Fat-soluble oligomeric procyanidine as well as preparation method and application thereof

Abstract

The invention discloses fat-soluble oligomeric procyanidine as well as a preparation method and application thereof, and belongs to the technical field of high-value processing of food byproducts. Oleic anhydride is utilized to modify the proanthocyanidin oligomer, and a fatty acid chain is introduced to the proanthocyanidin oligomer, so that the proanthocyanidin oligomer obtains good fat solubility, and meanwhile, the oxidation resistance of the proanthocyanidin oligomer is also improved. The modified procyanidine oligomer has the advantages of large particle size, high fat solubility, good in-vitro digestion stability, difficult degradation and easy absorption, overcomes the problem of low bioavailability of the existing procyanidine oligomer, and has good medical value and application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of high-value processing of food by-products, and particularly relates to a fat-soluble oligomeric proanthocyanidin, a preparation method and application thereof. Background Art

[0002] Peanuts are primarily valuable as an oil crop, providing humans with oil. Peanut skins, however, are rarely utilized as a byproduct of oil and fat processing. The proanthocyanidin content in peanut skins is 15-17%. The proanthocyanidins crudely extracted from peanut skins are primarily polymers, primarily type A proanthocyanidins containing catechin and epicatechin monomers. The bioavailability of proanthocyanidins of varying degrees of polymerization varies significantly. Due to their large molecular weight, polymers are difficult for the body to absorb and utilize, thus reducing their bioavailability. Therefore, it is necessary to depolymerize polymeric proanthocyanidins into smaller, more readily absorbed oligomeric proanthocyanidins, thereby reducing their molecular weight and steric hindrance and improving the bioavailability of proanthocyanidins. Peanut proanthocyanidins are readily soluble in water and also in polar organic solvents such as methanol, ethanol, and acetone. However, their polyhydroxy structure makes them hydrophilic but less lipophilic, making them poorly absorbed by the small intestine. Therefore, the development of peanut red skin fat-soluble oligomeric proanthocyanidins has broad market prospects. The esterification reaction of hydroxyl functional groups with fatty acids can improve the hydrophobicity of proanthocyanidins and fat-soluble flavonoids. In addition, the esterification products can easily bind to cell membranes, thereby eliminating fat-soluble "garbage" attached to the cell surface. When fatty chains are connected to proanthocyanidins, their stability is increased and the antioxidant effect is prolonged.

[0003] At present, the main methods for esterification of proanthocyanidins are enzymatic and chemical methods (including acyl chloride and acyl anhydride). For example, patent CN106755162 A uses enzymatic method to prepare fat-soluble grape seed proanthocyanidins; patent CN 118203514 A uses chemical method to prepare fat-soluble grape seed proanthocyanidins using linoleoyl chloride. Although the above-mentioned existing technologies can produce fat-soluble proanthocyanidins, they still have the following disadvantages: (1) The enzymatic method has mild conditions, but it takes a long time, is inefficient, and is costly; (2) The types of enzymes are limited and expensive, which is not conducive to industrial production; (3) The acyl chloride reaction is highly active and requires strict isolation from moisture to avoid hydrolysis side reactions; (4) The acyl chloride derivatives will generate HCl, which can destroy the phenolic hydroxyl groups in the proanthocyanidins; (5) Since the acyl chloride byproducts will generate HCl and salt byproducts, the purification steps are complicated and the operation is inconvenient. Summary of the Invention

[0004] The present invention provides a method for preparing fat-soluble oligomeric proanthocyanidins, comprising the following steps: The oligomeric proanthocyanidins and oleic anhydride are mixed, a solvent is added, and the mixture is stirred evenly; a catalyst and a molecular sieve are then added to carry out an esterification reaction; after the reaction is completed, the precipitate is removed, and then the solvent is removed to obtain a crude esterification product; the crude esterification product is extracted, the extract is chromatographed, and the organic solvent is removed to obtain a fat-soluble oligomeric proanthocyanidin.

[0005] In the above preparation method, each raw material is selected from the following parts: 0.05-0.15 parts of oligomeric proanthocyanidins, 0.05-0.5 parts of oleic anhydride, 10-100 parts of solvent, and 0.01-0.1 parts of catalyst.

[0006] In a specific embodiment, each raw material is selected from the following parts: 0.1 parts of oligomeric proanthocyanidins, 0.3 parts of oleic anhydride, 50 parts of solvent, and 0.075 parts of catalyst.

[0007] In the above preparation method, the solvent is selected from one of acetone, ethyl acetate and tetrahydrofuran.

[0008] In the above preparation method, the catalyst is selected from one of sodium acetate, sodium carbonate and sodium bicarbonate.

[0009] In the above preparation method, the esterification reaction conditions are selected from: reaction at 35-60°C for 3-18 hours; preferably: reaction at 50°C for 12 hours.

[0010] In the above preparation method, the extraction solvent used in the extraction is a water / ethyl acetate mixed solvent, and the volume ratio of the two is 1:1.

[0011] In the above preparation method, the specific step of the extraction is: extracting the crude esterification product with water / ethyl acetate (1:1, v / v), and collecting the ethyl acetate layer.

[0012] In the above preparation method, the chromatography is performed by gradient elution using silica gel column chromatography to remove oleic acid and separate and purify the product, and the eluent is n-hexane (70%-0%): ethyl acetate (30%-100%).

[0013] In the above preparation method, the purpose of adding molecular sieves is to remove moisture from the reaction system.

[0014] In the above preparation method, when the component is a solid component, the portion number represents grams; when the component is a liquid component, the portion number represents milliliters; in actual application, the portion number can be enlarged or reduced proportionally.

[0015] The present invention provides fat-soluble oligomeric proanthocyanidins prepared by the method.

[0016] The present invention provides the use of the fat-soluble oligomeric proanthocyanidins in the preparation of medicines with antioxidant and / or fat-soluble effects.

[0017] The beneficial effects of the present invention are: The present invention uses oleic anhydride to modify proanthocyanidin oligomers, introducing fatty acid chains into the proanthocyanidin oligomers. This improves the proanthocyanidin oligomers' fat solubility and antioxidant capacity. The modified proanthocyanidin oligomers have a large particle size, high fat solubility, good in vitro digestion stability, are not easily degraded, and are easily absorbed. This overcomes the low bioavailability of existing proanthocyanidin oligomers and offers promising medical value and application prospects.

[0018] In addition, the preparation method of the present invention uses oleic anhydride as a chemical reaction reagent, which has the following advantages: (1) Compared with the enzymatic method, the reaction time is relatively short, the cost is low, it is conducive to industrial production, the universality is stronger, and there is no need to consider the problem of enzyme inactivation; (2) Compared with oleoyl chloride, acyl chloride has higher reaction activity and needs to be strictly isolated from water to avoid hydrolysis side reactions. In addition, oleoyl chloride reacts violently and also generates HCl, which can destroy the phenolic hydroxyl groups in proanthocyanidins, while oleic anhydride can react at mild temperatures without the need for strict anhydrous conditions. The byproduct is oleic acid, which is easy to purify later; (3) In addition, oleoyl chloride is highly active and easy to react with other heteroatoms in proanthocyanidins, while oleic anhydride has higher selectivity and preferentially esterifies phenolic hydroxyl groups, reducing structural damage; (4) Oleic anhydride is more stable and easy to store, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the content of total phenols and proanthocyanidins in fat-soluble oligomeric proanthocyanidins; Figure 2 is the oil-water partition coefficient of fat-soluble oligomeric proanthocyanidins; Figure 3 This is the infrared spectrum analysis diagram of fat-soluble oligomeric proanthocyanidins; Figure 4 Scanning electron microscope images; A is OPSP, B is OPSP-O; Figure 5 For particle size and zeta potential determination; where A is particle size and B is zeta potential; Figure 6 It is the thermal stability analysis diagram; Figure 7 For antioxidant activity analysis; A is DPPH scavenging rate, B is hydroxyl radical scavenging rate, and C is β-carotene bleaching scavenging rate; Figure 8 In vitro digestion stability. DETAILED DESCRIPTION

[0020] In the present invention, peanut red skin fat-soluble oligomeric proanthocyanidins are prepared from peanut red skin oligomeric proanthocyanidins (OPSP) and oleic anhydride, and modified fat-soluble oligomeric proanthocyanidins (OPSP-O) are prepared by modifying the proanthocyanidin oligomers and introducing fatty acid chains.

[0021] In the present invention, peanut red coat oligomeric proanthocyanidins were purchased from Shandong Jinsheng Biotechnology Co., Ltd.

[0022] In the present invention, the 4A molecular sieve needs to be activated before use (oven at 150° C. for 24 h). After activation, the 4A molecular sieve is placed in an acetone solution for 5 days to remove the moisture therein.

[0023] In the present invention, oleic anhydride is used to modify oligomeric proanthocyanidins. Its byproducts are weakly acidic, have little impact on the system, are low-cost, and are easily separated and relatively simple to operate. Acetone is used as a solvent because of its high polarity, which can effectively dissolve proanthocyanidins, thereby increasing the product yield. Sodium acetate is used as a catalyst because it is an organic base with high solubility in organic solvents, which can effectively exert its acid-binding effect and improve the yield.

[0024] The other materials used in the present invention, unless otherwise stated, can be obtained through commercial channels. Unless otherwise specified, other terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below with reference to specific examples and data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0025] Example 1 The steps for preparing peanut red skin fat-soluble oligomeric proanthocyanidins are as follows: 0.1 g of oligomeric proanthocyanidins (i.e., water-soluble oligomeric proanthocyanidins) and 0.1 g of oleic anhydride were added to a screw-cap glass bottle containing 50 mL of acetone. After thorough dissolution by magnetic stirring, 0.05 g of sodium acetate was added to catalyze the reaction, and 100 g of 4A molecular sieves were added to remove moisture. The reaction was heated at 50°C for 12 h. After completion of the reaction, the precipitate was removed by filtration using filter paper to terminate the reaction. The solvent was removed by rotary evaporation to obtain the crude esterification product. The crude product was extracted three times with water / ethyl acetate (1:1, v / v). The ethyl acetate layers were combined and separated and purified using a silica gel column (300-400 mesh) using a gradient elution system containing n-hexane (70%-0%) and ethyl acetate (30%-100%) per 100 mL of eluent to remove the oleic acid. The organic solvent was removed by rotary evaporation, and the product was dried under vacuum to obtain fat-soluble oligomeric proanthocyanidins from peanut red skin.

[0026] Comparative Example 1 The steps for preparing peanut red skin fat-soluble oligomeric proanthocyanidins are as follows: 0.1 g of oligomeric proanthocyanidins (i.e., water-soluble oligomeric proanthocyanidins) and 0.1 g of oleic acid were added to a screw-capped glass bottle containing 50 mL of acetone. After thorough dissolution by magnetic stirring, 0.05 g of sodium acetate was added to catalyze the reaction, and 100 g of 4A molecular sieves were added to remove moisture. The reaction was heated at 50°C for 12 h. After completion of the reaction, the precipitate was removed by filtration using filter paper to terminate the reaction. The solvent was removed by rotary evaporation to obtain the crude esterification product. The crude product was extracted three times with water / ethyl acetate (1:1, v / v). The ethyl acetate layers were combined and separated and purified using a silica gel column (300-400 mesh) using a gradient elution system containing n-hexane (70%-0%) and ethyl acetate (30%-100%) per 100 mL of eluent to remove the oleic acid. The organic solvent was removed by rotary evaporation, and the product was dried under vacuum to obtain fat-soluble oligomeric proanthocyanidins from peanut red skin.

[0027] 1. Effect of different pretreatment conditions on the yield of fat-soluble oligomeric proanthocyanidins Taking the preparation conditions in Example 1 as a standard, the effects of different reaction temperatures, reaction times, catalyst amounts, and reaction substrate ratios on the yield of fat-soluble oligomeric proanthocyanidins were studied.

[0028] The experimental design is shown in Table 1: Table 1 Pretreatment test design The test results are as follows: The conditions for the preparation process of fat-soluble oligomeric proanthocyanidins screened out from the above single-factor experiment are: reaction temperature 50°C, reaction time 12h, catalyst dosage 0.075g / 0.1g OPSP, and oleic anhydride: peanut red skin oligomeric proanthocyanidins ratio 3:1.

[0029] Under the preparation process conditions, using the method of Example 1, the reaction conversion rate of oligomeric proanthocyanidins is as high as 92.04%, and the yield of the reaction product (fat-soluble oligomeric proanthocyanidins) is as high as 59%; using the method of Comparative Example 1, the reaction conversion rate of oligomeric proanthocyanidins is 8.6%, and the product reaction yield is only 2%.

[0030] 2. Response Surface Experimental Design Based on the results of the single-factor experiment, a three-factor, three-level response surface experiment was designed for the reaction substrate ratio (A), reaction temperature (B), and reaction time (C). The optimal reaction conditions were determined using the proanthocyanidin conversion rate (Y) as an indicator. The experimental results were subjected to a multivariate regression analysis using Design Expert 13, and the binary multivariate regression equation for the proanthocyanidin conversion rate (Y) and the reaction substrate ratio (A), reaction temperature (B), and reaction time (C) was obtained: Y=93.58+2.48A+0.0025B+0.0538C+0.1750AB+0.4025AC+0.1450BC-3.04A 2 -4.87B 2 -3.28C 2 .

[0031] The conversion rate of fat-soluble oligomeric proanthocyanidins was determined as follows: The proanthocyanidin content in OPSP-O was determined using the vanillin-hydrochloric acid method. 2 g of vanillin was dissolved in a 1:2 (v / v) solution of hydrochloric acid and methanol to obtain a final volume of 100 mL of a 2% vanillin / hydrochloric acid-methanol solution. 1.0 mL of the sample solution and 9.0 mL of the 2% vanillin / hydrochloric acid-methanol solution were added to a brown tube and vortexed. The tube was then incubated in a 30°C water bath for 30 minutes. The absorbance was measured at 500 nm using a UV spectrophotometer. Results are expressed as mg catechins (CE) / g of sample.

[0032] The calculation formula for GSP conversion rate is: OPSP conversion rate (%) = (A0–A1) / A0×100; Wherein, A0 is the amount of proanthocyanidins added to the initial reaction solution; A1 is the content of proanthocyanidins in OPSP-O.

[0033] OPSP-O yield calculation: The initial total mass of peanut skin and oleic anhydride is recorded as A1; After the esterification of peanut red skin oligomeric proanthocyanidins was complete, the reaction was terminated by filtering the precipitate with filter paper. The solvent was removed by rotary evaporation to obtain the crude esterified product. The crude product was extracted three times with water / ethyl acetate (1:1, v / v). The ethyl acetate layers were combined, the organic solvent was removed by rotary evaporation, and vacuum drying was performed to obtain the fat-soluble oligomeric proanthocyanidins from peanut red skin, designated A2.

[0034] Conversion rate EE (%) = A2 / A1 × 100%; The results of the three-factor three-level response surface experiment are shown in Table 2: Table 2 Response surface experimental design and results Analysis using Design-Expert 13 software revealed the following optimal reaction conditions: reaction temperature of 50.10°C, reaction time of 12.21 h, substrate-to-reaction ratio of 3:1, and optimal conversion of 93.04%. Taking into account the accuracy of the instrument and the operability of the experiment, the optimal conversion conditions for fat-soluble proanthocyanidins were revised to: temperature of 50°C, reaction time of 12 h, and substrate-to-reaction ratio of 3:1. Under these optimal conditions (catalyst dosage of 0.075 g / 0.1 g OPSP), a conversion of 92.38% was obtained for fat-soluble oligomeric proanthocyanidins.

[0035] 3. Characterization of fat-soluble oligomeric proanthocyanidins The fat-soluble oligomeric proanthocyanidins prepared under the optimal conditions of the above response surface methodology were purified by silica gel column chromatography and then structurally characterized.

[0036] 1. Determination of proanthocyanidins and polyphenols content The proanthocyanidin content can be found in the above-mentioned fat-soluble oligomeric proanthocyanidin conversion rate determination method.

[0037] The total phenolic content of OPSP-O was determined using the Folin-phenol method, using gallic acid as a reference. 100 μL of sample was added to 0.5 mL of Folin-phenol reagent and a 4.25% sodium carbonate solution to make a 10 mL volume. The solution was allowed to stand for 30 minutes, and the absorbance was measured at 760 nm. The total phenolic content was expressed as g gallic acid equivalents per 100 g of sample using a gallic acid calibration curve.

[0038] The test results are as follows Figure 1 shown OPSP-O had lower total phenolic and proanthocyanidin contents than OPSP. The OPSP sample contained 6.45 mg / mg of total phenolic and 0.92 mg / mg of proanthocyanidin, respectively. The esterified OPSP-O sample contained 0.94 mg / mg of total phenolic and 0.26 mg / mg of proanthocyanidin, respectively. These results indicate that some of the hydroxyl groups in OPSP were esterified by oleic anhydride, reducing the number of hydroxyl groups in the esterified OPSP-O, leading to lower total phenolic and proanthocyanidin contents in GSP-O.

[0039] 2. Lipophilicity Determination Preparation of an n-octanol-water solution: 50 mL of n-octanol and 150 mL of double-distilled water were placed in a stoppered Erlenmeyer flask. Oscillate the solution at 120 rpm in a thermostatic shaker (37°C) for 24 hours. Allow to stand overnight to separate the layers, then separate them in a separatory funnel. The upper layer is the oil phase (water-saturated n-octanol solution) and the lower layer is the aqueous phase (n-octanol-saturated aqueous solution). 5 mg of each of OPSP and OPSP-O was dissolved in the oil phase and diluted to 10 mL to prepare a 0.5 mg / mL water-saturated n-octanol solution. The absorbance (Ax) was measured at 280 nm. Separately, 5.0 mL of each of the water-saturated n-octanol solution containing 0.5 mg / mL OPSP and OPSP-O and the water-saturated n-octanol solution were measured. Oscillate the solution at a constant temperature for 24 hours, then centrifuge at 5000 rpm for 20 minutes. The oil phase was separated and the absorbance (A0) was measured at 280 nm. The oil-water partition coefficient was calculated using the following formula: Log P = Log Ax / (A0-Ax); The test results are as follows Figure 2 As shown: A high log p value indicates a compound with high lipophilicity. Esterification with oleic anhydride results in increased lipophilicity. The log p value of OPSP-O (0.70) is higher than that of its parent compound, OPSP (-0.70), indicating increased lipophilicity.

[0040] 3. Fourier transform infrared spectroscopy detection FTIR spectra of liposoluble oligomeric proanthocyanidins (OPSP-O) and their raw materials (OPSP) were recorded on an iS50 FTIR spectrometer at room temperature in the wavenumber range of 500–4000 cm −1 , with a resolution of 4 cm −1。 Test results such as Figure 3 As shown: FTIR analysis provides information on the molecular structure and chemical bonding of molecules of any size. OPSP and OPSP-O have a hydroxyl group corresponding to phenol (3309 cm -1 ) and benzene ring (1440 cm -1 、1605 cm -1 ) absorption band. This indicates that the modification process involves a local change in the OPSP molecular structure, but does not destroy its overall structure. OPSP-O exhibits an unsaturated CH (3009 cm -1 ), methyl (2903 cm -1 ), methylene (2853 cm -1 ) and carbonyl (1709 cm -1 ) and the strong absorption peak of -CH2 in the fatty acid chain (722 cm -1). It was concluded that the long carbon chain of oleic acid was successfully introduced into the molecular structure of OPSP, thus obtaining OPSP-O.

[0041] 4. NMR analysis of esterification products The flavantriol esterification product was dissolved in deuterated methanol and the 1 The structure was confirmed by H NMR 500 MHz.

[0042] The test results are as follows: Proanthocyanidin B ring (H-2, H-5, H-6, δ 6.84-7.15 ppm), proanthocyanidin A ring (H-2, H-6, δ 6.05 ppm), C ring (H-2, H-3, δ 3.63-4.84 ppm), compared with the parent proanthocyanidin, B ring H-2, H-5, H-6 has a downfield shift (δ 6.81-7.71 ppm), indicating that the B ring is the esterification site, while the A ring (δ 6.08 ppm) and the C ring (δ 3.88-4.62 ppm) have no significant changes, indicating that the A ring and the C ring are not esterification sites. 1 The H NMR results showed that δ 5.34 ppm was the H on the unsaturated double bonds C1 and C2 of oleic acid, δ 0.90-2.78 ppm was the saturated methyl and methylene groups in oleic acid, and a new triple peak appeared at 2.27 ppm, which once again proved the successful esterification of oleic acid with proanthocyanidins.

[0043] 5. Microscopic morphology detection The morphology of OPSP and OPSP-O was observed using a scanning electron microscope. The samples were attached to the laboratory bench with double-sided conductive tape and then sputtered with gold. Scanning electron microscopy was performed at 10 kV, a working distance of 9.5 mm, and a magnification of 800x.

[0044] The test results are as follows Figure 4 As shown: OPSP is spherical or hemispherical with a smooth surface. The disappearance of the particle structure of OPSP-O is caused by temperature changes during the synthesis process.

[0045] 6. Particle size and zeta potential determination Particle size and potential determination: OPSP and OPSP-O were dissolved in a trace amount of methanol in deionized water and the supernatant was collected. The collected supernatant was added to a dynamic light scattering instrument to measure the particle size and potential.

[0046] The results of the test are as follows Figure 5 As shown: The larger particle size of OPSP-O than OPSP may be due to the significant increase in molecular hydrophobicity caused by the introduction of oleic acid, which leads to hydrophobic aggregation in water, forming larger particles or micelles. The flexible structure of the oleic acid chain may enhance intermolecular interactions (such as hydrophobic interactions and van der Waals forces).

[0047] Zeta potential reflects the stability of the particle surface charge. The larger the absolute value, the more stable the system. The absolute value of zeta potential of OPSP-O is higher than that of OPSP, which may be due to the dissociation of the carboxyl group (-COOH) of oleic acid into -COO in water. - , increasing the surface negative charge density. And hydrophobic particles can absorb free ions (such as OH - ) or form a double layer to enhance the surface charge.

[0048] 7. OPSP-O thermal stability analysis TG curves were recorded between 25°C and 800°C using a Mettler Toledo STARe System TGA2 thermogravimetric analyzer. 5 mg of sample was weighed and the temperature was increased at 10°C / min under a nitrogen flow rate of 50 mL / min.

[0049] Test results such as Figure 6 As shown: All samples showed obvious mass loss in two stages. The first stage was from 26 to 200°C. The weight loss of the samples in this stage can be attributed to the loss of adsorbed water in the samples. The second stage was from 200 to 800°C. The mass loss in this stage was mainly due to the decomposition of proanthocyanidins. It was found that the decomposition peak of OPSP-O appeared earlier than that of OPSP. This may be because the formation of ester bonds reduced the thermal stability of OPSP-O, and the ester bonds would break at lower temperatures.

[0050] IV. Biological properties of OPSP-O 1. Determination of OPSP-O antioxidant activity (1) DPPH clearance rate OPSP-O (40 mg) was mixed with deionized water (10 mL) by ultrasonication (400 W) for 1 min at room temperature, and then centrifuged at 1611 g for 10 min. The supernatant of each sample (2 mL) was mixed with DPPH (2 mL, 1 mmol / L, anhydrous ethanol) and incubated in the dark for 30 min at room temperature with the same volume of water as the control. Finally, the absorbance at 517 nm was measured using a UV-visible spectrophotometer with ethanol as a blank. The above experiment was repeated using VC as a positive control, and the DPPH free radical scavenging activity (SA) was calculated. DPPH ), calculated as follows: SA DPPH(%)=1−(A 样品 –A 对照 ) / A 空白 ×100%.

[0051] (2) Hydroxyl ion free radical scavenging rate Add 2 mL of the sample to be tested and 2 mL of distilled water to each of the three test tubes, and then add 2 mL of FeSO4 (6 mmol / L) and 2 mL of H2O2 (6 mmol / L) to each of the three test tubes. Mix the test tubes evenly and let them stand for 10 minutes. Add 2 mL of salicylic acid (6 mmol / L), 2 mL of distilled water, and 2 mL of salicylic acid (6 mmol / L) to each of the three test tubes, mix the test tubes evenly, and let them stand for 30 minutes. Adjust the "0" point with distilled water and measure the absorbance at 510 nm. The absorbance values ​​of the three reaction solutions are Ai, Aj, and A0, respectively. Calculate the scavenging rate of the sample to be tested for hydroxyl radicals according to the following formula: Clearance rate (%) = [1-(Ai-Aj) / A0] × 100%.

[0052] (3) β-carotene bleaching test Prepare a mixture containing 4 mL of β-carotene (0.1 mg / mL in chloroform), 40 mg of linoleic acid, and 400 mg of Tween 40. Remove the chloroform using a rotary evaporator under vacuum at 50°C. Add 100 mL of distilled water and shake vigorously until a β-carotene-linoleic acid suspension forms. Add 25 μL of the sample (4 mg / mL concentration) and 225 μL of the β-carotene-linoleic acid suspension to the wells of a 96-well flat-bottom microtiter plate. Add 25 μL of methanol and 225 μL of the β-carotene-linoleic acid suspension to the wells of a 96-well flat-bottom microtiter plate as a control. Immediately record the absorbance at 450 nm, denoting As0 and Ac0, respectively. After incubation in a 50°C water bath for 90 minutes, immediately record the absorbance at 450 nm. Denote Ast and Act, respectively.

[0053] The antioxidant activity (AA) of β-carotene / linoleic acid oxidation was evaluated using the following formula: Where As0 and Ast are the corrected absorbance values ​​of the test sample at time zero and after incubation, respectively; Ac0 and Act are the corrected absorbance values ​​of the control at time zero and after incubation, respectively.

[0054] Test results such as Figure 7 As shown: OPSP-O exhibits a higher DPPH radical scavenging ability than OPSP, likely due to electronic and steric effects on the benzene ring of proanthocyanidins caused by oleic acid acylation, which enhances their free radical scavenging capacity. However, its hydroxyl radical scavenging rate is lower than that of OPSP, as hydroxyl radical scavenging relies primarily on direct hydrogen donation or electron transfer from the phenolic hydroxyl group. Oleic acid acylation consumes some of the phenolic hydroxyl groups (forming ester bonds), resulting in a reduction in active sites and decreased scavenging ability. Furthermore, ·OH groups are primarily generated in the aqueous phase, while oleic acid acylation reduces the hydrophilicity of the molecules, resulting in poor solubility or dispersibility in water, reducing their exposure to ·OH groups. β-carotene bleaching experiments revealed that OPSP-O exhibited a higher lipid peroxidation inhibitory ability than OPSP. This result may be due to OPSP-O's increased lipid solubility, which draws antioxidant groups toward the lipid oxidation interface. Consequently, esterified proanthocyanidins are more effective than native proanthocyanidins in inhibiting lipid peroxidation. However, the DPPH scavenging rate, hydroxyl radical scavenging rate, and β-carotene bleaching scavenging rate of UNOPSP-O were lower than those of OPSP and OPSP-O. This is because UNOPSP-O contains a large amount of oleic acid, which leads to low antioxidant effect.

[0055] 2. In vitro digestion stability of OPSP-O Weigh 200 mg of each sample and dilute to 25 mL with PBS to prepare a 4 mg / mL OPSP or OPSP-O solution. Preheat the solution at 37°C for 10 minutes. Adjust the pH to 2 with 1 mol / L HCl. Once the pH stabilizes, immediately add 2% (w / w) pepsin. Digest the solution in a constant-temperature shaking incubator at 37°C and 120 rpm. At 0, 10, 30, 60, 90, and 120 minutes, take 1 mL of sample and dilute it to 4 mL with methanol. Measure and record the proanthocyanidin content of each sample.

[0056] After digestion for 2 h at 37 ° C and 120 r / min in a constant temperature shaking incubator, the pH of the solution was adjusted to 7.5 with NaOH (1 mol / L), and then 2% (w / w) trypsin and 2.5 mL of 4% pig bile were added. Digestion was carried out in a constant temperature shaking incubator at 37 ° C and 120 rpm. At 30 min, 60 min, 120 min, 180 min, and 240 min, 1 mL of sample was taken and diluted to 4 mL with methanol. The proanthocyanidin content in the samples was measured and recorded.

[0057] Test results such as Figure 8 As shown: At the beginning of the reaction, the contents of OPSP and OPSP-O proanthocyanidins were measured to be 82.21 mg / 100 mg and 27.88 mg / 100 mg, respectively.

[0058] OPSP and OPSP-O were digested in a simulated stomach (pH 2) and small intestine (pH 7-8). Esterified proanthocyanidins were significantly more stable than proanthocyanidins. In the simulated gastric environment, due to the acidic conditions, the proanthocyanidin content in OPSP initially increased and then decreased. The increase in proanthocyanidin content from 0 to 10 min may be due to the hydrolysis of oligomers in OPSP during gastric digestion, resulting in an increase in the concentration of monomers and dimers. The decrease in proanthocyanidin content from 10 to 120 min may be due to the hydrolysis of proanthocyanidins in OPSP due to exposure to the acidic environment, resulting in a decrease in proanthocyanidin content. The greater stability of OPSP-O in the simulated gastric environment may be due to the introduction of oleic acid chains, which shield some active hydroxyl groups, reducing acid sensitivity, and the increased hydrophobicity, which reduces the free concentration in gastric fluid.

[0059] When simulating the intestinal environment, it was found that the content of proanthocyanidins in OPSP was greatly reduced, which may be because the intestinal environment is alkaline, accelerating the oxidation of OPSP phenolic hydroxyl groups, while the content of OPSP-O proanthocyanidins hardly changed and showed an upward trend, which may be because pancreatic enzymes can specifically hydrolyze oleic acid ester bonds to release free proanthocyanidins and oleic acid.

[0060] In summary, by simulating the in vitro gastrointestinal environment, it was found that the in vitro digestion resistance of fat-soluble oligomeric proanthocyanidins was enhanced.

[0061] 3. Effect of OPSP-O on Caco-2 cell activity The cells in the logarithmic growth phase were digested with trypsin to prepare a concentration of 1×10 5 Cell suspension of 1×10 cells / mL was inoculated into a 96-well plate. 4 100 μL of each solution was added to each well and cultured in a 37°C, 5% CO2 incubator until cells adhered. 100 μL of various concentrations of OPSP, OPSP-O, UNOPSP-O (unpurified lipid-soluble oligomeric proanthocyanidins), Oil, and OPSP+O (a mixed solution of proanthocyanidins and oleic acid) (20, 40, 80, 100, 200, and 400 μg / mL) were added, with six replicate wells set for each concentration. A corresponding blank well was also set for zeroing. Culture was continued for 24 hours. 10 μL of MTT solution was added to all wells and the cells were incubated in the incubator for 4 hours. The supernatant was aspirated, and 200 μL of DMSO was added to each well. The cells were shaken evenly and the absorbance at 570 nm was measured with a microplate reader to calculate cell viability.

[0062] The test results are as follows: The MTT assay was used to evaluate the effects of different concentrations of OPSP, OPSP-O, UNOPSP-O, Oil, and OPSP+O solutions (20, 40, 80, 100, 200, and 400 μg / mL) on the relative proliferation rate of Caco-2 cells. The results showed that the relative proliferation rates of OPSP, OPSP-O, UNOPSP-O, Oil, and OPSP+O were all above 80%. This indicates that within the tested concentration range, OPSP, OPSP-O, UNOPSP-O, Oil, and OPSP+O had no significant cytotoxic effects on Caco-2 cells, making them suitable for cellular trafficking and uptake.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A method for preparing fat-soluble oligomeric proanthocyanidins, characterized in that: The steps include: The oligomeric proanthocyanidins and oleic anhydride are mixed, a solvent is added, and the mixture is stirred evenly; a catalyst and a molecular sieve are then added to carry out an esterification reaction; after the reaction is completed, the precipitate is removed, and then the solvent is removed to obtain a crude esterification product; the crude esterification product is extracted, the extract is chromatographed, and the organic solvent is removed to obtain a fat-soluble oligomeric proanthocyanidin.

2. The preparation method according to claim 1, characterized in that The raw materials are selected from the following quantities: 0.05-0.15 parts of oligomeric proanthocyanidins, 0.05-0.5 parts of oleic anhydride, 10-100 parts of solvent, and 0.01-0.1 parts of catalyst.

3. The preparation method according to claim 1, characterized in that The solvent is selected from one of acetone, ethyl acetate and tetrahydrofuran.

4. The preparation method according to claim 1, characterized in that The catalyst is selected from one of sodium acetate, sodium carbonate and sodium bicarbonate.

5. The preparation method according to claim 1, characterized in that The esterification reaction conditions are selected from: reaction at 35-60° C. for 3-18 hours.

6. The preparation method according to claim 1, characterized in that The extraction solvent used in the extraction is a water / ethyl acetate mixed solvent, and the volume ratio of the two is 1:

1.

7. The preparation method according to claim 1, characterized in that The specific steps of the extraction are: extracting the crude esterification product with water / ethyl acetate (1:1, v / v), and collecting the ethyl acetate layer.

8. The preparation method according to claim 1, characterized in that The chromatography is performed by gradient elution using silica gel column chromatography to remove oleic acid and separate and purify the product, and the eluent is n-hexane (70%-0%): ethyl acetate (30%-100%).

9. The fat-soluble oligomeric proanthocyanidins prepared by the method according to any one of claims 1 to 8.

10. Use of the fat-soluble oligomeric proanthocyanidins according to claim 9 in the preparation of a drug having antioxidant and / or fat-soluble effects.

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