An antioxidant compound based on grape seed active peptide, and a preparation method and application thereof

Abstract

This invention specifically relates to an antioxidant compound based on grape seed active peptides, its preparation method, and its application, belonging to the field of health food technology. The compound comprises 70-90 parts grape seed active peptides, 10-18 parts cinnamon water extract, 8-15 parts hawthorn water extract, 5-10 parts rose water extract, and 0.5-3 parts vitamin C. The preparation method of the grape seed active peptides includes ultrasound-assisted enzymatic hydrolysis, ultrafiltration separation, macroporous resin purification, and spray drying. Experimental verification shows that the components of the compound of this invention have significant synergistic effects, significantly increasing SOD and GSH-Px activity, reducing MDA content, and exhibiting better antioxidant effects than single components. Furthermore, it has excellent taste and high safety. This compound can be used as an active ingredient in pharmaceuticals or health foods for the prevention and adjuvant improvement of various oxidative stress-related diseases. It has advantages such as synergistic effects, good taste, high safety, and suitability for industrial production, and has broad market application prospects.

Description

Technical Field

[0001] This invention belongs to the field of health food technology, specifically relating to an antioxidant compound based on grape seed active peptides, its preparation method, and its application. Background Technology

[0002] Oxidative stress refers to a state of cell damage caused by an imbalance between the body's oxidation and antioxidant systems and the excessive accumulation of free radicals. It is closely related to various pathological processes such as aging, cardiovascular disease, neurodegenerative diseases, diabetes and its complications. Currently, most commonly used antioxidants in clinical practice are chemically synthesized, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Although they have certain antioxidant effects, long-term use poses potential risks of liver and kidney toxicity, teratogenicity, and carcinogenicity, limiting their application in the food and pharmaceutical fields. Therefore, the development of efficient, safe, and natural antioxidants has become a current research hotspot.

[0003] Grape seeds, a byproduct of winemaking, are rich in protein and polyphenols. Grape seed bioactive peptides are small-molecule peptides obtained from grape seed protein through enzymatic hydrolysis, characterized by their small molecular weight, easy absorption, and significant antioxidant activity. However, the antioxidant effects of single grape seed bioactive peptide products often target only one specific site, making it difficult to comprehensively eliminate multiple free radicals in the body. Furthermore, they suffer from problems such as strong bitterness, poor taste, and insufficient stability, limiting their market application. In addition, medicinal and edible plants such as cinnamon, hawthorn, and rose have also been proven to possess excellent antioxidant effects.

[0004] Therefore, the development of a multi-target, highly effective, palatable, and safe antioxidant compound by combining grape seed active peptides with cinnamon water extract, hawthorn water extract, rose water extract, and vitamin C through scientific compounding technology, and utilizing the synergistic effects of each component, can overcome the limitations of single-component formulations and provide a safe and effective antioxidant solution for people under oxidative stress. This has significant practical implications and market value. Summary of the Invention

[0005] To address the significant side effects of existing chemical antioxidants, the limited antioxidant targets and efficacy of single grape seed active peptides, as well as their unpleasant taste and insufficient market adaptability, this invention provides a natural antioxidant compound with multi-target synergy, strong antioxidant efficacy, excellent taste, and high safety, aiming to achieve safe and effective prevention and adjunctive intervention for oxidative stress-related diseases.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides an antioxidant compound based on grape seed active peptides.

[0008] The raw materials of the compound include grape seed active peptides, cinnamon water extract, hawthorn water extract, rose water extract, and vitamin C. Grape seed active peptides, as the core antioxidant component, provide direct free radical scavenging ability; cinnamon water extract is rich in polyphenols, which have synergistic antioxidant and metabolic-improving effects; hawthorn water extract contains flavonoids and organic acids, which can enhance antioxidant enzyme activity; rose water extract is rich in vitamin C derivatives and anthocyanins, possessing both antioxidant and flavor-enhancing functions; vitamin C, as a classic antioxidant, can form a redox cycle with other components, synergistically enhancing the antioxidant effect.

[0009] Preferably, the compound comprises the following raw materials in parts by weight: 70-90 parts grape seed active peptides, 10-18 parts cinnamon water extract, 8-15 parts hawthorn water extract, 5-10 parts rose water extract, and 0.5-3 parts vitamin C. Within this ratio range, the components can fully exert their synergistic effect, ensuring a high level of antioxidant efficacy while also considering taste and safety.

[0010] More preferably, the compound comprises the following raw materials in parts by weight: 85 parts grape seed active peptides, 15 parts cinnamon water extract, 12 parts hawthorn water extract, 8 parts rose water extract, and 2 parts vitamin C. In vitro and in vivo antioxidant activity experiments verified that this ratio of compound exhibits the best antioxidant effect, with significantly better DPPH free radical scavenging rate, ABTS free radical scavenging rate, and total reducing power compared to other ratios. Furthermore, it has a mild taste, no obvious sourness or astringency, and demonstrates excellent overall performance.

[0011] Preferably, the average molecular weight of the grape seed active peptide is 300-600 Da. Active peptides in this molecular weight range are easily absorbed by the human intestine, can quickly enter the bloodstream, and act on antioxidant processes. Its amino acid composition includes 17.5-18.2% proline, 10.3-11.0% glycine, 8.5-9.0% histidine, 7.2-7.8% cysteine, 6.1-6.6% tyrosine, and 5.3-5.8% arginine. This specific amino acid composition endows the active peptide with excellent antioxidant activity. The sulfur-containing amino acid (cysteine) provides thiol groups to directly scavenge free radicals, the imidazole ring of histidine has the ability to chelate metal ions, and proline and glycine help maintain the conformational stability of the peptide.

[0012] In some embodiments, the method for preparing the grape seed active peptide includes the following steps:

[0013] (1) Raw material pretreatment: Grape seed meal was defatted with petroleum ether, washed with deionized water and dried, crushed and passed through an 80-mesh standard sieve, and 0.10-0.20 mol / L phosphate buffer (pH 6.0-7.0) was added at a weight-volume ratio of 1 g: 10-15 mL. The mixture was magnetically stirred for 30 min to prepare a uniform grape seed protein suspension.

[0014] (2) Ultrasonic-assisted enzymatic hydrolysis: The grape seed protein suspension was pretreated in a high-energy ultrasonic instrument with an ultrasonic power of 500-800 W, a treatment time of 10-20 min, and an ultrasonic temperature of 40-50℃. Ultrasonic pretreatment can destroy the protein aggregation structure, increase the contact area between the enzyme and the substrate, and significantly improve the subsequent enzymatic hydrolysis efficiency. After pretreatment, the suspension was stirred and preheated at 35-45℃ for 15-20 min, and then a compound enzyme system was added at 1.8-2.5% of the grape seed meal raw material mass, and enzymatic hydrolysis was carried out at 35-45℃ for 4-6 h. The compound enzyme system is composed of neutral protease, papain and cellulase in a mass ratio of 1:(0.7-1.1):(0.9-1.4). Among them, neutral protease and papain work synergistically to cleave grape seed protein from different sites to generate small molecule peptides with antioxidant activity; cellulase can destroy the residual cell wall structure in grape seed meal, release more protein and active ingredients, and further improve the enzymatic hydrolysis efficiency and product yield.

[0015] (3) Separation and purification: After enzymatic hydrolysis, heat to 90-95℃ for 8-12 min to inactivate the enzyme protein by denaturation; cool to room temperature and centrifuge at 4000-5000 r / min for 15-20 min to remove the precipitate and collect the supernatant to obtain the enzymatic hydrolysate. Ultrafiltration the hydrolysate through a 1-3 kDa ultrafiltration membrane to collect components smaller than the corresponding molecular weight, remove unhydrolyzed macromolecular proteins and polysaccharides, and prepare a solution with a concentration of 8-12 mg / mL. Chromatographic purification is performed using a D101 macroporous resin column. The ultrafiltration hydrolysate is added to the column at a ratio of 1:12-16 (column volume). First, elute with double-distilled water for 3 column volumes to remove water-soluble impurities and salts, then elute with 40-60% ethanol solution and collect the ethanol eluent. This step effectively enriches peptides with antioxidant activity, removes bitter peptides and impurities, and significantly improves the product's taste. The eluent was concentrated under low pressure at 35-40℃ to remove ethanol, and finally spray-dried (inlet air temperature 180-200℃, outlet air temperature 80-90℃) to obtain grape seed active peptide powder.

[0016] Secondly, the present invention provides a method for preparing the antioxidant complex.

[0017] The specific steps include: weighing grape seed active peptides, cinnamon water extract, hawthorn water extract, rose water extract, and vitamin C according to the above-mentioned weight proportions, adding an appropriate amount of double-distilled water, and stirring at 30-40℃ for 30-45 minutes until uniformly mixed to form a homogeneous mixture; sending the mixture into a spray dryer, setting the inlet air temperature to 170-190℃ and the outlet air temperature to 75-85℃, and spray drying to obtain the compound powder. This method is simple to operate, low in cost, and suitable for industrial production. The obtained compound powder has good flowability and solubility, facilitating subsequent formulation processing.

[0018] Thirdly, the present invention provides a pharmaceutical or health food composition.

[0019] The composition comprises the above-mentioned antioxidant complex and a pharmaceutically or food-grade acceptable carrier. The carrier may be one or more, depending on the dosage form requirements, and includes, but is not limited to: fillers (such as microcrystalline cellulose, lactose, mannitol, starch), binders (such as hydroxypropyl methylcellulose, povidone, sodium carboxymethyl cellulose), disintegrants (such as crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose), lubricants (such as magnesium stearate, talc, micronized silica), flow aids (such as silica), flavoring agents (such as aspartame, steviol glycosides, citric acid), etc.

[0020] The composition can be in the form of tablets, dispersible tablets, instant tablets, powders, granules, or capsules. Appropriate dosage forms can be selected according to the needs of different populations (such as the elderly, children, and those with swallowing difficulties), ensuring convenient administration and good compliance. For example, the compound powder can be mixed with fillers and disintegrants and then directly compressed into tablets; or it can be mixed with excipients, granulated, and then filled into capsules; or it can be directly packaged into granules or powders.

[0021] Fourthly, the present invention provides the application of the aforementioned antioxidant complexes or compositions.

[0022] This refers to its application in the preparation of products for the prevention and / or adjunctive improvement of oxidative stress-related conditions, including pharmaceuticals, health foods, functional foods, and special medical foods. Oxidative stress-related conditions include, but are not limited to, aging and related degenerative diseases, cardiovascular diseases, diabetes and its complications, neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), liver damage, kidney damage, and inflammatory diseases. The compound of this invention can exert its preventive and adjunctive improvement effects on oxidative stress-related conditions through multiple mechanisms, such as scavenging various free radicals, enhancing the activity of endogenous antioxidant enzymes, and inhibiting lipid peroxidation.

[0023] The compound of this invention exerts its antioxidant effect through multiple targets and pathways: its active ingredients can directly scavenge various free radicals such as superoxide anions and hydroxyl radicals; specific amino acids (such as cysteine ​​and histidine) can chelate transition metal ions and inhibit the Fenton reaction; simultaneously, it can enhance the activity of endogenous antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and inhibit the formation of malondialdehyde (MDA), a lipid peroxidation product. Through the above synergistic mechanism, it achieves effective prevention and adjunctive improvement of oxidative stress-related diseases.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) Synergistic effect, multi-target antioxidant. This invention achieves a synergistic effect of "1+1>2" among the five components through scientifically optimized formulation. In vitro and in vivo experiments show that the complete formula can significantly enhance SOD and GSH-Px activity and reduce MDA content, with effects far superior to those of a single component. Its mechanism of action covers direct scavenging of free radicals, chelation of metal ions to inhibit Fenton reaction, enhancement of endogenous antioxidant enzyme activity, and inhibition of lipid peroxidation, achieving multi-target, all-round antioxidant effects.

[0026] (2) The preparation process is scientific, reasonable and efficient. The preparation of grape seed active peptides adopts high-energy ultrasonic pretreatment combined with enzymatic hydrolysis, which effectively destroys the protein molecular structure, promotes the conversion of protein into peptides, and improves the yield and purity of active ingredients; the water extract adopts heating reflux combined with freeze drying to retain the active ingredients to the greatest extent; the overall process steps are simple, easy to industrialize, and will not increase the metabolic burden on the liver and kidneys, making it suitable for long-term use.

[0027] (3) Excellent taste and high safety. Grape seed active peptides have small molecular weight and are easily absorbed. They can not only exert a highly effective antioxidant effect, but also supplement the amino acids needed by the human body. Vitamin C can directly remove free radicals and work synergistically with other ingredients to enhance the body's antioxidant system and reduce oxidative stress damage.

[0028] (4) Wide range of applications and outstanding market value. The compound of this invention can be used as an active ingredient in pharmaceuticals, health foods, functional foods and special medical foods to prevent and assist in the improvement of various oxidative stress-related diseases such as aging, cardiovascular diseases, diabetic complications, and neurodegenerative diseases. It can be prepared into various dosage forms such as tablets, capsules and granules to meet the needs of different groups of people and has significant market application value. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0030] After reading the contents disclosed in this invention, those skilled in the art can make appropriate adjustments or substitutions to the process parameters of the methods and applications described in this invention without departing from the spirit and scope of this invention. Such obvious adjustments, substitutions or combinations should be included within the protection scope of this invention.

[0031] Unless otherwise specified, the materials, reagents, instruments and testing methods used in the following embodiments can be obtained commercially or prepared, operated and implemented with reference to conventional methods disclosed in the art.

[0032] It should be noted that all technical parameters described in this document as numerical ranges (such as temperature, ratio, time, content, etc.) should be understood as encompassing all possible sub-ranges and specific numerical points within that range, regardless of whether the specific numerical value or sub-range is explicitly listed. Unless otherwise specified, the technical terms used in this document have the meanings commonly understood by those skilled in the art.

[0033] The cinnamon water extract, hawthorn water extract, and rose water extract used in this invention were prepared according to the following methods:

[0034] Cinnamon water extract: Take dried cinnamon, pulverize it, pass it through a 40-mesh standard sieve, and store it in a sealed, light-protected container for later use. Accurately weigh 10.00 g of cinnamon powder and place it in a 500 mL round-bottom flask. Add purified water at a material-to-liquid ratio of 1:20 (g:mL), incubate at room temperature for 90 min, and then extract by reflux in a 90℃ constant temperature water bath for 2 h. After extraction, centrifuge at 3500 r / min for 10 min and collect the filtrate. Transfer the filtrate to a rotary evaporator and concentrate it under reduced pressure at a vacuum of 0.07 MPa and a 50℃ water bath until it becomes a thick paste. Spread the paste evenly on a freeze-drying tray, place it in a freeze dryer, pre-freeze at -45℃ for 4 h, and then sublimate and dry at 30℃ for 12 h to obtain the cinnamon water extract.

[0035] Hawthorn water extract: Take dried hawthorn, remove the pits, pulverize, pass through a 40-mesh standard sieve, and store in a sealed, light-protected container for later use. Accurately weigh 10.00 g of hawthorn powder and place it in a 500 mL round-bottom flask. Add purified water at a solid-liquid ratio of 1:22 (g:mL), incubate at room temperature for 80 min, and then reflux in a 90℃ constant temperature water bath for 2.0 h. After extraction, centrifuge at 3500 r / min for 10 min and collect the filtrate. Subsequent concentration and freeze-drying steps are the same as for cinnamon water extract to obtain hawthorn water extract.

[0036] Rose water extract: Take dried rose petals, pulverize them, and pass them through a 40-mesh standard sieve. Store in a sealed container away from light for later use. Accurately weigh 10.00 g of rose petal powder and place it in a 500 mL round-bottom flask. Add purified water at a material-to-liquid ratio of 1:20 (g:mL). After soaking at room temperature for 70 min, extract by reflux in an 80℃ water bath for 1.5 h. After extraction, centrifuge at 3500 r / min for 10 min and collect the filtrate. Subsequent concentration and freeze-drying steps are the same as for cinnamon water extract to obtain the rose water extract.

[0037] Example 1

[0038] An antioxidant compound based on grape seed active peptides, the raw materials and their weight parts are: 80 parts grape seed active peptides, 12 parts cinnamon water extract, 10 parts hawthorn water extract, 6 parts rose water extract, and 1.5 parts vitamin C.

[0039] The preparation method of grape seed bioactive peptides is as follows:

[0040] (1) After defatting the grape seed meal, crush it through an 80-mesh sieve and add 0.15mol / L phosphate buffer (pH 6.5) at a weight-volume ratio of 1 g: 12 mL to prepare a grape seed protein suspension.

[0041] (2) The suspension was pretreated by focused ultrasound (power 650 W, time 15 min, temperature 45℃), and then preheated by stirring at 40℃ for 18 min; a compound enzyme system (neutral protease: papain: cellulase = 1:0.9:1.1, w / w) was added at 2.0% of the mass of grape seed meal raw material, and enzymatically hydrolyzed at 40℃ for 5 h.

[0042] (3) After enzymatic hydrolysis, the temperature was raised to 92℃ for 10 min to inactivate the enzyme. After cooling to room temperature, the solution was centrifuged at 4500 r / min for 18 min to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was ultrafiltered through a 2 kDa ultrafiltration membrane, and the fractions smaller than 2 kDa were collected to prepare a solution with a concentration of 10 mg / mL. The ultrafiltration enzymatic hydrolysate was added to a D101 macroporous resin column at a ratio of 1:14 of the macroporous resin column volume. After eluting with double-distilled water for 3 column volumes, the solution was eluted with 50% ethanol solution, and the ethanol eluent was collected. The solution was concentrated under low pressure at 38℃ and finally spray-dried (inlet air temperature 190℃, outlet air temperature 85℃) to obtain grape seed active peptides. The obtained grape seed active peptides were analyzed by SEC-HPLC, which showed that the average molecular weight was 450 Da, with proline content of 17.8%, glycine content of 10.6%, histidine content of 8.7%, cysteine ​​content of 7.5%, tyrosine content of 6.3%, and arginine content of 5.5%.

[0043] Preparation of compound: Weigh each raw material according to the above weight parts, add double distilled water and stir at 35℃ for 40 min until the mixture is uniform, spray dry (inlet air temperature 185℃, outlet air temperature 82℃) to obtain compound powder, and make into capsules of 0.4 g / capsule.

[0044] Example 2

[0045] An antioxidant compound based on grape seed active peptides, the raw materials and their weight parts are: 70 parts grape seed active peptides, 10 parts cinnamon water extract, 8 parts hawthorn water extract, 5 parts rose water extract, and 0.5 parts vitamin C.

[0046] The preparation method of grape seed bioactive peptides is as follows:

[0047] (1) After defatting the grape seed meal, crush it through an 80-mesh sieve and add 0.10 mol / L phosphate buffer (pH 6.0) at a weight-volume ratio of 1 g: 10 mL to prepare a grape seed protein suspension.

[0048] (2) The suspension was pretreated by focused ultrasound (power 500 W, time 10 min, temperature 40℃), and then preheated by stirring at 35℃ for 15 min; a compound enzyme system (neutral protease: papain: cellulase = 1:0.7:0.9, w / w) was added at 1.8% of the mass of grape seed meal raw material, and enzymatically hydrolyzed at 35℃ for 4 h.

[0049] (3) After enzymatic hydrolysis, the temperature was raised to 90℃ for 8 min to inactivate the enzyme. After cooling to room temperature, the solution was centrifuged at 4000 r / min for 15 min to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was ultrafiltered through a 1 kDa ultrafiltration membrane, and the fractions smaller than 1 kDa were collected to prepare a solution with a concentration of 8 mg / mL. The ultrafiltration enzymatic hydrolysate was added to a D101 macroporous resin column at a ratio of 1:12 of the macroporous resin column volume. After eluting with double-distilled water for 3 column volumes, the solution was eluted with 40% ethanol solution, and the ethanol eluent was collected. The solution was concentrated under low pressure at 35℃ and finally spray-dried (inlet air temperature 180℃, outlet air temperature 80℃) to obtain grape seed active peptides. The obtained grape seed active peptides were analyzed by SEC-HPLC, which showed that the average molecular weight was 380 Da, with proline content of 17.5%, glycine content of 10.3%, histidine content of 8.5%, cysteine ​​content of 7.2%, tyrosine content of 6.1%, and arginine content of 5.3%.

[0050] The preparation steps for the compound are the same as in Example 1.

[0051] Example 3

[0052] An antioxidant compound based on grape seed active peptides, the raw materials and their weight parts are: 85 parts grape seed active peptides, 15 parts cinnamon water extract, 12 parts hawthorn water extract, 8 parts rose water extract, and 2 parts vitamin C.

[0053] The preparation method of grape seed bioactive peptides is as follows:

[0054] (1) After defatting the grape seed meal, crush it through an 80-mesh sieve and add 0.20 mol / L phosphate buffer (pH 7.0) at a weight-volume ratio of 1 g: 15 mL to prepare a grape seed protein suspension.

[0055] (2) The suspension was pretreated by focused ultrasound (power 800 W, time 20 min, temperature 50℃), and then preheated by stirring at 45℃ for 20 min; a compound enzyme system (neutral protease: papain: cellulase = 1:1.1:1.4, w / w) was added at 2.5% of the mass of grape seed meal raw material, and enzymatic hydrolysis was carried out at 45℃ for 6 h.

[0056] (3) After enzymatic hydrolysis, the temperature was raised to 95℃ for 12 min to inactivate the enzyme. After cooling to room temperature, the solution was centrifuged at 5000 r / min for 20 min to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was ultrafiltered through a 3 kDa ultrafiltration membrane, and the fractions smaller than 3 kDa were collected to prepare a solution with a concentration of 12 mg / mL. The ultrafiltration enzymatic hydrolysate was added to a D101 macroporous resin column at a ratio of 1:16 of the macroporous resin column volume. After eluting with double-distilled water for 3 column volumes, the solution was eluted with 60% ethanol solution, and the ethanol eluent was collected. The solution was concentrated under low pressure at 40℃ and finally spray-dried (inlet air temperature 200℃, outlet air temperature 90℃) to obtain grape seed active peptides. The obtained grape seed active peptides were analyzed by SEC-HPLC, which showed that the average molecular weight was 550 Da, with proline content of 18.2%, glycine content of 11.0%, histidine content of 9.0%, cysteine ​​content of 7.8%, tyrosine content of 6.6%, and arginine content of 5.8%.

[0057] The preparation steps for the compound are the same as in Example 1.

[0058] Comparative Example 1: Compound lacking a single component

[0059] To investigate the contribution and synergistic effect of each key component to the overall antioxidant activity of the compound, the following four comparative examples were set up. Each comparative example was based on the formulation of Example 3, except that one core component was missing, while the remaining components and their weight parts remained unchanged.

[0060] Comparative Example 1-1: Grape seed active peptide group without grape seed active peptide. Grape seed active peptide was not added to the formula, and the remaining components (cinnamon water extract, hawthorn water extract, rose water extract, vitamin C) and their weight parts were the same as in Example 3.

[0061] Comparative Examples 1-2: Cinnamon water extract-deficient group. Cinnamon water extract was not added to the formulation, and the remaining components (grape seed active peptides, hawthorn water extract, rose water extract, vitamin C) and their weight parts were the same as in Example 3.

[0062] Comparative Examples 1-3: Hawthorn water extract-free group. Hawthorn water extract was not added to the formula, and the remaining components (grape seed active peptide, cinnamon water extract, rose water extract, vitamin C) and their weight parts were the same as in Example 3.

[0063] Comparative Examples 1-4: Rose extract-free group. Rose extract was not added to the formulation, and the remaining components (grape seed active peptide, cinnamon extract, hawthorn extract, vitamin C) and their weight parts were the same as in Example 3.

[0064] Comparative Example 2: Single-Component Control Group

[0065] To evaluate the antioxidant activity of each individual component, four comparative examples were set up. Each comparative example contained only one active ingredient from the compound, and its content and preparation method were consistent with those described in Example 3.

[0066] Comparative Example 2-1: Grape seed active peptide alone. Containing only grape seed active peptide, with a content of 85 parts, prepared using the same method as in Example 3.

[0067] Comparative Example 2-2: Cinnamon water extract alone. This group contained only cinnamon water extract, with a content of 15 parts, and was prepared using the same method as in Example 3.

[0068] Comparative Examples 2-3: Hawthorn aqueous extract alone. Containing only hawthorn aqueous extract, with a content of 12 parts, prepared using the same method as in Example 3.

[0069] Comparative Examples 2-4: Rose petal water extract alone. These consisted of only rose petal water extract, with a content of 8 parts, prepared using the same method as in Example 3.

[0070] Comparative Example 3: Compounds with different weight ranges

[0071] To verify the rationality of the raw material weight range defined in this invention and the optimality of the synergistic effect, two sets of compound compounds with weights exceeding the protection scope of this invention were set as comparative examples.

[0072] Comparative Example 3-1: High Addition Group. 60 parts grape seed active peptide, 20 parts cinnamon water extract, 20 parts hawthorn water extract, 12 parts rose water extract, and 4 parts vitamin C. In this formulation, the content of all components except grape seed active peptide is higher than in Example 3.

[0073] Comparative Example 3-2: Low Addition Group. Grape seed active peptide 100 parts, cinnamon water extract 8 parts, hawthorn water extract 5 parts, rose water extract 3 parts, vitamin C 0.3 parts. In this formulation, the content of all components except grape seed active peptide is lower than that in Example 3.

[0074] Comparative Example 4: The compound in the preparation of grape seed active peptides omitting ultrasonic pretreatment

[0075] To verify the necessity of the focused ultrasonic pretreatment process in improving the yield and activity of grape seed active peptides, this comparative example was set up. The types and weight parts of the raw materials of this compound are exactly the same as those in Example 3. The difference is that in the preparation process of grape seed active peptides, the "focused ultrasonic pretreatment" operation is omitted in step (2), and the rest of the preparation steps are consistent with those in Example 3.

[0076] Comparative Example 5: The complex compound eluted by chromatographic elution was omitted in the preparation of grape seed active peptides.

[0077] To verify the key role of the D101 macroporous resin chromatography elution process in purifying grape seed active peptides and enhancing their activity, this comparative example was set up. The types and weight parts of the raw materials of this compound are exactly the same as those in Example 3. The difference is that in the preparation process of grape seed active peptides, the "D101 macroporous resin column chromatography" operation is omitted in step (3). The enzymatic hydrolysate is directly concentrated under low pressure and spray-dried after ultrafiltration to obtain grape seed active peptides.

[0078] Example 1: Detection of antioxidant activity in vivo

[0079] 1.1 Test Materials

[0080] The experimental samples were the contents of capsules prepared in Examples 1-3 and Comparative Examples 1-5. A high-dose group (1.35 g / kg bw) was included in each group. The positive control drug was vitamin E solution (0.1 g / mL). The normal control group and the model control group were given an equal volume of physiological saline. SPF-grade male SD rats, weighing 200-220 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with 10 rats per group. They were housed in an SPF-grade barrier system at an ambient temperature of 22-25℃ and a relative humidity of 45-55%, with a 12-hour light / 12-hour dark cycle and free access to food and water.

[0081] 1.2 Test Methods

[0082] Except for the normal control group, rats in the other groups were intraperitoneally injected with D-galactose (150 mg / kg bw) daily for 60 consecutive days to establish an oxidative stress model. Simultaneously with model establishment, each treatment group was administered the corresponding test substance once daily by gavage at a volume of 0.1 mL / 10 g bw for 42 consecutive days. After the last administration, the rats were fasted but allowed free access to water for 12 hours. Blood was collected from the orbital sinus to separate serum. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as the content of malondialdehyde (MDA), were measured in the serum according to the kit instructions, and the rate of change of each indicator compared to the model control group was calculated.

[0083] Table 1 Comparison of antioxidant effects in vivo among different groups (n=10)

[0084]

[0085] As shown in Table 1, the antioxidant effects of each group in the Examples were significantly better than those of all comparative examples. The effects of Examples 1 (without any component) were significantly lower than those of Example 3, but still better than the corresponding single component (Comparative Example 2), clearly demonstrating that the compound of the present invention has a synergistic antioxidant effect of "1+1>2". The antioxidant effect of Comparative Example 3 (with a weight fraction exceeding the scope of the present invention) was significantly lower than that of Example 3, proving that only components within the scope of protection of the present invention can synergistically exert optimal efficacy. The effects of Comparative Example 4 (omitting ultrasonic pretreatment) and Comparative Example 5 (omitting chromatographic elution) were both lower than those of Example 3, indicating that ultrasonic pretreatment and chromatographic elution processes play a key role in improving the yield and purity of grape seed active peptides, thereby enhancing the bioactivity of the compound.

[0086] Example 2: In vitro antioxidant activity assay

[0087] 2.1 Test Materials

[0088] The test samples were the compound powders prepared in Examples 1-3 and each comparative example, dissolved in deionized water to prepare sample solutions with a concentration of 1.0 mg / mL. Reagents included DPPH reagent, ABTS reagent, Trolox standard, and phosphate buffer (pH 7.4). The main instrument was an ELISA reader (detection wavelengths 517 nm, 734 nm, and 700 nm).

[0089] 2.2 Test Methods

[0090] DPPH free radical scavenging rate determination: Mix 2 mL of sample solution with 2 mL of 0.1 mmol / L DPPH ethanol solution, react in the dark for 30 min, and measure the absorbance at 517 nm to calculate the scavenging rate.

[0091] ABTS free radical scavenging rate determination: Prepare ABTS working solution, mix 0.1 mL of sample solution with 3.9 mL of working solution, react for 6 min, measure absorbance at 734 nm, and calculate scavenging rate.

[0092] Total reducing power determination: The absorbance of the reaction solution at 700 nm was measured using the potassium ferricyanide method, and the results are expressed as Trolox equivalents (mmol Trolox / g).

[0093] Table 2 Comparison of in vitro antioxidant activities among different groups (n=3)

[0094]

[0095] As shown in Table 2, Example 3 exhibited the highest scavenging rate and total reducing capacity against DPPH and ABTS free radicals, significantly outperforming all the other examples. This result is consistent with the trend observed in in vivo antioxidant experiments, further confirming that the compound of this invention can exert antioxidant effects through multiple pathways. Its synergistic mechanism and advanced preparation process were also verified in in vitro chemical models.

[0096] Example 3: In vitro safety evaluation

[0097] 3.1 Test Materials

[0098] The test samples were the compound powders prepared in Example 3 and Comparative Examples 3-5, and were prepared into sample solutions with concentrations of 0.25-4.0 mg / mL using DMEM medium. The test cells were human hepatocytes (LO2 cells, purchased from the Cell Bank of the Chinese Academy of Sciences). The main reagents included DMEM medium, MTT reagent, and DMSO.

[0099] 3.2 Test Methods

[0100] LO2 cells in the logarithmic growth phase were harvested at a rate of 4 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. After discarding the supernatant, sample solutions of different concentrations were added, and the cells were incubated for another 24 h, 48 h, and 72 h, respectively. After incubation, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated for 4 h. After discarding the supernatant, 150 μL of DMSO was added to each well, and the cells were shaken for 10 min to dissolve the crystals completely. The absorbance (OD value) was measured at 490 nm, and the cell viability was calculated.

[0101] Table 3. Effects of each experimental group on LO2 cell survival (n=3, concentration 2.0 mg / mL)

[0102]

[0103] As shown in Table 3, when the sample concentration was ≤2.0 mg / mL, the LO2 cell survival rate in Example 3 and each comparative group was ≥86%, and the half-maximal inhibitory concentration (IC50) was ≥86%. 50 All concentrations were ≥4.0 mg / mL, far exceeding the in vitro concentrations corresponding to the expected human intake (0.1-0.15 mg / mL). The results indicate that the compound of this invention has good safety and no significant cytotoxicity.

[0104] Example 4: Taste Evaluation Test

[0105] 4.1 Test Methods

[0106] Thirty healthy volunteers (aged 20-50, half male and half female) were recruited for a blinded taste evaluation. The sample was dissolved in 40℃ warm water to prepare a 10 g / L solution for volunteers to taste. Scoring was based on three dimensions: sourness, astringency, and palatability, using a 5-point scale. The specific scoring criteria are as follows:

[0107] Sourness: 1 point (too sour or no sourness), 3 points (moderate sourness), 5 points (mild sourness).

[0108] Astringency: 1 point (strong and unacceptable astringency), 3 points (slight astringency), 5 points (almost no astringency).

[0109] Palatability: 1 point (difficult to swallow), 3 points (acceptable), 5 points (smooth texture).

[0110] Calculate the average and standard deviation of the scores for each dimension. A higher score indicates a better taste.

[0111] Table 4. Taste evaluation results for each experimental group (n=30, 1-5 points)

[0112]

[0113] As shown in Table 4, Example 3 achieved the highest scores in all three dimensions of sourness, astringency, and palatability, indicating the best taste. Comparative Example 3 (outside the specified ratio) had a significantly enhanced sourness and astringency due to an imbalance in component proportions, resulting in a significantly lower score than Example 3. Comparative Example 4 (without ultrasonic pretreatment) and Comparative Example 5 (without chromatographic elution) had slightly lower taste scores than Example 3 due to lower purity of grape seed active peptides, but were still superior to Comparative Example 3. The results demonstrate that the optimized ratios and key preparation processes of this invention have a positive effect on improving the taste of the compound.

[0114] In summary, the antioxidant compound based on grape seed active peptides provided by this invention (preferably composed of 85 parts grape seed active peptides, 15 parts cinnamon water extract, 12 parts hawthorn water extract, 8 parts rose water extract, and 2 parts vitamin C) exhibits significant antioxidant effects. The components of this compound demonstrate a clear synergistic effect of "1+1>2," and the optimal synergistic effect is achieved only within the weight percentages protected by this invention. In the preparation process, focused ultrasonic pretreatment can improve the yield of active peptides, and chromatographic elution can improve the purity of active ingredients; both enhance the overall efficacy of the compound. Furthermore, this compound has high safety, a pleasant taste, and is suitable for long-term use, possessing broad market application prospects.

Claims

1. An antioxidant complex based on grape seed active peptides, characterized in that, The raw materials of the compound include grape seed active peptides, cinnamon water extract, hawthorn water extract, rose water extract, and vitamin C.

2. The antioxidant compound according to claim 1, characterized in that, The compound comprises the following raw materials in parts by weight: 70-90 parts grape seed active peptide, 10-18 parts cinnamon water extract, 8-15 parts hawthorn water extract, 5-10 parts rose water extract, and 0.5-3 parts vitamin C.

3. The antioxidant compound according to claim 2, characterized in that, The compound comprises the following raw materials in parts by weight: 85 parts grape seed active peptides, 15 parts cinnamon water extract, 12 parts hawthorn water extract, 8 parts rose water extract, and 2 parts vitamin C.

4. The antioxidant compound according to claim 1, characterized in that, The average molecular weight of the grape seed active peptide is 300-600 Da; the amino acid composition of the grape seed active peptide is as follows: proline content is 17.5-18.2%, glycine content is 10.3-11.0%, histidine content is 8.5-9.0%, cysteine ​​content is 7.2-7.8%, tyrosine content is 6.1-6.6%, and arginine content is 5.3-5.8%.

5. The antioxidant compound according to claim 1 or 4, characterized in that, The preparation method of the grape seed active peptide includes the following steps: (1) Raw material pretreatment: Grape seed meal was defatted, washed, dried and crushed, and then added to phosphate buffer to prepare grape seed protein suspension; (2) Ultrasonic-assisted enzymatic hydrolysis: The suspension obtained in step (1) is pretreated by ultrasound, and then a complex enzyme system is added for enzymatic hydrolysis; the complex enzyme system is composed of neutral protease, papain and cellulase in a mass ratio of 1:(0.7-1.1):(0.9-1.4); (3) Separation and purification: After the enzymatic hydrolysis is completed, the enzyme is inactivated and centrifuged. The supernatant is collected and then purified by ultrafiltration membrane ultrafiltration, macroporous resin column chromatography, elution, concentration and drying to obtain grape seed active peptide powder.

6. A method for preparing the antioxidant complex according to any one of claims 1-5, characterized in that, Includes the following steps: Weigh out grape seed active peptides, cinnamon water extract, hawthorn water extract, rose water extract and vitamin C according to the weight parts, add double-distilled water, stir and mix evenly to obtain a mixture; spray dry the mixture to obtain compound powder.

7. A pharmaceutical or health food composition, characterized in that, It comprises the antioxidant complex as described in any one of claims 1-5 and a pharmaceutically or food-grade acceptable carrier.

8. The composition according to claim 7, characterized in that, The composition is available in the form of tablets, dispersible tablets, powders, granules, or capsules.

9. The use of the antioxidant complex according to any one of claims 1-5 or the composition according to any one of claims 7-8 in the preparation of products for the prevention and / or adjunctive improvement of oxidative stress-related conditions.

10. The application according to claim 9, characterized in that, The oxidative stress-related conditions include aging and related degenerative diseases, cardiovascular diseases, diabetes and its complications, neurodegenerative diseases, liver damage, kidney damage, or inflammatory diseases.

Images