Oligomeric proanthocyanidin with anti-oxidation and anti-aging effects and preparation method thereof

Abstract

The present invention provides an anti-oxidative and anti-aging oligomeric proanthocyanidin, and a preparation method and application thereof. The preparation method of the oligomeric proanthocyanidin comprises the following steps: adding polymeric proanthocyanidin to a threonine aqueous solution, stirring evenly, then adding aspartic acid and glutamic acid, and ultrasonically reacting; dialysis and / or nanofiltration to obtain an oligomeric proanthocyanidin solution, and freeze-drying. Preferably, the concentration of the threonine is 20 g / L; the concentration of the glutamic acid is 2 g / L; the concentration of the aspartic acid is 1.5 g / L, the concentration of the polymeric proanthocyanidin is 5 g / L, and the polymeric proanthocyanidin is extracted from grape seeds, purple rice or black rice. By providing this milder degradation pathway, it is helpful to obtain a more active oligomeric proanthocyanidin product. These oligomers are structurally more suitable as antioxidants and anti-aging agents, thereby showing better biological activity than other methods.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and cosmetics, and in particular to an anti-oxidation and anti-aging oligomeric proanthocyanidin and an application thereof. Background Art

[0002] According to the degree of polymerization, proanthocyanidins can be divided into oligomeric proanthocyanidins (2-4 polymers) and polymeric proanthocyanidins (≥5 polymers). The degree of polymerization of proanthocyanidins has a great influence on their antioxidant activity. Some oligomeric proanthocyanidins have significant activity in antioxidant, free radical scavenging, anti-aging, etc., and are easily absorbed by the skin. The increase in the degree of polymerization not only reduces the antioxidant effect, but also reduces the skin uptake due to steric hindrance.

[0003] The corresponding polymeric proanthocyanidins have higher stability, and most of the plants and commercially available plant extracts are polymeric proanthocyanidins. Polymeric proanthocyanidins can be prepared into oligomeric proanthocyanidins by biological enzymes, acid hydrolysis, etc. Enzymatic hydrolysis will lead to an increase in the subsequent impurity removal costs. For example, patent CN201210311225.X uses hydrochloric acid, citric acid, acetic acid, ascorbic acid, malic acid, tartaric acid, gallic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, sulfurous acid, etc. for acid hydrolysis. On the one hand, improper control of hydrochloric acid concentration will lead to further degradation of the product; on the other hand, during the acid hydrolysis process, the active group may be attacked by nucleophiles, introducing unnecessary functional groups.

[0004] In recent years, the country's safety regulations on cosmetic raw materials have become increasingly stringent, while consumers have higher requirements for the efficacy of cosmetics. There is an urgent need for a safe oligomeric proanthocyanidin raw material that meets the antioxidant and anti-aging needs of cosmetics. Summary of the invention

[0005] The inventors have found that when acid hydrolyzing polymeric proanthocyanidins to prepare oligomeric proanthocyanidins, if strong acids such as hydrochloric acid are used, the hydrolyzate contains a large amount of anthocyanidins and has poor antioxidant activity. The inventors hypothesize that when acidic amino acids such as aspartic acid or glutamic acid are used instead of hydrochloric acid, the acidity is weaker than hydrochloric acid, and it is expected that both the pH value of acid hydrolysis and the excessive acid hydrolysis can be achieved. That is, it helps to break the macromolecular structure of polymeric proanthocyanidins and generate smaller oligomers. This acidic environment helps to control the degradation rate and degree of degradation, and may allow the final product to retain more beneficial structures. In addition, after testing, the inventors were surprised to find that threonine, a neutral amino acid, is very important for the reaction. When threonine, aspartic acid and glutamic acid are used alone or mixed to catalyze the hydrolysis of polymeric proanthocyanidins, the yield and antioxidant activity of the product have no obvious advantages, but the ternary system of threonine, aspartic acid and glutamic acid has significantly improved the yield and antioxidant activity of oligomeric proanthocyanidins when certain polymeric proanthocyanidins are used as reaction raw materials, thereby obtaining the present invention.

[0006] In a first aspect of the present invention, there is provided an oligomeric proanthocyanidin, characterized in that the preparation method of the oligomeric proanthocyanidin comprises the following steps:

[0007] (1) Add the polymerized proanthocyanidins to the threonine aqueous solution, stir evenly, then add aspartic acid and glutamic acid, and perform ultrasonic reaction;

[0008] (2) Dialysis and / or nanofiltration to obtain an oligomeric proanthocyanidin solution, and freeze-drying to obtain the product.

[0009] In some embodiments of the present invention, the concentration of threonine is 0.1-300 g / L. In some embodiments of the present invention, the concentration of glutamic acid is 0.1-300 g / L. In some embodiments of the present invention, the concentration of aspartic acid is 0.1-300 g / L.

[0010] In some embodiments of the present invention, the concentration of threonine is 10-30 g / L. In some embodiments of the present invention, the concentration of glutamic acid is 1-3 g / L. In some embodiments of the present invention, the concentration of aspartic acid is 1-2 g / L.

[0011] In some embodiments of the present invention, the concentration of threonine is 10-30 g / L, the concentration of glutamic acid is 1-3 g / L, and the concentration of aspartic acid is 1-2 g / L. That is, the concentration of threonine after being dispersed (dissolved) in water is 10-30 g / L, the concentration of glutamic acid after being dispersed (dissolved) in water is 1-3 g / L, and the concentration of aspartic acid after being dispersed (dissolved) in water is 1-2 g / L. The same applies below.

[0012] In some embodiments of the present invention, the concentration of the polymeric proanthocyanidins in step (1) after being dispersed in water is 3-7 g / L.

[0013] In some embodiments of the present invention, when the ultrasonic reaction in step (1) starts, the concentration of threonine is 20 g / L; the concentration of glutamic acid is 2 g / L; the concentration of aspartic acid is 1.5 g / L, and the concentration of polymeric proanthocyanidins is 5 g / L. That is, in some embodiments of the present invention, the concentration of threonine is 20 g / L; the concentration of glutamic acid is 2 g / L; the concentration of aspartic acid is 1.5 g / L, and the concentration of polymeric proanthocyanidins is 5 g / L.

[0014] In some embodiments of the present invention, the polymeric proanthocyanidins are extracted from one, two or more of grape seeds, grapes, apples, purple rice, black rice, gardenia, rosemary, papaya, litchi, hops, longan, sea buckthorn, emblica, cinnamon, sorghum, buckwheat, loquat, cherry, pomegranate, purple sweet potato, lingonberry, lotus seed pod, kiwi, bayberry, hazelnut, albizzia, ginkgo, walnut, macadamia, cypress, Bruguiera gymnorrhiza, Schima superba, birch, eucalyptus, poplar, camphor, chestnut, oak and black wattle.

[0015] In some embodiments of the present invention, the polymeric proanthocyanidins are extracted from grape seeds, purple rice or black rice.

[0016] In some embodiments of the present invention, the polymeric proanthocyanidins are extracted from grape seeds. In some embodiments of the present invention, the polymeric proanthocyanidins are extracted from purple rice. In some embodiments of the present invention, the polymeric proanthocyanidins are extracted from black rice. In some embodiments of the present invention, the grape seeds are from Vitis vinifera.

[0017] In some embodiments of the present invention, the molecular weight cutoff of the dialysis and / or nanofiltration is 1500-2000Da, 300Da. In some embodiments of the present invention, the molecular weight cutoff of the dialysis is 1500-2000Da. In some embodiments of the present invention, the molecular weight cutoff of the dialysis is 1500Da. In some embodiments of the present invention, the molecular weight cutoff of the dialysis is 2000Da. In some embodiments of the present invention, the molecular weight cutoff of the dialysis is 300Da.

[0018] In some embodiments of the present invention, the nanofiltration is filtration using a nanofiltration membrane.

[0019] In some embodiments of the present invention, the molecular weight cutoff of the nanofiltration is 1500-2000Da. In some embodiments of the present invention, the molecular weight cutoff of the nanofiltration is 1500Da. In some embodiments of the present invention, the molecular weight cutoff of the nanofiltration is 2000Da. In some embodiments of the present invention, the molecular weight cutoff of the nanofiltration is 300Da.

[0020] In some embodiments of the present invention, the nanofiltration is carried out using RisingSun NF7 nanofiltration membrane. In some embodiments of the present invention, the nanofiltration is carried out using RisingSun NF2 nanofiltration membrane.

[0021] In some embodiments of the present invention, the dialysis and / or nanofiltration is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-1500Da. In some embodiments of the present invention, the dialysis is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-1500Da. In some embodiments of the present invention, the nanofiltration is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-1500Da.

[0022] In some embodiments of the present invention, the dialysis and / or nanofiltration is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-2000Da. In some embodiments of the present invention, the dialysis is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-2000Da. In some embodiments of the present invention, the nanofiltration is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-2000Da.

[0023] In some embodiments of the present invention, the nanofiltration is treated with a filter membrane so that the solution only retains a solute of a molecular weight of 300-1500Da. In some embodiments of the present invention, the nanofiltration is treated with a filter membrane so that the solution only retains a solute of a molecular weight of 300-2000Da. In some embodiments of the present invention, the dialysis is treated with a filter membrane so that the solution only retains a solute of a molecular weight of 300-1500Da. In some embodiments of the present invention, the dialysis is treated with a filter membrane so that the solution only retains a solute of a molecular weight of 300-2000Da.

[0024] In some embodiments of the present invention, the ultrasonic power is 300-700 W. Preferably, it is 500 W. In some embodiments of the present invention, the ultrasonic power is 500 W.

[0025] In some embodiments of the present invention, the ultrasonic reaction is performed in an ice water bath.

[0026] In some embodiments of the present invention, the ultrasonic reaction time is 0.5-3h. In some embodiments of the present invention, the ultrasonic reaction time is 0.5-2h. In some embodiments of the present invention, the ultrasonic reaction time is 1h. In some embodiments of the present invention, the ultrasonic reaction time is 2h. In some embodiments of the present invention, the ultrasonic reaction time is 0.5h.

[0027] In some embodiments of the present invention, the preparation method of the polymeric proanthocyanidins comprises the following steps: weighing the plant raw material, crushing and sieving, soaking with petroleum ether for defatting, and filtering; adding 60% ethanol (V / V) and standing, filtering, removing the solvent of the filtrate, adding water thereto, stirring thoroughly to disperse evenly, and then adding ethanol to precipitate and remove impurities such as protein and polysaccharide; filtering, removing the solvent of the filtrate, adding water thereto at 5°C, stirring thoroughly to disperse evenly, and then adding ethyl acetate for extraction, taking the aqueous phase, and evaporating the solvent to obtain the polymeric proanthocyanidins.

[0028] In some embodiments of the present invention, the polymeric proanthocyanidins are prepared according to the following steps: weighing the plant raw material, crushing and sieving, soaking with petroleum ether to defatted, filtering; adding 60% ethanol (V / V) and standing, filtering, removing the solvent of the filtrate, adding water thereto, stirring thoroughly to disperse evenly, and then adding ethanol to precipitate and remove impurities such as protein and polysaccharides; filtering, removing the solvent of the filtrate, adding water thereto at 5°C, stirring thoroughly to disperse evenly, and then adding ethyl acetate to extract, taking the aqueous phase, and evaporating the solvent to obtain the polymeric proanthocyanidins.

[0029] In some embodiments of the present invention, the method for preparing oligomeric proanthocyanidins comprises the following steps:

[0030] (a) Preparation of polymeric proanthocyanidins: Weigh 20 g of grape seeds, purple rice or black rice, crush and sieve, soak in 1000 mL of petroleum ether for 30 h to defat; filter, and air-dry the residue; add 800 mL of 60% ethanol (V / V) thereto, and let stand at 25°C for 3 days under closed conditions; filter, and spin-dry the filtrate under reduced pressure at 30°C; add 100 mL of water thereto, stir well to disperse evenly, and then add 500 mL of ethanol to precipitate and remove impurities such as protein and polysaccharide; filter, and spin-dry the filtrate under reduced pressure at 30°C; add 1000 mL of water thereto at 5°C, stir well to disperse evenly, and then add 200 mL of ethyl acetate for extraction, take the aqueous phase, and spin-dry under reduced pressure at 30°C to obtain polymeric proanthocyanidins from grape seeds, purple rice or black rice;

[0031] (b) Preparation of oligomeric proanthocyanidins from polymeric proanthocyanidins: 5 g of the polymeric proanthocyanidins prepared as described in (a) was added to 1000 mL of an aqueous solution of threonine and stirred evenly. Then, 2.0 g of glutamic acid and 1.5 g of aspartic acid were added and stirred to dissolve. The mixture was subjected to ultrasonic reaction at 500 W in an ice-water bath for 1 h. The mixture was filtered using a nanofiltration membrane with a molecular weight cutoff of 1500-2000 Da, and the filtrate was then filtered using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The residue was freeze-dried to obtain an oligomeric proanthocyanidin product.

[0032] In some embodiments of the present invention, the preparation method comprises the following steps or the preparation method steps are as follows:

[0033] (a) Preparation of polymeric proanthocyanidins: Weigh 20 g of grape seeds, crush and sieve, soak in 1000 mL of petroleum ether for 30 h to defat; filter, and air-dry the residue; add 800 mL of 60% ethanol (V / V) thereto, and let stand at 25°C for 3 days under closed conditions; filter, and spin-dry the filtrate under reduced pressure at 30°C; add 100 mL of water thereto, stir well to disperse evenly, and then add 500 mL of ethanol to precipitate and remove impurities such as protein and polysaccharide; filter, and spin-dry the filtrate under reduced pressure at 30°C; add 1000 mL of water thereto at 5°C, stir well to disperse evenly, and then add 200 mL of ethyl acetate for extraction, take the aqueous phase, and spin-dry under reduced pressure at 30°C to obtain polymeric proanthocyanidins from grape seeds;

[0034] (b) Preparation of oligomeric proanthocyanidins from polymeric proanthocyanidins: 5 g of the polymeric proanthocyanidins prepared as described in (a) was added to 1000 mL of an aqueous solution of threonine and stirred evenly. Then, 2.0 g of glutamic acid and 1.5 g of aspartic acid were added and stirred to dissolve. The mixture was subjected to ultrasonic reaction at 500 W in an ice-water bath for 1 h. The mixture was filtered using a nanofiltration membrane with a molecular weight cutoff of 1500-2000 Da, and the filtrate was then filtered using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The residue was freeze-dried to obtain an oligomeric proanthocyanidin product.

[0035] In some embodiments of the present invention, the preparation method comprises the steps described in Example 1.

[0036] In the second aspect of the present invention, a method for preparing oligomeric proanthocyanidins is provided, characterized in that the preparation method comprises the steps described in the first invention of the present invention.

[0037] In some embodiments of the present invention, a method for preparing oligomeric proanthocyanidins is provided, wherein the method comprises the following steps:

[0038] (1) Add the polymerized proanthocyanidins to the threonine aqueous solution, stir evenly, then add aspartic acid and glutamic acid, and perform ultrasonic reaction;

[0039] (2) Dialysis and / or nanofiltration to obtain an oligomeric proanthocyanidin solution, and freeze-drying to obtain the product.

[0040] In some embodiments of the present invention, the concentration of threonine is 10-30 g / L, the concentration of glutamic acid is 1-3 g / L, and the concentration of aspartic acid is 1-2 g / L. That is, the concentration of threonine after being dispersed (dissolved) in water is 10-30 g / L, the concentration of glutamic acid after being dispersed (dissolved) in water is 1-3 g / L, and the concentration of aspartic acid after being dispersed (dissolved) in water is 1-2 g / L. The same applies below.

[0041] In some embodiments of the present invention, the concentration of the polymeric proanthocyanidins in step (1) after being dispersed in water is 3-7 g / L.

[0042] In some embodiments of the present invention, when the ultrasonic reaction in step (1) starts, the concentration of threonine is 20 g / L; the concentration of glutamic acid is 2 g / L; the concentration of aspartic acid is 1.5 g / L, and the concentration of polymeric proanthocyanidins is 5 g / L. That is, in some embodiments of the present invention, the concentration of threonine is 20 g / L; the concentration of glutamic acid is 2 g / L; the concentration of aspartic acid is 1.5 g / L, and the concentration of polymeric proanthocyanidins is 5 g / L.

[0043] In some embodiments of the present invention, the polymeric proanthocyanidins are extracted from one, two or more of grape seeds, grapes, apples, purple rice, black rice, gardenia, rosemary, papaya, litchi, hops, longan, sea buckthorn, emblica, cinnamon, sorghum, buckwheat, loquat, cherry, pomegranate, purple sweet potato, lingonberry, lotus seed pod, kiwi, bayberry, hazelnut, albizzia, ginkgo, walnut, macadamia, cypress, Bruguiera gymnorrhiza, Schima superba, birch, eucalyptus, poplar, camphor, chestnut, oak and black wattle.

[0044] In some embodiments of the present invention, the polymeric proanthocyanidins are extracted from grape seeds, purple rice or black rice.

[0045] In some embodiments of the present invention, the polymeric proanthocyanidins are extracted from grape seeds. In some embodiments of the present invention, the polymeric proanthocyanidins are extracted from purple rice. In some embodiments of the present invention, the polymeric proanthocyanidins are extracted from black rice. In some embodiments of the present invention, the grape seeds are from Vitis vinifera.

[0046] In some embodiments of the present invention, the molecular weight cutoff of the dialysis and / or nanofiltration is 1500-2000Da, 300Da. In some embodiments of the present invention, the molecular weight cutoff of the dialysis is 1500-2000Da. In some embodiments of the present invention, the molecular weight cutoff of the dialysis is 1500Da. In some embodiments of the present invention, the molecular weight cutoff of the dialysis is 2000Da. In some embodiments of the present invention, the molecular weight cutoff of the dialysis is 300Da.

[0047] In some embodiments of the present invention, the nanofiltration is filtration using a nanofiltration membrane.

[0048] In some embodiments of the present invention, the molecular weight cutoff of the nanofiltration is 1500-2000Da. In some embodiments of the present invention, the molecular weight cutoff of the nanofiltration is 1500Da. In some embodiments of the present invention, the molecular weight cutoff of the nanofiltration is 2000Da. In some embodiments of the present invention, the molecular weight cutoff of the nanofiltration is 300Da.

[0049] In some embodiments of the present invention, the nanofiltration is carried out using RisingSun NF7 nanofiltration membrane. In some embodiments of the present invention, the nanofiltration is carried out using RisingSun NF2 nanofiltration membrane.

[0050] In some embodiments of the present invention, the dialysis and / or nanofiltration is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-1500Da. In some embodiments of the present invention, the dialysis is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-1500Da. In some embodiments of the present invention, the nanofiltration is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-1500Da.

[0051] In some embodiments of the present invention, the dialysis and / or nanofiltration is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-2000Da. In some embodiments of the present invention, the dialysis is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-2000Da. In some embodiments of the present invention, the nanofiltration is treated with a filter membrane to obtain a solution of a solute with a molecular weight of 300-2000Da.

[0052] In some embodiments of the present invention, the nanofiltration is treated with a filter membrane so that the solution only retains a solute of a molecular weight of 300-1500Da. In some embodiments of the present invention, the nanofiltration is treated with a filter membrane so that the solution only retains a solute of a molecular weight of 300-2000Da. In some embodiments of the present invention, the dialysis is treated with a filter membrane so that the solution only retains a solute of a molecular weight of 300-1500Da. In some embodiments of the present invention, the dialysis is treated with a filter membrane so that the solution only retains a solute of a molecular weight of 300-2000Da.

[0053] In some embodiments of the present invention, the ultrasonic power is 300-700 W, preferably 500 W.

[0054] In some embodiments of the present invention, the ultrasonic reaction is performed in an ice water bath.

[0055] In some embodiments of the present invention, the ultrasonic reaction time is 0.5-3h. In some embodiments of the present invention, the ultrasonic reaction time is 0.5-2h. In some embodiments of the present invention, the ultrasonic reaction time is 1h. In some embodiments of the present invention, the ultrasonic reaction time is 2h. In some embodiments of the present invention, the ultrasonic reaction time is 0.5h.

[0056] In some embodiments of the present invention, the preparation method of the polymeric proanthocyanidins comprises the following steps: weighing the plant raw material, crushing and sieving, soaking with petroleum ether for defatting, and filtering; adding 60% ethanol (V / V) and standing, filtering, removing the solvent of the filtrate, adding water thereto, stirring thoroughly to disperse evenly, and then adding ethanol to precipitate and remove impurities such as protein and polysaccharide; filtering, removing the solvent of the filtrate, adding water thereto at 5°C, stirring thoroughly to disperse evenly, and then adding ethyl acetate for extraction, taking the aqueous phase, and evaporating the solvent to obtain the polymeric proanthocyanidins.

[0057] In some embodiments of the present invention, the polymeric proanthocyanidins are prepared according to the following steps: weighing the plant raw material, crushing and sieving, soaking with petroleum ether to defatted, filtering; adding 60% ethanol (V / V) and standing, filtering, removing the solvent of the filtrate, adding water thereto, stirring thoroughly to disperse evenly, and then adding ethanol to precipitate and remove impurities such as protein and polysaccharides; filtering, removing the solvent of the filtrate, adding water thereto at 5°C, stirring thoroughly to disperse evenly, and then adding ethyl acetate to extract, taking the aqueous phase, and evaporating the solvent to obtain the polymeric proanthocyanidins.

[0058] In some embodiments of the present invention, the preparation method comprises the following steps:

[0059] (a) Preparation of polymeric proanthocyanidins: Weigh 20 g of grape seeds, purple rice or black rice, crush and sieve, soak in 1000 mL of petroleum ether for 30 h to defat; filter, and air-dry the residue; add 800 mL of 60% ethanol (V / V) thereto, and let stand at 25°C for 3 days under closed conditions; filter, and spin-dry the filtrate under reduced pressure at 30°C; add 100 mL of water thereto, stir well to disperse evenly, and then add 500 mL of ethanol to precipitate and remove impurities such as protein and polysaccharide; filter, and spin-dry the filtrate under reduced pressure at 30°C; add 1000 mL of water thereto at 5°C, stir well to disperse evenly, and then add 200 mL of ethyl acetate for extraction, take the aqueous phase, and spin-dry under reduced pressure at 30°C to obtain polymeric proanthocyanidins from grape seeds, purple rice or black rice;

[0060] (b) Preparation of oligomeric proanthocyanidins from polymeric proanthocyanidins: 5 g of the polymeric proanthocyanidins prepared as described in (a) was added to 1000 mL of an aqueous solution of threonine and stirred evenly. Then, 2.0 g of glutamic acid and 1.5 g of aspartic acid were added and stirred to dissolve. The mixture was subjected to ultrasonic reaction at 500 W in an ice-water bath for 1 h. The mixture was filtered using a nanofiltration membrane with a molecular weight cutoff of 1500-2000 Da, and the filtrate was then filtered using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The residue was freeze-dried to obtain an oligomeric proanthocyanidin product.

[0061] In some embodiments of the present invention, the preparation method comprises the following steps:

[0062] (a) Preparation of polymeric proanthocyanidins: Weigh 20 g of grape seeds, crush and sieve, soak in 1000 mL of petroleum ether for 30 h to defat; filter, and air-dry the residue naturally; add 800 mL of 60% ethanol (V / V) thereto, and let stand at 25°C for 3 days under closed conditions; filter, and spin-dry the filtrate under reduced pressure at 30°C; add 100 mL of water thereto, stir well to disperse evenly, and then add 500 mL of ethanol to precipitate and remove protein and polysaccharide; filter, and spin-dry the filtrate under reduced pressure at 30°C; add 1000 mL of water thereto at 5°C, stir well to disperse evenly, and then add 200 mL of ethyl acetate for extraction, take the aqueous phase, and spin-dry under reduced pressure at 30°C to obtain polymeric proanthocyanidins from grape seeds;

[0063] (b) Preparation of oligomeric proanthocyanidins from polymeric proanthocyanidins: 5 g of the polymeric proanthocyanidins prepared as described in (a) was added to 1000 mL of an aqueous solution of threonine and stirred evenly. Then, 2.0 g of glutamic acid and 1.5 g of aspartic acid were added and stirred to dissolve. The mixture was subjected to ultrasonic reaction at 500 W in an ice-water bath for 1 h. The mixture was filtered using a nanofiltration membrane with a molecular weight cutoff of 1500-2000 Da, and the filtrate was then filtered using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The residue was freeze-dried to obtain an oligomeric proanthocyanidin product.

[0064] In some embodiments of the present invention, the preparation method comprises the steps described in Example 1.

[0065] In the third aspect of the present invention, there is provided use of the oligomeric proanthocyanidins as described in the first aspect in the preparation of anti-oxidation or anti-aging drugs or cosmetics.

[0066] In some embodiments of the present invention, there is provided the use of the oligomeric proanthocyanidins as described in the first aspect in the preparation of anti-aging drugs. In some embodiments of the present invention, there is provided the use of the oligomeric proanthocyanidins as described in the first aspect in the preparation of antioxidant drugs. In some embodiments of the present invention, there is provided the use of the oligomeric proanthocyanidins as described in the first aspect in the preparation of drugs for reducing free radicals. In some embodiments of the present invention, there is provided the use of the oligomeric proanthocyanidins as described in the first aspect in the preparation of drugs for delaying aging.

[0067] In some embodiments of the present invention, there is provided the use of the oligomeric proanthocyanidins as described in the first aspect in the preparation of anti-aging cosmetics. In some embodiments of the present invention, there is provided the use of the oligomeric proanthocyanidins as described in the first aspect in the preparation of antioxidant cosmetics. In some embodiments of the present invention, there is provided the use of the oligomeric proanthocyanidins as described in the first aspect in the preparation of free radical reducing cosmetics. In some embodiments of the present invention, there is provided the use of the oligomeric proanthocyanidins as described in the first aspect in the preparation of anti-aging cosmetics.

[0068] The technical effect of the technical solution of the present invention is that the materials used in the reaction process are highly safe and the impurity removal cost is low.

[0069] The use of a ternary system of glutamic acid, aspartic acid, and threonine helps to obtain more active oligomeric proanthocyanidin products by providing this milder degradation pathway. These oligomers are structurally more suitable as antioxidants and anti-aging agents, thus showing better biological activities than oligomeric proanthocyanidin products from other methods. DETAILED DESCRIPTION

[0070] The present disclosure is further illustrated by way of examples below, but the present disclosure is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product instructions. Unless otherwise specified, the reagents and raw materials used in the present disclosure are all commercially available.

[0071] Example 1. Preparation of oligomeric proanthocyanidins

[0072] Preparation method of polyproanthocyanidins: Weigh 20g of grape seeds (Golden Fragrant Grapes), crush and sieve, soak in 1000mL petroleum ether for 30h to defat. Filter, and air-dry the residue naturally. Add 800mL of 60% ethanol (V / V) to it, and let it stand at 25℃ for 3d under closed conditions. Filter, and spin-dry the filtrate under reduced pressure at 30℃. Add 100mL of water to it, stir it thoroughly to disperse it evenly, and then add 500mL of ethanol to precipitate and remove impurities such as protein and polysaccharide. Filter, and spin-dry the filtrate under reduced pressure at 30℃. Add 1000mL of water to it at 5℃, stir it thoroughly to disperse it evenly, and then add 200mL of ethyl acetate to extract, take the aqueous phase, and spin-dry it under reduced pressure at 30℃ to obtain polyproanthocyanidins from grape seeds.

[0073] The same method is used to prepare gardenia, seabuckthorn seed meal, purple rice and black rice as raw materials to prepare gardenia polyanthocyanidins, seabuckthorn seed meal polyanthocyanidins, purple rice polyanthocyanidins and black rice polyanthocyanidins.

[0074] Preparation of oligomeric proanthocyanidins from polymeric proanthocyanidins: 5 g of polymeric proanthocyanidins from grape seeds prepared as described above were added to 1000 mL of threonine aqueous solution, stirred evenly, then aspartic acid and glutamic acid were added, stirred to dissolve, and ultrasonically reacted at 500W in an ice-water bath (Jinghao, Hangzhou, JH500W-20 ultrasonic equipment) for 1 h. Filtered using a nanofiltration membrane with a molecular weight cutoff of 1500-2000Da (RisingSun, NF7), and the filtrate was then filtered using a nanofiltration membrane with a molecular weight cutoff of 300Da (RisingSun, NF2), and the residue was freeze-dried to obtain the oligomeric proanthocyanidin product PT-OPC-01. The other oligomeric proanthocyanidin products in Table 1 were prepared using basically the same method (for example, the only difference in preparing PT-OPC-06 was that alanine was used instead of the above-mentioned threonine; the only difference in preparing PT-OPC-02 was that 5 g of polyproanthocyanidin from grape seeds was added to 1000 mL of water without threonine; the only difference in preparing PT-OPC-03 was that aspartic acid was not added when glutamic acid was added). The mass and amount of oligomeric proanthocyanidin in the oligomeric proanthocyanidin product were determined by the traditional vanillin method and the improved vanillin method, respectively, and then the average degree of polymerization was calculated. The mass proportion of oligomeric proanthocyanidin in the oligomeric proanthocyanidin product was calculated using the vanillin method. The results are shown in Table 1.

[0075] Table 1. Preparation of oligomeric proanthocyanidins by reaction of polymeric proanthocyanidins

[0076]

[0077] Proanthocyanidin B2 from grape seeds was prepared according to the literature method (Xin Libo et al. Study on chemical composition and antioxidant properties of oligomeric proanthocyanidins from grape seeds [J]. Food Research and Development, 2012, 33(8): 40-43.

[11] ) and was denoted as WX-OPC.

[0078] Example 2: DPPH free radical scavenging activity test

[0079] A 0.2 mmol / L methanol solution of DPPH was prepared, and a 0.1 mg / mL methanol solution of the oligomeric proanthocyanidin product prepared by the method of Example 1 was prepared.

[0080] Mix 2 mL of DPPH methanol solution with 2 mL of methanol and measure the absorbance value A at 517 nm. D Take different volumes of methanol solution of oligomeric proanthocyanidins, add methanol to 4 mL and mix well, and measure the absorbance value A at 517 nm. 原 Take the corresponding volume of methanol solution of oligomeric proanthocyanidins, add 2 mL of DPPH methanol solution, add methanol to 4 mL and mix well, react at room temperature in the dark for 30 minutes, and measure the absorbance value A at 517 nm. To reduce the experimental error, each sample was tested 6 times at each concentration and the average value was taken.

[0081] DPPH free radical scavenging rate = [1-(AA 原 ) / A D ] ×100%.

[0082] Through linear fitting of the clearance rate of each specific product at different concentrations, the IC50 value of each product for scavenging DPPH was calculated. As shown in Table 2. The oligomeric proanthocyanidin product PT-OPC-01 obtained by the reaction in the glutamic acid-aspartic acid-threonine mixed system has the strongest free radical scavenging activity, which is significantly better than PT-OPC-02 to PT-OPC-06. When the same glutamic acid-aspartic acid-threonine ternary mixed system is used, the free radical scavenging activity of the grape seed oligomeric proanthocyanidin product is significantly better than that of the purple rice and black rice oligomeric proanthocyanidin products, and the free radical scavenging activity of the latter two is significantly better than that of the gardenia and sea buckthorn seed meal oligomeric proanthocyanidin products, showing the influence of polymeric proanthocyanidin reaction raw materials from different sources on the oligomeric proanthocyanidin reaction products.

[0083] Table 2. DPPH free radical scavenging test results

[0084]

[0085] Without being bound by theory, threonine may play a specific chemical role in the hydrolysis process, such as acting as a hydrogen bond donor or participating in certain specific reaction pathways, thereby affecting the structure and function of the final product. In addition, in the presence of the ternary system of aspartic acid, glutamic acid and threonine, certain sensitive components in anthocyanidins, such as antioxidant phenolic structures, may be more effectively protected, reducing the loss of these beneficial components during acid hydrolysis.

[0086] Example 3: Cell anti-aging experiment

[0087] Human fibroblast WI-38 cells (BLUEFBIO, BFN60800671) were cultured in DMEM medium (containing 10% fetal bovine serum) at 37°C, 5% CO2. The cells were exposed to low concentrations (about 400 μM) of H 2 O 2 The oligomeric proanthocyanidins product prepared by the method of Example 1 was dissolved in an equal volume of DMEM medium to obtain solutions of different concentrations, which were added to the induced aged cells for 24 hours (the negative control group used an equal volume of DMEM medium). The activity of β-galactosidase in the cells was measured using the β-galactosidase staining method using the cell senescence β-galactosidase staining kit (Beyotime, C0602), and the number of senescent cells was recorded under a microscope to evaluate the degree of cell aging. The cells that were not exposed to H were also tested. 2 O 2 Human fibroblast WI-38 cells not treated with oligomeric proanthocyanidins were used as controls. The experiment was repeated 5 times and the results were averaged.

[0088] The results showed that no exposure to H 2 O 2 The blue-stained cell ratio (i.e., senescent cell ratio) of cells was only 2.4%. 2 O 2 The ratio of blue-stained cells (i.e., senescent cells) in cells treated and not treated with oligomeric proanthocyanidin products was as high as 54.9%, indicating that the cells had aged.

[0089] The senescence rate of cells treated with PT-OPC-01 (1μg / mL) was only 7.6%. The senescence rate of cells treated with PT-OPC-02 to PT-OPC-06 (4μg / mL) was between 25.3% and 59.7%. The senescence rate of cells treated with WX-OPC (2μg / mL) was 24.5%. The senescence rate of cells treated with PT-OPC-01 was significantly lower than that of the other groups, with statistically significant differences (P<0.01).

[0090] Although the embodiments of the present disclosure have been described above, it is understood that the above embodiments are exemplary and do not constitute limitations to the present disclosure. A person skilled in the art may make changes, modifications and variations to the above embodiments within the scope of the present disclosure.

Claims

1. A method for preparing oligomeric proanthocyanidins, characterized in that: The preparation method comprises the following steps: (a) Weigh 20 g of grape seeds, crush and sieve, soak in 1000 mL of petroleum ether for 30 h to defat; filter, and air-dry the residue; add 800 mL of 60% ethanol (V / V) thereto, and let it stand at 25°C for 3 days under closed conditions; filter, and spin-dry the filtrate under reduced pressure at 30°C; add 100 mL of water thereto, stir well to disperse evenly, and then add 500 mL of ethanol to precipitate and remove protein and polysaccharide; filter, and spin-dry the filtrate under reduced pressure at 30°C; add 1000 mL of water thereto at 5°C, stir well to disperse evenly, and then add 200 mL of ethyl acetate to extract, take the aqueous phase, and spin-dry under reduced pressure at 30°C to obtain polyanthocyanidins from grape seeds; (b) 5 g of the polymeric proanthocyanidins prepared by the method described in (a) was added to 1000 mL of a threonine aqueous solution, and stirred evenly. Then, 2.0 g of glutamic acid and 1.5 g of aspartic acid were added, and stirred to dissolve. The mixture was ultrasonically reacted in an ice-water bath for 1 h. The mixture was filtered using a nanofiltration membrane with a molecular weight cutoff of 1500-2000 Da, and the filtrate was then filtered using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The residue was freeze-dried to obtain an oligomeric proanthocyanidin product. The concentration of the threonine is 10-30 g / L; The ultrasonic power is 300-700W.

2. The preparation method according to claim 1, characterized in that The concentration of threonine is 20 g / L, and the ultrasonic power is 500 W.

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