seed residue; (2) Mix the defatted grape seed residue with a citric acid-ethanol solution containing 2wt% poloxamer 407 (pH is 3.5-4.5, and the volume concentration of ethanol is 50%), and perform ultrasonic extraction at 50℃, the ultrasonic power is 450w, and the extraction is performed twice, each time for 8min, to obtain an extract liquid rich in proanthocyanidin oligomers after filtration; (3) After membrane filtration of the extract liquid, perform functional resin chromatography on the extract liquid by using the β-cyclodextrin functional resin provided in the preparation example 1, and perform gradient elution by using an ethanol solution as an eluent (the concentration of the ethanol solution is 30%, 50% and 70% in turn, and each concentration is washed for 2 column volumes), to obtain an eluate; (4) After filtration of the eluate, add 0.06wt% ascorbic acid protective agent, and perform vacuum drying at 55℃, to obtain grape seed proanthocyanidin extract.

[0034] Example 2 The difference between the present embodiment and example 1 is that the process parameters of the supercritical fluid extraction are as follows: the extraction temperature is 23℃, the extraction pressure is 12Mpa, the extraction time is 26min, the extraction agent flow rate is 12kg / h, and the addition amount of the entrainer is 6%.

[0035] Example 3 The difference between this example and example 1 is that the process parameters of supercritical fluid extraction are as follows: extraction temperature is 22°C, extraction pressure is 11 MPa, extraction time is 30 min, extractant flow rate is 14 kg / h, and the amount of entrainer added is 3%.

[0036] Example 4 The difference between this example and example 1 is that the acid alcohol solution used in the ultrasonic extraction of defatted grape seed residues is a citric acid-ethanol solution with a volume concentration of 40% and a pH of 4.5.

[0037] Example 5 The difference between this example and example 1 is that the amount of poloxamer 407 in the acid alcohol solution used in the ultrasonic extraction of defatted grape seed residues is 1 wt%.

[0038] Example 6 The difference between this example and example 1 is that the amount of ascorbic acid protective agent added during the post-treatment drying process of the eluate is 0.03%.

[0039] Comparative Example Comparative Example 1 The difference between this comparative example and example 1 is that step (3) uses a common conventional 101 type macroporous resin for purification.

[0040] Comparative Example 2 The difference between this comparative example and example 1 is that 50% ethanol is used for ultrasonic extraction in step (2).

[0041] Comparative Example 3 The difference between this comparative example and example 1 is that CO2 is used as the supercritical fluid in step (1), and the process parameters are as follows: extraction pressure is 28 MPa, extraction temperature is 45°C, I separation kettle pressure is 9 MPa, II separation kettle pressure is 7 MPa; I separation kettle temperature is 35°C, II separation kettle temperature is 35°C, and extraction time is 4 h.

[0042] Comparative Example 4 The difference between this comparative example and example 1 is that supercritical ethylene fluid is used as the extractant in step (1), and no entrainer, i.e. no acetone, is added.

[0043] Comparative Example 5 The difference between this comparative example and example 1 is that supercritical ethylene fluid is used as the extractant in step (1), and ethanol is used as the entrainer, with an addition amount of 4%.

[0044] Comparative Example 6 The difference between this comparative example and example 1 is that the acid alcohol solution used in the ultrasonic extraction process in step (2) does not contain poloxamer 407.

[0045] Comparative Example 7 The difference between this comparative example and Example 1 is that 0.06wt% trehalose is added as a protective agent in step (4).

[0046] Performance detection test Detection method / test method The content of total procyanidins in the extract was determined by the procyanidin determination method in the Chinese Pharmacopoeia 2020 edition, and the extract was determined by HPLC-MS, and the proportion of oligomers with a degree of polymerization of 2-4 was calculated. The results are shown in Table 2: Table 2. Performance determination results of the extract As can be seen from Table 2: The total procyanidin content in the extract provided in Examples 1-6 of the present application is more than 97%, and the proportion of oligomers is more than 86%. Therefore, it is shown that the extraction method provided in the present application can improve the extraction rate of oligomers.

[0047] It can be seen from Example 1 and Comparative Example 1 that the proportion of oligomers in the extract obtained by using a conventional ordinary 101 type macroporous resin for purification is significantly reduced, which shows that the use of a β-cyclodextrin functional resin for purification in the present application can realize specific adsorption of oligomers and improve the content of oligomers in the product.

[0048] It can be seen from Example 1 and Comparative Examples 2 and 6 that during the ultrasonic extraction process, the use of an acidic extraction solution can inhibit the oxidation of anthocyanins, and the addition of poloxamer can selectively solubilize oligomers and improve the extraction efficiency of oligomers.

[0049] It can be seen from Example 1 and Comparative Examples 3-5 that compared with the use of traditional CO2 supercritical fluid, the use of ethylene as a supercritical fluid can improve the extraction rate of oligomers; and the entrainer acetone also plays a key role in degreasing and removing gum.

[0050] It can be seen from Example 1 and Comparative Example 7 that compared with the use of trehalose as a protective agent, the use of ascorbic acid as a protective agent can cooperate with vacuum low-temperature drying technology to avoid the degradation of heat-sensitive substances, and the obtained product has a light color, meeting the high-end cosmetic raw material standard.

[0051] The specific embodiments are merely an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for efficiently extracting proanthocyanidins from grape seeds, characterized in that: It includes: (1) Grape seed powder is extracted by supercritical fluid extraction using ethylene as a supercritical fluid and acetone as an entrainer to obtain defatted grape seed residue; (2) mixing the defatted grape seed residue with an acidic alcohol solution containing 1-3 wt% of poloxamer, performing ultrasonic extraction at 45-55° C., and filtering to obtain an extract rich in proanthocyanidin oligomers; (3) grafting a macroporous resin with β-cyclodextrin to obtain a functionalized resin, filtering the extract through a membrane, and then performing functionalized resin chromatography, performing gradient elution using an ethanol solution as an eluent to obtain an eluate; (4) After filtering the eluate, ascorbic acid protective agent is added, and vacuum drying is performed at 50-60° C. to obtain grape seed proanthocyanidin extract.

2. The efficient extraction method of grape seed proanthocyanidins according to claim 1, characterized in that: The process parameters of the supercritical fluid extraction are: extraction temperature of 20-23° C., extraction pressure of 11-15 MPa, extraction time of 25-30 min, extraction agent flow rate of 12-14 kg / h, and entrainer addition amount of 3-6%.

3. The efficient extraction method of grape seed proanthocyanidins according to claim 1, characterized in that: In step (2), when ultrasonic extraction is performed on the defatted grape seed residue, the acidic alcohol solution is a citric acid-ethanol solution with a volume concentration of 40-50% and a pH of 3.5-4.

5.

4. The efficient extraction method of grape seed proanthocyanidins according to claim 1, characterized in that: In step (2), when ultrasonic extraction is performed on the defatted grape seed residue, the ultrasonic power is 400-500W and the total extraction time is 10-20 minutes.

5. The efficient extraction method of grape seed proanthocyanidins according to claim 1, characterized in that: In step (3), the step of grafting the macroporous resin with β-cyclodextrin includes: Select styrene-based macroporous resin and perform activation treatment; The activated resin is mixed with epichlorohydrin in an alkaline solution, and subjected to a shaking reaction in the presence of a catalytic amount of tetrabutylammonium bromide to obtain an epoxy resin; Beta-cyclodextrin is dissolved in an alkaline solution, and then the epoxy resin is added, and polyethylene glycol 400 is added as a phase transfer catalyst. A grafting reaction is carried out under a nitrogen atmosphere to obtain a functionalized resin grafted with beta-cyclodextrin.

6. The efficient extraction method of grape seed proanthocyanidins according to claim 5, characterized in that: The molar ratio of the beta-cyclodextrin to the epoxy resin is 1.1-1.3:

1.

7. The efficient extraction method of grape seed proanthocyanidins according to claim 5, characterized in that: The grafting reaction temperature is 60-70° C., and the reaction time is 18-24 hours.

8. The efficient extraction method of grape seed proanthocyanidins according to claim 5, characterized in that: The volume ratio of the activated resin to epichlorohydrin is 2.5-3.5:

1.

9. The efficient extraction method of grape seed proanthocyanidins according to claim 5, characterized in that: During the preparation of the epoxy resin, the reaction temperature is 53-57° C. and the reaction time is 4-8 hours.

10. The efficient extraction method of grape seed proanthocyanidins according to claim 5, characterized in that: In step (4), the mass concentration of ascorbic acid protective agent added is 0.05-0.07wt%, and the vacuum drying temperature is 55-60°C.

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