Anti-aging theaflavin composition as well as preparation method and application thereof

Abstract

The invention provides an anti-aging theaflavin composition as well as a preparation method and application thereof, and belongs to the technical field of biology. The composition comprises a coated and modified nanocrystalline theaflavin-metal organic framework compound and a mitochondrial targeting coenzyme Q10-L-selenium-methyl selenocysteine compound. The nanocrystalline theaflavin-metal organic framework is prepared by adopting a special method, the stability and the bioavailability of theaflavin are improved by adopting the gamma-cyclodextrin-tannic acid copolymer for coating, and the stability and the bioavailability of theaflavin are improved by being matched with the mitochondrial targeting coenzyme Q10-L-selenium-methyl selenocysteine compound for reasonable compatibility and use, so that the anti-aging effect can be well realized; the effect of theaflavin is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an anti-aging theaflavin composition and a preparation method and application thereof. Background Art

[0002] The mechanisms of aging are complex and diverse, involving multiple levels. The free radical theory posits that cells produce free radicals during metabolism, which attack intracellular biomolecules such as DNA, proteins, and lipids, leading to cell damage and aging. The telomere shortening theory states that the telomeres at the ends of chromosomes gradually shorten with cell division. When telomeres shorten to a certain extent, cells enter senescence or apoptosis. Furthermore, factors such as oxidative stress, inflammatory responses, abnormal regulation of gene expression, and mitochondrial dysfunction are also closely related to aging.

[0003] Based on these theories, numerous anti-aging technologies and products have emerged. In drug development, rapamycin and its analogs inhibit the mTOR signaling pathway, regulating cell growth and metabolism and extending lifespan. Metformin improves glucose metabolism and activates the AMPK signaling pathway, exhibiting certain anti-aging effects. In the health supplement sector, antioxidants such as vitamin C and vitamin E scavenge free radicals and reduce oxidative damage. Coenzyme Q10 participates in cellular energy metabolism and antioxidant processes, helping to maintain normal cell function. Substances such as hyaluronic acid and collagen can improve skin condition and reduce the appearance of wrinkles. However, existing anti-aging drugs and health supplements still present numerous challenges. Long-term use of some medications can lead to a range of adverse reactions. For example, rapamycin may affect immune system function and increase the risk of infection. The efficacy of anti-aging products varies widely, with ineffective results being a common problem.

[0004] Theaflavins are a class of polyphenolic compounds extracted from black tea. They are the product of the oxidative polymerization of catechins during the fermentation process and possess a unique chemical structure and diverse biological activities. Theaflavins contain multiple phenolic hydroxyl groups and possess strong antioxidant properties, capable of scavenging free radicals in the body. Theaflavins can reduce lipid peroxidation, protect cell membrane integrity, and slow the aging process. Theaflavins have significant anti-inflammatory effects, protecting cells from inflammatory damage and delaying aging. Theaflavins can regulate lipid and sugar metabolism and improve insulin resistance. By regulating metabolic function, theaflavins help maintain normal physiological functions and slow the aging process.

[0005] However, the use of theaflavins still has the following problems:

[0006] (1) Poor stability: Theaflavin has active chemical properties and is prone to oxidation, isomerization and other reactions under conditions such as light, heat, and oxygen, resulting in a decrease in its content and activity.

[0007] (2) Low bioavailability: The absorption of theaflavins in the gastrointestinal tract is relatively poor after oral administration. The polyphenolic hydroxyl groups in its molecular structure easily combine with proteins, metal ions, etc. in the gastrointestinal tract to form complexes, thereby reducing the bioaccessibility and absorption rate of theaflavins, making it difficult for them to fully exert their anti-aging and other physiological effects in the body.

[0008] (3) There may be incompatibility: In some cases, theaflavins alone may not achieve the desired comprehensive effect and need to be used in combination with other drugs or health products. However, theaflavins may interact with certain drugs or ingredients, affecting their absorption, distribution, metabolism and excretion. For example, when used with iron-containing drugs, they may form insoluble complexes, affecting iron absorption.

[0009] Therefore, it is necessary to develop more stable theaflavins combination ingredients that are not easily oxidized, have high absorption and utilization rates, and are safe and efficient, so as to improve the utilization of theaflavins and enhance the anti-aging effect. Summary of the Invention

[0010] The efficacy of existing anti-aging products varies widely, with some commonly experiencing ineffective results. The use of theaflavins also suffers from poor stability, low bioavailability, and poor compatibility. The present invention provides an anti-aging theaflavin composition, its preparation method, and its application. Nanocrystalline theaflavin-metal organic frameworks are prepared and coated with a γ-cyclodextrin-tannic acid copolymer to improve the stability and bioavailability of theaflavins. Combined with a mitochondrially targeted coenzyme Q10-L-selenium-methylselenocysteine complex, the composition exhibits synergistic anti-aging effects, effectively enhancing the efficacy of theaflavins. The specific technical solution is as follows:

[0011] An anti-aging theaflavin composition comprises the following raw materials in parts by mass: 20 to 25 parts of a coated modified nanocrystalline theaflavin-metal organic framework complex and 5 to 8 parts of a mitochondrial targeted coenzyme Q10-L-selenium-methylselenocysteine complex; the coated modified nanocrystalline theaflavin-metal organic framework complex comprises a nanocrystalline theaflavin-metal organic framework complex having its surface coated with a γ-cyclodextrin-tannic acid copolymer.

[0012] In the above composition, the preparation of the nanocrystalline theaflavin-metal organic framework complex includes: adding theaflavin to an ethanol aqueous solution and stirring to mix; then adding a Zn(NO3)2·6H2O aqueous solution and a 2-methylimidazole aqueous solution, stirring to react, and forming a reaction solution with a white precipitate; centrifuging to collect the white precipitate, washing with a washing solution, and drying under reduced pressure to a constant weight to obtain the nanocrystalline theaflavin-metal organic framework complex.

[0013] In the preparation of the nanocrystalline theaflavin-metal organic framework complex, the amount of theaflavin is 20g to 30g; the amount of the ethanol aqueous solution is 200mL to 250mL; the volume concentration of the ethanol aqueous solution is 65% to 75%; the amount of the Zn(NO3)2·6H2O aqueous solution is 50mL to 60mL; the concentration of the Zn(NO3)2·6H2O aqueous solution is 0.1mol / L to 0.15mol / L; the amount of the 2-methylimidazole aqueous solution is 50mL to 60mL. mL; the concentration of the 2-methylimidazole aqueous solution is 0.4 mol / L to 0.6 mol / L; the stirring reaction is carried out at 45°C to 50°C at a speed of 150 r / min to 200 r / min for 10 h to 15 h; the centrifugation is carried out at 6000 r / min to 8000 r / min for 15 min to 20 min; the washing solution is an ethanol aqueous solution with a volume concentration of 50% to 60%; the number of washing times is 3 to 5 times; and the temperature of the reduced pressure drying is 40°C to 50°C.

[0014] In the above-mentioned composition, the preparation of the γ-cyclodextrin-tannic acid copolymer includes: adding γ-cyclodextrin and tannic acid into deionized water and stirring and mixing; adjusting the pH to 5.0±0.2 with a hydrochloric acid aqueous solution and stirring for reaction; maintaining the pH at 5.0±0.2 by dropwise adding a sodium hydroxide aqueous solution during the reaction; after the reaction is completed, removing the solvent by evaporation under reduced pressure, and drying under reduced pressure to a constant weight to obtain the γ-cyclodextrin-tannic acid copolymer.

[0015] In the preparation of the above-mentioned γ-cyclodextrin-tannic acid copolymer, the amount of γ-cyclodextrin used is 8g to 10g; the amount of tannic acid used is 2g to 2.5g; the amount of deionized water used is 100mL to 150mL; the concentration of the hydrochloric acid aqueous solution is 0.1mol / L to 0.15mol / L; the stirring reaction is carried out at 60°C to 65°C and a speed of 100r / min to 150r / min for 2h to 3h; the concentration of the sodium hydroxide aqueous solution is 0.1mol / L to 0.15mol / L; the temperature of the reduced pressure evaporation is 40°C to 50°C; and the temperature of the reduced pressure drying is 40°C to 50°C.

[0016] In the above composition, the preparation of the mitochondrial targeted coenzyme Q10-L-seleno-methylselenocysteine complex includes: under nitrogen protection, adding coenzyme Q10 and L-seleno-methylselenocysteine to dichloromethane and stirring to mix; then adding N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine in sequence, stirring to react, and obtaining a reaction solution; filtering to remove the white precipitate (including the by-product dicyclohexylurea), concentrating the filtrate to obtain a concentrated solution; adding the concentrated solution dropwise to cold ether to precipitate; centrifuging, taking the precipitate, washing with detergent, and drying under reduced pressure to constant weight to obtain a mitochondrial targeted coenzyme Q10-L-seleno-methylselenocysteine complex.

[0017] In the preparation of the above-mentioned mitochondrial targeted coenzyme Q10-L-seleno-methylselenocysteine complex, the amount of coenzyme Q10 is 4g to 6g; the amount of L-seleno-methylselenocysteine is 2g to 3g; the amount of dichloromethane is 100mL to 120mL; the amount of N,N-dicyclohexylcarbodiimide is 2g to 3g; the amount of 4-dimethylaminopyridine is 0.3g to 0.5g; the stirring reaction is carried out at 60°C to 65°C and a speed of 100r / min to 150r / min for 6h ~8h; the concentration is to concentrate by reduced pressure evaporation at 40℃~50℃ to 30%~40% of the original volume; the temperature of the cold ether is 4℃~10℃; the amount of the cold ether is 500mL~600mL; the centrifugation is to centrifuge at 5000r / min~6000r / min for 10min~15min; the detergent is 4℃~10℃ cold ether and deionized water; the number of washings with the cold ether is 3 times to 5 times, and the number of washings with the deionized water is 3 times to 5 times; the temperature of the reduced pressure drying is 35℃~40℃.

[0018] The method for preparing the anti-aging theaflavins composition comprises the following steps:

[0019] S1: Dispersing the nanocrystalline theaflavin-metal organic framework complex in phosphate buffer containing 2 wt% to 3 wt% of γ-cyclodextrin-tannic acid copolymer at 8 to 10 times its mass to form a suspension; first ultrasonically treating, then high-pressure homogenizing to obtain the nanocrystalline theaflavin-metal organic framework complex with the surface uniformly coated with the γ-cyclodextrin-tannic acid copolymer; evaporating under reduced pressure to remove the solvent; and drying under reduced pressure to constant weight to obtain the coated modified nanocrystalline theaflavin-metal organic framework complex;

[0020] S2: Mixing the coated modified nanocrystalline theaflavin-metal organic framework complex and the mitochondria-targeted coenzyme Q10-L-selenium-methylselenocysteine complex according to parts by mass to obtain a composition.

[0021] In the preparation method of the above-mentioned composition, the pH value of the phosphate buffer is 5.0±0.2; the ultrasonic treatment is performed at a power of 250W to 300W for 10min to 15min; the high-pressure homogenization is performed at a high-pressure homogenization cycle of 100MPa to 120MPa for 4 to 6 times, with an interval of 30s to 40s between each cycle; the temperature of the reduced pressure evaporation is 40°C to 50°C; and the temperature of the reduced pressure drying is 40°C to 50°C.

[0022] The application of the anti-aging theaflavin composition in medicines and foods with anti-aging effects.

[0023] The anti-aging theaflavins composition of the present invention, its preparation method and application, have the following beneficial effects:

[0024] 1. Theaflavins themselves have anti-aging effects such as antioxidant, anti-inflammatory, and metabolic regulation, but there are problems such as low stability and bioavailability. The coated modified nanocrystalline theaflavins-metal organic framework complex solves these problems and can better exert the efficacy of theaflavins. In the mitochondrial-targeted coenzyme Q10-L-selenium-methylselenocysteine complex, coenzyme Q10 is involved in cellular energy metabolism and antioxidant, L-selenium-methylselenocysteine supplements selenium and enhances antioxidant capacity, and the complex achieves mitochondrial targeting and can play a more precise role in the cell's energy factory. The two work synergistically to fight cellular aging from multiple aspects. In terms of antioxidant, they can not only scavenge free radicals but also enhance the cell's own antioxidant capacity; in terms of cell function maintenance, they can not only protect cell membrane integrity but also ensure mitochondrial energy supply, thereby achieving a better anti-aging effect.

[0025] If too little of the mitochondrial-targeted Coenzyme Q10-L-Selenium-Methylselenocysteine complex is added, the improvement in cellular energy metabolism and antioxidant capacity will be limited, preventing it from fully exerting its role in enhancing cell vitality and reducing oxidative damage. This will result in a weak overall anti-aging effect, limiting the ability of the theaflavins complex to maintain normal cellular function and slow the aging process. Excessive addition can cause adverse reactions, including disruption of normal metabolic balance within cells, as excess Coenzyme Q10 and selenium can affect the function or metabolic pathways of other biomolecules. Furthermore, excessive amounts of the complex can increase the body's metabolic burden.

[0026] Second, metal-organic frameworks (MOFs) possess a porous structure and large specific surface area, capable of supporting large quantities of theaflavins, thereby protecting them. Embedding theaflavins within MOFs reduces their exposure to external light, heat, and oxygen, lowering the likelihood of oxidation and isomerization reactions, thereby enhancing their stability. Furthermore, the MOF structure helps alter the form of theaflavins in the gastrointestinal tract, reducing their binding to proteins and metal ions. This in turn improves the bioaccessibility and absorption of theaflavins, enabling them to better exert their anti-aging and other physiological effects.

[0027] Third, a γ-cyclodextrin-tannic acid copolymer is coated on the surface of the theaflavin-MOF complex, further enhancing its stability. The copolymer coating acts as a physical barrier, preventing damage to the theaflavin-MOF complex from external factors and reducing oxidation and other reactions. Furthermore, this copolymer improves the complex's dispersibility and solubility in the body, facilitating its absorption in the gastrointestinal tract and thus increasing its bioavailability.

[0028] γ-cyclodextrin has a unique cyclic structure, and its inner cavity can accommodate small molecules such as theaflavins, acting as an inclusion complex, further protecting theaflavins from environmental influences and enhancing their stability. γ-cyclodextrin also improves the solubility and bioavailability of the included substances, aiding the absorption and transport of the theaflavins-MOF complex within the body. Tannic acid, with its multiple phenolic hydroxyl groups, possesses considerable antioxidant capacity, synergizing with theaflavins to enhance the antioxidant effect of the complex. Furthermore, tannic acid forms a copolymer with γ-cyclodextrin, and through intermolecular interactions, the coating becomes tighter and more stable, further enhancing the protective effect of the theaflavins-MOF complex.

[0029] Fourth, the Coenzyme Q10-L-Selenium-Methylselenocysteine complex offers advantages over a mixture of Coenzyme Q10 and L-Selenium-Methylselenocysteine: the chemical reaction that forms the complex alters its molecular structure and properties. The complex achieves mitochondrial targeting, enabling more precise delivery of Coenzyme Q10 and L-Selenium-Methylselenocysteine to the mitochondria. In a mixture, the random distribution of the two makes it difficult for them to be concentrated in the mitochondria. Furthermore, the synergistic effects between the components of the complex make its antioxidant and cellular energy metabolism-enhancing effects more pronounced than a simple mixture, resulting in a more effective anti-aging effect. DETAILED DESCRIPTION

[0030] Example 1

[0031] An anti-aging theaflavin composition comprises the following raw materials in parts by mass: 23 parts of a coated modified nanocrystalline theaflavin-metal organic framework complex and 7 parts of a mitochondrial targeted coenzyme Q10-L-selenium-methylselenocysteine complex; the coated modified nanocrystalline theaflavin-metal organic framework complex is a nanocrystalline theaflavin-metal organic framework complex having a surface coated with a γ-cyclodextrin-tannic acid copolymer.

[0032] In the above composition, the preparation of the nanocrystalline theaflavin-metal organic framework complex includes: adding 22g of theaflavin to 230mL of 70% volume concentration ethanol aqueous solution and stirring to mix; then adding 55mL of 0.12mol / L concentration of Zn(NO3)2·6H2O aqueous solution and 55mL of 0.5mol / L concentration of 2-methylimidazole aqueous solution, stirring and reacting at 48°C and 180r / min speed for 12h to form a reaction solution with a white precipitate; centrifuging at 7000r / min for 18min, collecting the white precipitate, washing it 4 times with 55% volume concentration ethanol aqueous solution, and drying under reduced pressure at 45°C to constant weight to obtain the nanocrystalline theaflavin-metal organic framework complex.

[0033] In the above-mentioned composition, the preparation of the γ-cyclodextrin-tannic acid copolymer includes: adding 9g of γ-cyclodextrin and 2.3g of tannic acid to 120mL of deionized water and stirring and mixing; adjusting the pH to 5.0 with a 0.12mol / L hydrochloric acid aqueous solution, and stirring and reacting at 62°C and a speed of 120r / min for 2.5h; maintaining the pH at 5.0±0.2 by dropwise adding a 0.12mol / L sodium hydroxide aqueous solution during the reaction; after the reaction is completed, removing the solvent by evaporation under reduced pressure at 45°C, and drying under reduced pressure at 45°C to constant weight to obtain the γ-cyclodextrin-tannic acid copolymer.

[0034] In the above composition, the preparation of the mitochondrial targeted coenzyme Q10-L-selenium-methylselenocysteine complex comprises: adding 5g of coenzyme Q10 and 2.5g of L-selenium-methylselenocysteine to 110ml of water under nitrogen protection; L dichloromethane and stirred to mix; then, 2.5 g N,N-dicyclohexylcarbodiimide and 0.4 g 4-dimethylaminopyridine were added in sequence, and the mixture was stirred at 62°C and 120 r / min for 7 hours to obtain a reaction solution; the white precipitate (including the by-product dicyclohexylurea) was removed by filtration, and the filtrate was concentrated by evaporation under reduced pressure at 45°C to 35% of the original volume to obtain a concentrated solution; the concentrated solution was added dropwise to 550 mL 6°C cold ether to precipitate; centrifuged at 5500 r / min for 12 minutes, the precipitate was taken, washed four times with 6°C cold ether, then washed four times with deionized water, and dried under reduced pressure at 38°C to constant weight to obtain a mitochondrial-targeted coenzyme Q10-L-seleno-methylselenocysteine complex.

[0035] The method for preparing the anti-aging theaflavins composition comprises the following steps:

[0036] S1: Disperse the nanocrystalline theaflavin-metal organic framework complex in 9 times the mass of phosphate buffer (pH 5.0) containing 2.5wt% of γ-cyclodextrin-tannic acid copolymer to form a suspension; first ultrasonically treat at 250W power for 12 minutes, then homogenize under 100MPa high pressure for 5 cycles, with an interval of 35s between each cycle; obtain the nanocrystalline theaflavin-metal organic framework complex with the surface uniformly coated with γ-cyclodextrin-tannic acid copolymer, evaporate under reduced pressure at 45°C to remove the solvent, and dry under reduced pressure at 45°C to constant weight to obtain the coated modified nanocrystalline theaflavin-metal organic framework complex;

[0037] S2: Mixing the coated modified nanocrystalline theaflavin-metal organic framework complex and the mitochondria-targeted coenzyme Q10-L-selenium-methylselenocysteine complex according to parts by mass to obtain a composition.

[0038] The application of the anti-aging theaflavin composition in medicines and foods with anti-aging effects.

[0039] Example 2

[0040] An anti-aging theaflavin composition comprises the following raw materials in parts by mass: 20 parts of a coated modified nanocrystalline theaflavin-metal organic framework complex and 8 parts of a mitochondrial-targeted coenzyme Q10-L-selenium-methylselenocysteine complex; the coated modified nanocrystalline theaflavin-metal organic framework complex comprises a nanocrystalline theaflavin-metal organic framework complex having its surface coated with a γ-cyclodextrin-tannic acid copolymer.

[0041] In the above composition, the preparation of the nanocrystalline theaflavin-metal organic framework complex includes: adding 20g of theaflavin to 250mL of 65% volume concentration ethanol aqueous solution and stirring to mix; then adding 60mL of 0.1mol / L concentration of Zn(NO3)2·6H2O aqueous solution and 60mL of 0.4mol / L concentration of 2-methylimidazole aqueous solution, stirring and reacting at 50°C and 150r / min speed for 15h to form a reaction solution with a white precipitate; centrifuging at 6000r / min for 20min, collecting the white precipitate, washing it 5 times with 50% volume concentration ethanol aqueous solution, and drying under reduced pressure at 40°C to constant weight to obtain the nanocrystalline theaflavin-metal organic framework complex.

[0042] In the above composition, the preparation of the γ-cyclodextrin-tannic acid copolymer includes: adding 8g of γ-cyclodextrin and 2.5g of tannic acid to 100mL of deionized water and stirring and mixing; adjusting the pH to 4.8 with a 0.15mol / L hydrochloric acid aqueous solution, and stirring and reacting at 65°C and a speed of 100r / min for 3h; maintaining the pH at 5.0±0.2 by dropwise adding a 0.1mol / L sodium hydroxide aqueous solution during the reaction; after the reaction is completed, removing the solvent by evaporation under reduced pressure at 40°C, and drying under reduced pressure at 50°C to constant weight to obtain the γ-cyclodextrin-tannic acid copolymer.

[0043] In the above composition, the preparation of the mitochondrial targeted coenzyme Q10-L-seleno-methylselenocysteine complex includes: under nitrogen protection, adding 6g coenzyme Q10 and 2g L-seleno-methylselenocysteine to 120mL of dichloromethane and stirring to mix; then adding 2g N,N-dicyclohexylcarbodiimide and 0.5g 4-dimethylaminopyridine in sequence, stirring and reacting at 60°C and 150r / min for 6h to obtain a reaction solution; filtering to remove the white precipitate (including the by-product dicyclohexylurea), and concentrating the filtrate by reduced pressure evaporation at 50°C to 40% of the original volume to obtain a concentrated solution; the concentrated solution is added dropwise to 500mL of 10°C cold ether, and a precipitate is precipitated; centrifugation is performed at 5000r / min for 15min, the precipitate is taken, washed 5 times with 10°C cold ether, then washed 3 times with deionized water, and dried at 35°C under reduced pressure to constant weight to obtain a mitochondrial targeted coenzyme Q10-L-seleno-methylselenocysteine complex.

[0044] The method for preparing the anti-aging theaflavins composition comprises the following steps:

[0045] S1: Disperse the nanocrystalline theaflavin-metal organic framework complex in phosphate buffer (pH 4.8) containing 3 wt% of γ-cyclodextrin-tannic acid copolymer by 8 times its mass to form a suspension; firstly ultrasonicate at 300W power for 10 min, then homogenize under 120MPa high pressure for 4 cycles, with an interval of 40s between each cycle; obtain the nanocrystalline theaflavin-metal organic framework complex with the surface uniformly coated with γ-cyclodextrin-tannic acid copolymer, evaporate under reduced pressure at 40°C to remove the solvent, and dry under reduced pressure at 50°C to constant weight to obtain the coated modified nanocrystalline theaflavin-metal organic framework complex;

[0046] S2: Mixing the coated modified nanocrystalline theaflavin-metal organic framework complex and the mitochondria-targeted coenzyme Q10-L-selenium-methylselenocysteine complex according to parts by mass to obtain a composition.

[0047] The application of the anti-aging theaflavin composition in medicines and foods with anti-aging effects.

[0048] Example 3

[0049] An anti-aging theaflavin composition comprises the following raw materials in parts by mass: 25 parts of a coated modified nanocrystalline theaflavin-metal organic framework complex and 5 parts of a mitochondrial-targeted coenzyme Q10-L-selenium-methylselenocysteine complex; the coated modified nanocrystalline theaflavin-metal organic framework complex comprises a nanocrystalline theaflavin-metal organic framework complex having its surface coated with a γ-cyclodextrin-tannic acid copolymer.

[0050] In the above composition, the preparation of the nanocrystalline theaflavin-metal organic framework complex includes: adding 30g of theaflavin to 200mL of 75% volume concentration ethanol aqueous solution and stirring to mix; then adding 50mL of 0.15mol / L concentration of Zn(NO3)2·6H2O aqueous solution and 50mL of 0.6mol / L concentration of 2-methylimidazole aqueous solution, stirring and reacting at 45°C and 200r / min speed for 10h to form a reaction solution with a white precipitate; centrifuging at 8000r / min for 15min, collecting the white precipitate, washing it three times with 60% volume concentration ethanol aqueous solution, and drying under reduced pressure at 50°C to constant weight to obtain the nanocrystalline theaflavin-metal organic framework complex.

[0051] In the above-mentioned composition, the preparation of the γ-cyclodextrin-tannic acid copolymer includes: adding 10g of γ-cyclodextrin and 2g of tannic acid to 150mL of deionized water and stirring and mixing; adjusting the pH to 5.2 with a 0.1mol / L hydrochloric acid aqueous solution, and stirring and reacting at 60°C and a speed of 150r / min for 2h; maintaining the pH at 5.0±0.2 by dropwise adding a 0.15mol / L sodium hydroxide aqueous solution during the reaction; after the reaction is completed, removing the solvent by evaporation under reduced pressure at 50°C, and drying under reduced pressure at 40°C to constant weight to obtain the γ-cyclodextrin-tannic acid copolymer.

[0052] In the above composition, the preparation of the mitochondrial targeted coenzyme Q10-L-seleno-methylselenocysteine complex includes: under nitrogen protection, adding 4g coenzyme Q10 and 3g L-seleno-methylselenocysteine to 100mL of dichloromethane and stirring to mix; then adding 3g N,N-dicyclohexylcarbodiimide and 0.3g 4-dimethylaminopyridine in sequence, stirring and reacting at 65°C and 100r / min for 8h to obtain a reaction solution; filtering to remove the white precipitate (including the by-product dicyclohexylurea), and concentrating the filtrate by reduced pressure evaporation at 40°C to 30% of the original volume to obtain a concentrated solution; the concentrated solution is added dropwise to 600mL of 4°C cold ether, and a precipitate is precipitated; centrifugation is performed at 6000r / min for 10min, the precipitate is taken, washed 3 times with 4°C cold ether, then washed 5 times with deionized water, and dried at 40°C under reduced pressure to constant weight to obtain a mitochondrial targeted coenzyme Q10-L-seleno-methylselenocysteine complex.

[0053] The method for preparing the anti-aging theaflavins composition comprises the following steps:

[0054] S1: Disperse the nanocrystalline theaflavin-metal organic framework complex in phosphate buffer (pH 5.2) containing 2 wt% of γ-cyclodextrin-tannic acid copolymer at 10 times the mass to form a suspension; firstly ultrasonicate at 250W power for 15 minutes, then homogenize under 100MPa high pressure for 6 cycles, with an interval of 30 seconds between each cycle; obtain the nanocrystalline theaflavin-metal organic framework complex with the surface uniformly coated with γ-cyclodextrin-tannic acid copolymer, evaporate under reduced pressure at 50°C to remove the solvent, and dry under reduced pressure at 40°C to constant weight to obtain the coated modified nanocrystalline theaflavin-metal organic framework complex;

[0055] S2: Mixing the coated modified nanocrystalline theaflavin-metal organic framework complex and the mitochondria-targeted coenzyme Q10-L-selenium-methylselenocysteine complex according to parts by mass to obtain a composition.

[0056] The application of the anti-aging theaflavin composition in medicines and foods with anti-aging effects.

[0057] In the above embodiments, the purity of theaflavins is 60 wt%; γ-cyclodextrin is sourced from Aisen Biotechnology (Shanxi) Co., Ltd., hydroxypropyl gamma-cyclodextrin; tannic acid is sourced from Anhui Weimao Biotechnology Co., Ltd., with a purity of 95 wt%; coenzyme Q10 is sourced from Shaanxi Yunhe Biotechnology Co., Ltd., and is food grade; L-selenium-methylselenocysteine is sourced from Shandong Tangzheng Biotechnology Co., Ltd., and is food grade.

[0058] Comparative Example 1

[0059] In the composition, the amount of the mitochondrial-targeted coenzyme Q10-L-seleno-methylselenocysteine complex added is 2 parts; other parameters and methods are the same as in Example 1.

[0060] Comparative Example 2

[0061] In the composition, the amount of the mitochondrial-targeted coenzyme Q10-L-seleno-methylselenocysteine complex added is 15 parts; other parameters and methods are the same as in Example 1.

[0062] Comparative Example 3

[0063] In the composition, the coated modified nanocrystalline theaflavin-metal organic framework complex is replaced by theaflavin; other parameters and methods are the same as in Example 1.

[0064] Comparative Example 4

[0065] In the composition, the coated modified nanocrystalline theaflavin-metal organic framework complex is replaced by the nanocrystalline theaflavin-metal organic framework complex (without γ-cyclodextrin-tannic acid copolymer coating); other parameters and methods are the same as in Example 1.

[0066] Comparative Example 5

[0067] No γ-cyclodextrin was added to the coated modified nanocrystalline theaflavin-metal organic framework complex; other parameters and methods were the same as in Example 1.

[0068] Comparative Example 6

[0069] In the coated modified nanocrystalline theaflavin-metal organic framework complex, no tannic acid was added; other parameters and methods were the same as in Example 1.

[0070] Comparative Example 7

[0071] The coenzyme Q10-L-seleno-methylselenocysteine complex was replaced by a mixture of coenzyme Q10 and L-seleno-methylselenocysteine; other parameters and methods were the same as in Example 1.

[0072] 1. Stability testing

[0073] The coated modified nanocrystalline theaflavin-metal organic framework complexes in Examples 1 to 3 are recorded as C1 to C3; the theaflavin in Comparative Example 3 is recorded as D1; the nanocrystalline theaflavin-metal organic framework complex in Comparative Example 4 is replaced (without γ-cyclodextrin-tannic acid copolymer coating) and recorded as D2; the coated modified nanocrystalline theaflavin-metal organic framework complex in Comparative Example 5 (without adding γ-cyclodextrin) is recorded as D3; the coated modified nanocrystalline theaflavin-metal organic framework complex in Comparative Example 6 (without adding tannic acid) is recorded as D4.

[0074] 5g of each of substances C1 to C3 and D1 to D4 was placed in a 50mL brown wide-mouth glass bottle and sealed. The bottles were placed in a constant temperature and humidity incubator with a light intensity of 3000 lux and a temperature of 40°C. On the 7th day, 0.1g of sample was accurately weighed from each sample bottle. The theaflavin content was determined by high-performance liquid chromatography (HPLC). Theaflavin retention rate = (theaflavin content at each time point / initial theaflavin content) × 100%. The test results are shown in Table 1 below.

[0075] Table 1 Theaflavin 7-day retention rate results

[0076] Sample Theaflavins 7-day retention rate (%) Sample Theaflavins 7-day retention rate (%) C1 89.64 D1 61.82 C2 88.38 D2 69.53 C3 91.21 D3 78.39 D4 75.65

[0077] 2. Bioavailability (Theaflavins content in animal blood)

[0078] A total of 80 healthy male BALB / c mice aged 6-8 weeks and weighing 18-25g were selected and randomly divided into 10 groups (3 groups for each embodiment and 7 groups for each comparative example), with 8 mice in each group. The compositions of each embodiment and comparative example were respectively formulated into a suspension with a concentration of 10 mg / mL, and the mice were gavage-administered at a dose of 10 mL / kg. Blood was collected from the orbital venous plexus at 0.5h, 1h, 2h, and 4h after administration, with about 0.3 mL of blood collected each time. The blood was placed in an anticoagulant tube containing sodium heparin, centrifuged at 4°C, 3000r / min for 10min, and the plasma was separated and stored at -80°C for testing. Liquid chromatography-mass spectrometry (LC-MS) was used to detect the concentration of theaflavins in plasma. Quantitative analysis was performed in multiple reaction monitoring (MRM) mode, and the quantitative ion pair of theaflavins was m / z 568.2-406.1. The test results are shown in Table 2 below.

[0079] Table 2 Theaflavin content results (average value)

[0080]

[0081] 3. Anti-aging effect testing

[0082] Human skin fibroblasts (HSF) were revived and placed in DMEM medium containing 10% fetal bovine serum and 1% double-antibody (penicillin-streptomycin) and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were digested with 0.25% trypsin and plated at 5×10 4 Cells were seeded into 24-well plates at a concentration of 100 μg / mL per well and cultured for 24 hours to allow the cells to adhere. The compositions of the Examples and Comparative Examples were diluted with culture medium to a 100 μg / mL solution. The original culture medium in the 24-well plates was replaced. Three replicate wells were set up for each sample. A blank control group (containing only culture medium) and a positive control group (treated with 10 μmol / L resveratrol) were also set up. Culture was continued for 48 hours.

[0083] Reactive oxygen species (ROS) level assay: Aspirate the culture medium from the wells, wash twice with PBS, add 10 μmol / L DCFH-DA fluorescent probe working solution, and incubate at 37°C in the dark for 20 min. Wash three times with PBS, add 200 μL of trypsin to digest the cells, collect the cell suspension, and analyze the ROS level using flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The mean fluorescence intensity is used to represent the relative ROS level.

[0084] Mitochondrial membrane potential assay: Aspirate the culture medium from the wells, wash twice with PBS, add 10 μg / mL JC-1 fluorescent probe working solution, and incubate at 37°C in the dark for 20 min. Wash twice with PBS, add 200 μL of trypsin to digest the cells, collect the cell suspension, and analyze by flow cytometry. The excitation wavelength for JC-1 monomer is 488 nm, and the emission wavelength is 530 nm; the excitation wavelength for JC-1 aggregate is 514 nm, and the emission wavelength is 585 nm. The relative level of mitochondrial membrane potential is expressed as the ratio of the fluorescence intensity of JC-1 aggregate to that of monomer.

[0085] Cell apoptosis rate detection: aspirate the culture medium in the wells, wash twice with PBS, add 0.25% trypsin to digest the cells, collect the cell suspension, centrifuge at 4°C, 1500 rpm for 5 minutes, discard the supernatant, and resuspend the cells in a Binding Buffer to a concentration of 1×10 6 Cells were measured at 400 μL / mL. 100 μL of cell suspension was added with 5 μL of Annexin V-FITC and 5 μL of PI. The cells were incubated at room temperature in the dark for 15 min. 400 μL of Binding Buffer was added and the cells were analyzed by flow cytometry. Cells that were positive for Annexin V-FITC and negative for PI were considered to be in the early stage of apoptosis, while cells that were positive for both Annexin V-FITC and PI were considered to be in the late stage of apoptosis. The cell apoptosis rate (%) = (number of early apoptotic cells + number of late apoptotic cells) / total number of cells × 100%. The test results are shown in Table 3 below.

[0086] Table 3 Anti-aging test results

[0087] Sample ROS relative level (%) Relative level of mitochondrial membrane potential (%) Cell apoptosis rate (%) Example 1 49.74 79.83 9.80 Example 2 51.91 78.25 11.71 Example 3 48.37 82.14 8.56 Comparative Example 1 55.32 74.68 14.90 Comparative Example 2 53.63 75.81 13.27 Comparative Example 3 70.15 59.32 20.34 Comparative Example 4 60.21 69.74 17.85 Comparative Example 5 64.50 64.85 15.93 Comparative Example 6 62.13 67.57 16.80 Comparative Example 7 54.85 75.62 13.09

[0088] From the above results, it can be seen that the coated modified nanocrystalline theaflavin-metal organic framework complex has better stability and protects theaflavin from oxidation, isomerization, etc.; the compositions of Examples 1 to 3 have good theaflavin utilization rate and obvious anti-aging effect.

[0089] In Comparative Example 1, the amount of the mitochondrially targeted Coenzyme Q10-L-Selenium-Methylselenocysteine complex added was only 2 parts, a significant reduction compared to Example 1. From a synergistic mechanism perspective, this complex exhibits a synergistic relationship with the coated modified nanocrystalline theaflavin-metal organic framework complex in exerting its anti-aging effects. However, if added at too low a level, it would not fully synergize with the theaflavin complex, resulting in insufficient activation of the intracellular antioxidant system and a decrease in various indices.

[0090] In Comparative Example 2, the addition amount of mitochondrial-targeted coenzyme Q10-L-seleno-methylselenocysteine complex was increased to 15 parts. The excessive amount of complex broke the balance between the complex and the theaflavins, which affected the stability of the system to a certain extent. This caused the theaflavins content to be doubled, and the synergistic imbalance resulted in an insignificant improvement in bioavailability.

[0091] Comparative Example 3 directly uses theaflavins instead of coating modified nanocrystalline theaflavins-MOF complexes. Regarding bioavailability, the polyphenolic hydroxyl groups in theaflavins' molecular structure readily bind to proteins and metal ions in the gastrointestinal tract, forming complexes that reduce their bioavailability. Without the protection of the MOF and coating copolymer, various biochemical indicators decline.

[0092] Comparative Example 4 did not coat the nanocrystalline theaflavin-metal-organic framework complex with γ-cyclodextrin-tannic acid copolymer. By forming an inclusion structure and physical barrier, the γ-cyclodextrin-tannic acid copolymer effectively isolates the theaflavins from the effects of light, oxygen, and other factors. Furthermore, the copolymer competes with substances in the gastrointestinal tract for binding sites, reducing theaflavins' binding to other substances, promoting their absorption, and increasing their bioavailability. Without the γ-cyclodextrin-tannic acid copolymer coating, the availability of elemental theaflavins decreased, leading to a decrease in other biomarkers.

[0093] Comparative Example 5 did not include γ-cyclodextrin. γ-cyclodextrin, with its unique cyclic structure, forms a stable inclusion complex with theaflavins, increasing their water solubility and stability. However, without γ-cyclodextrin, theaflavins are susceptible to aggregation and oxidation in solution. Furthermore, its solubilization of theaflavins is impaired, impairing their dispersion and absorption in the gastrointestinal tract and reducing their bioavailability. Due to the insufficient effective intracellular theaflavins, other antioxidant indicators decrease.

[0094] Comparative Example 6 did not include tannic acid. Tannic acid contains multiple phenolic hydroxyl groups, which bind to theaflavins through hydrogen bonds and hydrophobic interactions, enhancing their stability. Tannic acid also has a certain antiprotease effect, reducing theaflavins' degradation by proteases in the gastrointestinal tract and promoting their absorption. The absence of tannic acid reduces theaflavins' stability and bioavailability, leading to a decrease in various bioavailability indicators.

[0095] Comparative Example 7 uses a mixture of Coenzyme Q10 and L-seleno-methylselenocysteine in place of the mitochondrially targeted Coenzyme Q10-L-seleno-methylselenocysteine complex. This complex, formed through chemical bonds to form a specific structure, has clear mitochondrial targeting, enabling precise delivery of Coenzyme Q10 and selenocysteine to the mitochondria, exerting a synergistic antioxidant and mitochondrial protective effect. However, the random distribution of the components in the mixture prevents effective mitochondrial targeting, reducing the anti-aging properties of theaflavins.

Claims

1. An anti-aging theaflavin composition, characterized in that: The composition comprises the following raw materials in parts by mass: 20 to 25 parts of a coated modified nanocrystalline theaflavin-metal organic framework complex and 5 to 8 parts of a mitochondrial targeted coenzyme Q10-L-selenium-methylselenocysteine complex; the coated modified nanocrystalline theaflavin-metal organic framework complex is a nanocrystalline theaflavin-metal organic framework complex whose surface is coated with a γ-cyclodextrin-tannic acid copolymer.

2. The anti-aging theaflavin composition according to claim 1, characterized in that: The preparation of the nanocrystalline theaflavin-metal organic framework complex comprises: adding theaflavin to an ethanol aqueous solution and stirring and mixing; then adding a Zn(NO3)2·6H2O aqueous solution and a 2-methylimidazole aqueous solution, stirring and reacting to form a reaction solution with a white precipitate; centrifuging to collect the white precipitate, washing with a washing solution, and drying under reduced pressure to a constant weight to obtain the nanocrystalline theaflavin-metal organic framework complex.

3. The anti-aging theaflavin composition according to claim 2, characterized in that: The amount of theaflavins used is 20g to 30g; the amount of the ethanol aqueous solution used is 200mL to 250mL; the volume concentration of the ethanol aqueous solution is 65% to 75%; the amount of the Zn(NO3)2·6H2O aqueous solution used is 50mL to 60mL; the concentration of the Zn(NO3)2·6H2O aqueous solution is 0.1mol / L to 0.15mol / L; the amount of the 2-methylimidazole aqueous solution used is 50mL to 60mL; the concentration of the 2-methylimidazole aqueous solution is 0.4mol / L to 0.6mol / L; the stirring reaction is carried out at 45℃ to 50℃ and a speed of 150r / min to 200r / min for 10h to 15h; the centrifugation is carried out at 6000r / min to 8000r / min for 15min to 20min; the washing solution is an ethanol aqueous solution with a volume concentration of 50% to 60%; The washing is performed 3 to 5 times; and the reduced pressure drying temperature is 40° C. to 50° C.

4. The anti-aging theaflavin composition according to claim 1, characterized in that: The preparation of the γ-cyclodextrin-tannic acid copolymer comprises: adding γ-cyclodextrin and tannic acid into deionized water and stirring and mixing; adjusting the pH to 5.0±0.2 with a hydrochloric acid aqueous solution and stirring for reaction; maintaining the pH at 5.0±0.2 by dropwise adding a sodium hydroxide aqueous solution during the reaction; and after completion of the reaction, removing the solvent by evaporation under reduced pressure, and drying under reduced pressure to a constant weight to obtain the γ-cyclodextrin-tannic acid copolymer.

5. The anti-aging theaflavin composition according to claim 4, characterized in that: The amount of γ-cyclodextrin used is 8g to 10g; the amount of tannic acid used is 2g to 2.5g; the amount of deionized water used is 100mL to 150mL; the concentration of the hydrochloric acid aqueous solution is 0.1mol / L to 0.15mol / L; the stirring reaction is carried out at 60°C to 65°C and a speed of 100r / min to 150r / min for 2h to 3h; the concentration of the sodium hydroxide aqueous solution is 0.1mol / L to 0.15mol / L; the temperature of the reduced pressure evaporation is 40°C to 50°C; and the temperature of the reduced pressure drying is 40°C to 50°C.

6. The anti-aging theaflavin composition according to claim 1, characterized in that: The preparation of the mitochondrial-targeted coenzyme Q10-L-seleno-methylselenocysteine complex includes: adding coenzyme Q10 and L-seleno-methylselenocysteine to dichloromethane under nitrogen protection, and stirring to mix; then sequentially adding N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, stirring to react, and obtaining a reaction solution; filtering to remove a white precipitate, and concentrating the filtrate to obtain a concentrated solution; dropwise adding the concentrated solution to cold ether to precipitate; and centrifuging to obtain a precipitate, washing with a detergent, and drying under reduced pressure to a constant weight to obtain a mitochondrial-targeted coenzyme Q10-L-seleno-methylselenocysteine complex.

7. The anti-aging theaflavin composition according to claim 6, characterized in that: The amount of coenzyme Q10 is 4g to 6g; the amount of L-seleno-methylselenocysteine is 2g to 3g; the amount of dichloromethane is 100mL to 120mL; the amount of N,N-dicyclohexylcarbodiimide is 2g to 3g; the amount of 4-dimethylaminopyridine is 0.3g to 0.5g; the stirring reaction is carried out at 60℃ to 65℃ and a speed of 100r / min to 150r / min for 6h to 8h; the concentration is carried out at 40℃ to 50 ℃, concentrated by reduced pressure evaporation to 30% to 40% of the original volume; the temperature of the cold ether is 4℃ to 10℃; the amount of the cold ether is 500mL to 600mL; the centrifugation is performed at 5000r / min to 6000r / min for 10min to 15min; the detergent is 4℃ to 10℃ cold ether and deionized water; the number of washings with the cold ether is 3 to 5 times, and the number of washings with the deionized water is 3 to 5 times; the temperature of the reduced pressure drying is 35℃ to 40℃.

8. The method for preparing the anti-aging theaflavin composition according to claim 1, characterized in that: The steps include: S1: Dispersing the nanocrystalline theaflavin-metal organic framework complex in phosphate buffer containing 2 wt% to 3 wt% of γ-cyclodextrin-tannic acid copolymer at 8 to 10 times its mass to form a suspension; first ultrasonically treating, then high-pressure homogenizing to obtain the nanocrystalline theaflavin-metal organic framework complex with the surface uniformly coated with the γ-cyclodextrin-tannic acid copolymer; evaporating under reduced pressure to remove the solvent; and drying under reduced pressure to constant weight to obtain the coated modified nanocrystalline theaflavin-metal organic framework complex; S2: Mixing the coated modified nanocrystalline theaflavin-metal organic framework complex and the mitochondria-targeted coenzyme Q10-L-selenium-methylselenocysteine complex according to parts by mass to obtain a composition.

9. The method for preparing an anti-aging theaflavin composition according to claim 8, characterized in that: The pH value of the phosphate buffer is 5.0±0.2; the ultrasonic treatment is performed at a power of 250W to 300W for 10min to 15min; the high-pressure homogenization is performed at a high-pressure homogenization of 100MPa to 120MPa for 4 to 6 cycles, with an interval of 30s to 40s between each cycle; the temperature of the reduced-pressure evaporation is 40°C to 50°C; and the temperature of the reduced-pressure drying is 40°C to 50°C.

10. Use of the anti-aging theaflavin composition according to claim 1 in anti-aging medicines and foods.

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