A method for efficiently extracting lycopene

Abstract

This invention discloses a highly efficient extraction method for capsaicin. The method involves treating dried chili powder with low-pressure vacuum plasma, performing subcritical extraction using a deep eutectic solvent, and then specifically removing capsaicin from the extract using capsaicin magnetic composite molecularly imprinted microspheres. Capsaicin is then selectively enriched using capsaicin composite carbon dot molecularly imprinted microspheres, with further separation aided by an external magnetic field, ultimately yielding a high-purity capsaicin product with high color value. This invention utilizes high-energy electrons and reactive oxygen and nitrogen to disrupt the chili pepper cell wall structure through physical bombardment and chemical oxidation, facilitating the entry of the deep eutectic solvent into chili pepper tissue cells and promoting capsaicin dissolution. Simultaneously, the low-temperature anaerobic environment effectively inhibits the oxidative degradation of capsaicin. Furthermore, capsaicin can promote mitochondrial biosynthesis by activating the SIRT1-PGC-1α signaling pathway, maintain ATP levels in the cerebral cortex under hypoxic conditions, and significantly prolong the survival time of normobaric hypoxic model mice, demonstrating excellent hypoxia tolerance activity.

Description

Technical Field

[0001] This invention belongs to the field of bioactive ingredient extraction technology, specifically relating to a highly efficient extraction method for capsanthin. Background Technology

[0002] Capsaicin is a high-value natural carotenoid extracted from ripe red chili peppers. Its long-chain polyene structure endows it with strong coloring power and excellent antioxidant properties, leading to continuously growing demand in the food coloring, cosmetics, and functional food industries. Compared to β-carotene and vitamin E, capsaicin exhibits a more significant ability to scavenge free radicals and inhibit lipid peroxidation, demonstrating good health benefits in cardiovascular protection and blood lipid regulation. However, the content of capsaicin in natural chili pepper fruits is low, and it coexists with capsaicin, oils, waxes, and various carotenoids, making extraction and separation challenging.

[0003] Currently, the main extraction and purification technologies for capsaicin include solvent extraction, supercritical fluid extraction, column chromatography, and molecular distillation. Solvent extraction is simple, but its selectivity is poor, often resulting in a large amount of capsaicin residue in the crude extract, severely affecting the product's color and taste. Supercritical CO2 extraction has high extraction efficiency, but it requires significant equipment investment and operates under high pressure, and its separation effect on capsaicin and capsaicin is limited. Column chromatography offers high separation precision, but its versatile packing materials and insufficient specific recognition capabilities lead to long separation cycles and high solvent consumption.

[0004] In recent years, plasma technology has been introduced into the field of natural product extraction as an emerging green processing method. This technology generates high-energy active particles to gently disrupt the structure of plant cell membranes, promoting the release of intracellular active ingredients. It has advantages such as low processing temperature, short extraction time, and low solvent consumption. For example, Miheraban Sewaidula et al. used atmospheric pressure jet plasma pretreatment combined with a high-voltage electrostatic field to extract capsanthin from dried chili peppers. Although this method can improve the extraction rate and color value of capsanthin compared with traditional methods, existing plasma-assisted extraction technologies are mostly limited to single treatment under atmospheric pressure conditions. They fail to fully utilize the regulatory effect of different atmospheric pressure environments on plasma characteristics and extraction efficiency. Moreover, local overheating is prone to occur during the treatment process, leading to capsanthin degradation, which will adversely affect the quality of capsanthin.

[0005] Studies have shown that there are few technologies for specifically extracting capsanthin from chili pepper raw materials. For example, invention patent CN201410504416 discloses a three-dimensional backbone polymer based on melamine sponge modification. By introducing hexadecimal hydrophobic long chains on the backbone surface to generate hydrophobic interactions with capsanthin molecules, it achieves good adsorption capacity for capsanthin. However, capsanthin often contains impurities such as capsaicin and capsaicinine, and the spiciness threshold of these impurities is extremely low. This not only reduces the purity of capsanthin but also affects its flavor quality, making it difficult to prepare high-purity capsanthin. Molecular imprinting technology is a biomimetic technology for preparing polymers with specific recognition capabilities. It uses the target molecule as a template and forms molecularly imprinted polymers (MIPs) with specific recognition holes through non-covalent or covalent interactions between functional monomers and template molecules, after cross-linking polymerization and template elution. For example, invention patent CN2022105890201 discloses the use of harvestinoin and vitexin as dual-template molecules, with a natural eutectic solvent as the organic functional monomer and attapulgite clay as the carrier to prepare a magnetic dual-template molecularly imprinted polymer. This polymer can efficiently extract harvestinoin and vitexin from *Trollius chinensis*, demonstrating the good application potential of molecularly imprinted microspheres in the enrichment of natural products or the removal of impurities. However, their applications are mostly single-function; for example, when the core objective is the enrichment of the target product, the pretreatment of coexisting impurities is often neglected; or when the goal is the removal of specific impurities, high-purity recovery of the target product is not achieved. In actual production, capsanthin extract contains both the target product capsanthin and the residual impurity capsaicin. The two have similar structures and are difficult to separate, making efficient purification difficult using only single-function molecularly imprinted microspheres. Therefore, developing a method for the efficient and highly selective separation and purification of capsanthin is of great significance for improving the product quality of capsanthin and promoting the development of the active ingredient industry in chili peppers. Summary of the Invention

[0006] Technical Problem to be Solved: To address the aforementioned technical problems, the purpose of this invention is to disclose a highly efficient extraction method for capsaicin. This method involves treating dried chili powder with low-pressure vacuum plasma, performing subcritical extraction using a deep eutectic solvent, and then specifically removing capsaicin from the extract using capsaicin magnetic composite molecularly imprinted microspheres. Capsaicin is then selectively enriched using capsaicin composite carbon dot molecularly imprinted microspheres, with an external magnetic field assisting separation, ultimately yielding a high-purity capsaicin product with high color value. This invention utilizes high-energy electrons and reactive oxygen and nitrogen to disrupt the chili pepper cell wall structure through physical bombardment and chemical oxidation, facilitating the entry of the deep eutectic solvent into chili pepper tissue cells and promoting capsaicin dissolution. Simultaneously, the low-temperature anaerobic environment effectively inhibits the oxidative degradation of capsaicin. Furthermore, capsaicin can promote mitochondrial biosynthesis by activating the SIRT1-PGC-1α signaling pathway, maintain ATP levels in the cerebral cortex under hypoxic conditions, and significantly prolong the survival time of normobaric hypoxic model mice, demonstrating good hypoxia tolerance activity.

[0007] Technical solution: A highly efficient extraction method for capsanthin, comprising the following steps: S1. The dried chili powder was subjected to low-pressure vacuum plasma treatment, and then mixed with choline chloride-decanoic acid deep eutectic solvent for subcritical extraction. The extract was obtained by centrifugation and filtration. S2. Capsaicin magnetic composite molecularly imprinted microspheres and capsaicin composite carbon dot molecularly imprinted microspheres were added to the crude capsaicin extract for treatment. Then, the extract was removed by an external magnetic field, centrifuged, filtered, washed, and dried to obtain capsaicin.

[0008] Preferably, the addition ratio of dried chili powder and choline chloride-decanoic acid deep eutectic solvent in S1 is 1g:(5-20)mL, wherein the molar ratio of choline chloride to decanoic acid is 1:(2-3); the low-pressure vacuum plasma treatment conditions are discharge power 50-150W, vacuum degree 10-30Pa, treatment temperature 25-40℃, and treatment time 5-15min; the subcritical extraction conditions are extraction temperature 25-50℃, extraction pressure 1-3.5MPa, and extraction time 1-2.5h.

[0009] Preferably, the preparation method of capsaicin magnetic composite molecularly imprinted microspheres in S2 is as follows: first, capsaicin is dissolved in acetonitrile, then 2-vinylpyridine and methacrylic acid are added to prepare a pre-assembled solution, then carboxylated carbon nanotubes and Fe3O4 magnetic nanoparticles are added and ultrasonically mixed, then ethylene glycol dimethacrylate and azobisisobutyronitrile are added, followed by polymerization crosslinking, elution and drying to obtain capsaicin magnetic composite molecularly imprinted microspheres.

[0010] Preferably, the mass ratio of capsaicin, 2-vinylpyridine, and methacrylic acid is 1:(2-5):(2-5); the amount of carboxylated carbon nanotubes added is 5-25%, the amount of Fe3O4 magnetic nanoparticles added is 10-30%, and the amount of azobisisobutyronitrile added is 0.5-2%, all based on the total mass of 2-vinylpyridine and methacrylic acid; the amount of ethylene glycol dimethacrylate added is 1.5-5 times the total mass of 2-vinylpyridine and methacrylic acid.

[0011] Preferably, the polymerization and crosslinking conditions are a reaction temperature of 60-80℃ and a reaction time of 12-24 h; the elution method is to use a mixed solution of methanol and acetic acid prepared in a volume ratio of (8-9):(1-2) and elute at 30-50℃ for 2-6 h.

[0012] Preferably, the preparation method of the palmitic acid-β-cyclodextrin complex is as follows: β-cyclodextrin, palmitic acid, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine are mixed in a molar ratio of 1:(0.8-1.5):(1.0-1.5):(0.05-0.15) and reacted in a microwave at 200-350W and 30-40℃ for 2-4 hours. After filtration to remove impurities, purification and drying are performed to obtain a palmitic acid-β-cyclodextrin complex with a degree of substitution of 1-3. The preparation method of the capsanthin composite carbon dot molecularly imprinted microspheres is as follows: capsanthin is first dissolved in acetonitrile, and then 2-vinylnaphthalene and N-allyl-2-hydroxybenzamide are added to prepare a pre-assembled solution. Then, the palmitic acid-β-cyclodextrin complex and chili waste derivative carbon dots are added and ultrasonically treated and mixed. Subsequently, divinylbenzene and azobisisoheptanenitrile are added for polymerization and cross-linking treatment, followed by elution and drying to obtain capsanthin composite carbon dot molecularly imprinted microspheres.

[0013] Preferably, the mass ratio of capsanthin, 2-vinylnaphthalene, and N-allyl-2-hydroxybenzamide is 1:(3-8):(2-5); the amount of palmitic acid-β-cyclodextrin complex added is 10-25%, the amount of carbon dots from chili waste derivatives added is 10-20%, and the amount of azobisisobutyronitrile added is 0.5-2%, all based on the total mass of 2-vinylnaphthalene and N-allyl-2-hydroxybenzamide; the amount of divinylbenzene added is 1-5 times the total mass of 2-vinylnaphthalene and N-allyl-2-hydroxybenzamide; the polymerization and crosslinking treatment conditions are a treatment temperature of 50-70℃ and a treatment time of 12-24h.

[0014] Preferably, the preparation method of the carbon dots of chili waste derivative is as follows: the red chili waste is dried and ground into powder at 50-80℃, water is added and stirred to disperse at a material-to-liquid ratio of 1:(15-30), and treated for 1-3 hours under microwave power of 500-700W and treatment temperature of 160-220°C, and then microfiltered and dried to obtain the carbon dots of chili waste derivative.

[0015] Preferably, the amount of capsaicin magnetic composite molecularly imprinted microspheres added in S2 is 0.1-0.5%, and the amount of capsanthin composite carbon dot molecularly imprinted microspheres added is 0.2-0.8%, both based on the volume of the crude capsanthin extract.

[0016] Capsaicin prepared by any of the methods described above.

[0017] Preferably, the capsanthin is used in the preparation of hypoxia-resistant health foods. Beneficial effects

[0018] 1. This invention employs low-pressure vacuum plasma coupled with subcritical deep eutectic solvent extraction to extract capsanthin. The plasma generated under low-pressure vacuum conditions is rich in high-energy particles, which can efficiently etch the cuticle and cellulose network on the surface of pepper cell walls through physical bombardment and chemical oxidation, creating micropores and cracks in the cell walls to significantly reduce mass transfer resistance. At the same time, the vacuum environment facilitates plasma penetration into the interior of pepper tissue cells, destroying the cell membrane structure and releasing bound capsanthin. The subcritical conditions allow the deep eutectic solvent to rapidly penetrate into the damaged pepper cell walls, forming strong hydrogen bonds and hydrophobic interactions with capsanthin, capsaicin, and other components, achieving targeted dissolution and efficient elution. Furthermore, it reduces the solvent viscosity and surface tension of the deep eutectic solvent to further improve the diffusion coefficient. The low temperature and anaerobic environment effectively inhibit the oxidative degradation of the conjugated double bonds contained in capsanthin, laying a good foundation for the extraction of high purity and high color value of capsanthin.

[0019] 2. This invention utilizes capsaicin magnetic composite molecularly imprinted microspheres to remove residual capsaicin from capsaicin crude extract, while simultaneously employing capsaicin composite carbon dot molecularly imprinted microspheres to specifically adsorb capsaicin. The capsaicin magnetic composite molecularly imprinted microspheres, utilizing the carboxyl groups, hydrophobic domains, and aromatic rings within their cavities, form hydrogen bonds, hydrophobic interactions, and π-π stacking interactions with the amide groups, hydroxyl groups, long-chain alkyl groups, and aromatic rings of capsaicin. Under the assistance of an external magnetic field, they preferentially capture and remove structurally similar capsaicin molecules in the crude capsaicin extract that easily cause competitive interference. This not only eliminates the cross-interference of capsaicin on the capsaicin composite carbon dot molecularly imprinted microspheres but also reduces the surface area of ​​capsaicin molecules. The spatial shielding effect provides a good foundation for the efficient enrichment of capsanthin. After capsanthin is removed, the amide and carboxyl groups in the imprinted cavity of the capsanthin composite carbon dot molecularly imprinted microspheres form multiple hydrogen bonds with the ketone and hydroxyl groups of capsanthin. The hydrophobic region on the inner wall of the cavity forms a strong hydrophobic bond with the long chain of conjugated double bonds of capsanthin. The benzene ring and the conjugated double bond system undergo π-π stacking. Moreover, the carbon dots can improve the surface roughness and porosity of the microspheres, so as to expose more imprinted cavities and make it easier for capsanthin molecules to access the cavities. This greatly improves the adsorption capacity and enrichment efficiency of capsanthin by the capsanthin composite carbon dot molecularly imprinted microspheres, thereby achieving the goal of efficient extraction of capsanthin from the crude capsanthin extract.

[0020] 3. Compared with traditional methods, the method described in this invention can improve the yield of capsanthin, and the extracted capsanthin has the characteristics of high purity and high color value. Animal experimental results show that the capsanthin described in this invention can increase the activity of SIRT1 enzyme in the liver tissue of hypoxic mice and upregulate the level of PGC-1α deacetylation, thereby significantly increasing the mitochondrial DNA copy number in the heart group of hypoxic mice to promote mitochondrial biosynthesis, thereby maintaining the ATP content level in the cerebral cortex of hypoxic mice and ensuring the energy metabolism efficiency of mice under hypoxic conditions. The capsanthin described in this invention can significantly prolong the survival time of mice in the normobaric hypoxia model group, and can enhance the hypoxia tolerance of mice by activating the SIRT1-PGC-1α pathway to maintain mitochondrial synthesis level and prevent excessive ATP depletion. Detailed Implementation

[0021] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: Example 1

[0022] A method for preparing capsaicin magnetic composite molecularly imprinted microspheres includes the following steps: Step 1. Dissolve 10.0g capsaicin in 1000mL acetonitrile and sonicate for 15min. Then add 20.0g 2-vinylpyridine and 20.0g methacrylic acid and stir at room temperature for 30min. Then add 2.0g carboxylated carbon nanotubes and 4.0g Fe3O4 magnetic nanoparticles and treat with ultrasonic power of 300W for 60min to obtain a pre-assembled dispersion. Step 2. Add 60.0 g of ethylene glycol dimethacrylate and 0.2 g of azobisisobutyronitrile to the pre-assembled dispersion, degas with sonication for 20 min to remove oxygen, and then seal and place at 60 °C for polymerization and crosslinking reaction for 24 h under nitrogen protection. Collect the mixture magnetically and elute with an eluent prepared from 900 mL of methanol and 100 mL of acetic acid at 30 °C for 6 h with shaking. Then wash with water until neutral and dry under vacuum at 50 °C to obtain capsaicin magnetic composite molecularly imprinted microspheres. Example 2

[0023] A method for preparing capsaicin magnetic composite molecularly imprinted microspheres includes the following steps: Step 1. Dissolve 10.0g capsaicin in 1500mL acetonitrile and sonicate for 15min. Then add 35.0g 2-vinylpyridine and 35.0g methacrylic acid and stir at room temperature for 30min. Then add 10.5g carboxylated carbon nanotubes and 14.0g Fe3O4 magnetic nanoparticles and treat with ultrasonic power of 350W for 90min to obtain a pre-assembled dispersion. Step 2. Add 210.0 g of ethylene glycol dimethacrylate and 0.84 g of azobisisobutyronitrile to the pre-assembled dispersion, degas with sonication for 25 min to remove oxygen, and then seal and place at 70 °C for polymerization and crosslinking reaction for 18 h under nitrogen protection. Collect the mixture magnetically and elute with an eluent prepared from 850 mL of methanol and 150 mL of acetic acid at 40 °C for 4 h with shaking. Then wash with water until neutral and dry under vacuum at 45 °C to obtain capsaicin magnetic composite molecularly imprinted microspheres. Example 3

[0024] A method for preparing capsaicin magnetic composite molecularly imprinted microspheres includes the following steps: Step 1. Dissolve 10.0g capsaicin in 1200mL acetonitrile and sonicate for 15min. Then add 40.0g 2-vinylpyridine and 25.0g methacrylic acid and stir at room temperature for 30min. Then add 13.0g carboxylated carbon nanotubes and 9.75g Fe3O4 magnetic nanoparticles and treat with ultrasonic power of 350W for 50min to obtain a pre-assembled dispersion. Step 2. Add 162.5g of ethylene glycol dimethacrylate and 0.65g of azobisisobutyronitrile to the pre-assembled dispersion, degas by sonication for 20min, then purge with nitrogen for protection, seal and place at 65℃ for polymerization and crosslinking reaction for 15h. Collect the mixture by magnetic separation and elute with an eluent prepared from 850mL of methanol and 150mL of acetic acid at 35℃ for 5h by shaking. Then wash with water until neutral and dry under vacuum at 50℃ to obtain capsaicin magnetic composite molecularly imprinted microspheres. Example 4

[0025] A method for preparing capsaicin magnetic composite molecularly imprinted microspheres includes the following steps: Step 1. Dissolve 10.0g capsaicin in 2000mL acetonitrile and sonicate for 15min. Then add 50.0g 2-vinylpyridine and 50.0g methacrylic acid and stir at room temperature for 30min. Then add 25.0g carboxylated carbon nanotubes and 30.0g Fe3O4 magnetic nanoparticles and treat with ultrasonic power of 400W for 60min to obtain a pre-assembled dispersion. Step 2. Add 300.0g of ethylene glycol dimethacrylate and 2.0g of azobisisobutyronitrile to the pre-assembled dispersion, degas with sonication for 25min to remove oxygen, and seal under nitrogen protection and place at 80℃ for polymerization and crosslinking reaction for 12h. Collect the mixture by magnetic separation and elute with an eluent prepared from 800mL of methanol and 200mL of acetic acid at 50℃ for 2h by shaking. Then wash with water until neutral and dry under vacuum at 40℃ to obtain capsaicin magnetic composite molecularly imprinted microspheres. Comparative Example 1

[0026] The difference between this comparative example and Example 3 is that carboxylated carbon nanotubes are not added; the rest are the same as in Example 3. Comparative Example 2

[0027] The difference between this comparative example and Example 3 is that methacrylic acid is not added; the rest is the same as in Example 3.

[0028] Example 5 A method for preparing capsanthin-based carbon dot molecularly imprinted microspheres includes the following steps: Step I. 100g of waste red chili pepper leaves were dried at 70℃ and ground into powder. 2L of water was added and stirred to disperse the powder. The powder was then treated for 2h under microwave power of 700W and treatment temperature of 190°C. After microfiltration and drying, carbon dots, a derivative of chili pepper waste, were obtained. 28.3g of β-cyclodextrin was dissolved in 100mL of anhydrous dimethyl sulfoxide and stirred to dissolve. Then, 9.6g of palmitic acid, 7.7g of N,N'-dicyclohexylcarbodiimide and 0.45g of 4-dimethylaminopyridine were added and mixed. The mixture was reacted for 4h under microwave power of 350W, 40℃ and nitrogen atmosphere. Byproducts were removed by filtration. The filtrate was then added to 500mL of anhydrous acetone pre-cooled to 4℃ for recrystallization. The precipitate was collected by suction filtration and washed three times with 60mL of anhydrous acetone. The precipitate was then dried under vacuum at 40℃ to obtain a palmitic acid-β-cyclodextrin complex with an average degree of substitution of 2.5. Step II. Dissolve 10.0g capsanthin in 2000mL acetonitrile, add 80.0g 2-vinylnaphthalene and 50.0g N-allyl-2-hydroxybenzamide and stir at room temperature for 20min. Then add 32.5g palmitic acid-β-cyclodextrin complex and 26.0g carbon dots of chili waste derivatives and sonicate at 400W for 60min to prepare a pre-assembled solution. Step III. Add 650.0 g of divinylbenzene and 2.6 g of azobisisoheptanenitrile to the pre-assembled solution, degas by sonication for 30 min, then purge with nitrogen and seal for polymerization and crosslinking reaction at 70 °C for 12 h. Centrifuge and filter, wash with 500 mL of methanol, then elute with a mixed eluent prepared from 800 mL of methanol and 200 mL of acetic acid at 50 °C with shaking for 2 h, then wash with water until neutral, and vacuum dry at 50 °C to obtain capsanthin composite carbon dot molecularly imprinted microspheres. Example 6

[0029] A method for preparing capsanthin-based carbon dot molecularly imprinted microspheres includes the following steps: Step I. 100g of waste red chili pepper leaves were dried at 60℃ and ground into powder. 1.5L of water was added and stirred to disperse the powder. The powder was then treated for 2.5h under microwave power of 600W and treatment temperature of 200°C. After microfiltration and drying, carbon dots, a derivative of chili pepper waste, were obtained. 11.3g of β-cyclodextrin was dissolved in 50mL of anhydrous dimethyl sulfoxide and stirred to dissolve. Then, 3.0g of palmitic acid, 2.2g of N,N'-dicyclohexylcarbodiimide and 0.1g of 4-dimethylaminopyridine were added and mixed. The mixture was reacted for 2h under microwave power of 200W, 30℃ and nitrogen atmosphere. Byproducts were removed by filtration. The filtrate was then added to 200mL of anhydrous acetone pre-cooled to 4℃ for recrystallization. The precipitate was collected by suction filtration and washed three times with 30mL of anhydrous acetone. The precipitate was then dried under vacuum at 40℃ to obtain a palmitic acid-β-cyclodextrin complex with an average degree of substitution of 1.0. Step II. Dissolve 10.0g capsanthin in 1000mL acetonitrile, add 30.0g 2-vinylnaphthalene and 20.0g N-allyl-2-hydroxybenzamide and stir at room temperature for 20min. Then add 5.0g palmitic acid-β-cyclodextrin complex and 5.0g carbon dots of chili waste derivatives and sonicate at 300W for 45min to prepare a pre-assembled solution. Step III. Add 50.0 g of divinylbenzene and 0.25 g of azobisisoheptanenitrile to the pre-assembled solution, degas by sonication for 30 min, then purge with nitrogen and seal for polymerization and crosslinking reaction at 50 °C for 15 h. Centrifuge and filter, wash with 500 mL of methanol, then elute with a mixed eluent prepared from 900 mL of methanol and 100 mL of acetic acid at 30 °C for 6 h with shaking, then wash with water until neutral, and vacuum dry at 50 °C to obtain capsanthin composite carbon dot molecularly imprinted microspheres. Example 7

[0030] A method for preparing capsanthin-based carbon dot molecularly imprinted microspheres includes the following steps: Step I. 100g of waste red chili pepper leaves were dried at 75℃ and ground into powder. 1.5L of water was added and stirred to disperse the powder. The powder was then treated for 2h under microwave power of 700W and treatment temperature of 200°C. After microfiltration and drying, carbon dots, a derivative of chili pepper waste, were obtained. 17g of β-cyclodextrin was dissolved in 60mL of anhydrous dimethyl sulfoxide and stirred to dissolve. Then, 4.5g of palmitic acid, 4.0g of N,N'-dicyclohexylcarbodiimide and 0.22g of 4-dimethylaminopyridine were added and mixed. The mixture was reacted for 3.5h under microwave power of 300W, 38℃ and nitrogen atmosphere. Byproducts were removed by filtration. The filtrate was then added to 300mL of anhydrous acetone pre-cooled to 4℃ for recrystallization. The precipitate was collected by suction filtration and washed three times with 40mL of anhydrous acetone. The precipitate was then dried under vacuum at 40℃ to obtain a palmitic acid-β-cyclodextrin complex with an average degree of substitution of 2. Step II. Dissolve 10.0g capsanthin in 1500mL acetonitrile, add 70.0g 2-vinylnaphthalene and 25.0g N-allyl-2-hydroxybenzamide and stir at room temperature for 30min. Then add 14.25g palmitic acid-β-cyclodextrin complex and 11.4g carbon dots of chili waste derivatives and sonicate at an ultrasonic power of 350W for 60min to prepare a pre-assembled solution. Step III. Add 237.5g of divinylbenzene and 0.95g of azobisisoheptanenitrile to the pre-assembled solution, degas by sonication for 20 min, then purge with nitrogen and seal for polymerization and crosslinking reaction at 55℃ for 20 h. Centrifuge and filter, wash with 500mL of methanol, then elute with a mixed eluent prepared from 800mL of methanol and 200mL of acetic acid at 35℃ for 5 h of shaking, then wash with water until neutral, and vacuum dry at 45℃ to obtain capsanthin composite carbon dot molecularly imprinted microspheres. Example 8

[0031] A method for preparing capsanthin-based carbon dot molecularly imprinted microspheres includes the following steps: Step I. 100g of waste red chili pepper leaves were dried at 75℃ and ground into powder. 3L of water was added and stirred to disperse the powder. The powder was then treated for 3h under microwave power of 650W and treatment temperature of 210°C. After microfiltration and drying, carbon dots, a derivative of chili pepper waste, were obtained. 22.7g of β-cyclodextrin was dissolved in 80mL of anhydrous dimethyl sulfoxide and stirred to dissolve. Then, 5.1g of palmitic acid, 5.0g of N,N'-dicyclohexylcarbodiimide and 0.25g of 4-dimethylaminopyridine were added and mixed. The mixture was reacted for 3h under microwave power of 250W, 35℃ and nitrogen atmosphere. By-products were removed by filtration. The filtrate was then added to 400mL of anhydrous acetone pre-cooled to 4℃ for recrystallization. The precipitate was collected by suction filtration and washed three times with 50mL of anhydrous acetone. The precipitate was then dried under vacuum at 40℃ to obtain a palmitic acid-β-cyclodextrin complex with an average degree of substitution of 1.5. Step II. Dissolve 10.0g capsanthin in 1500mL acetonitrile, add 50.0g 2-vinylnaphthalene and 30.0g N-allyl-2-hydroxybenzamide and stir at room temperature for 35min. Then add 12.0g palmitic acid-β-cyclodextrin complex and 12.0g carbon dots of chili waste derivatives and sonicate at 350W for 75min to prepare a pre-assembled solution. Step III. Add 160.0 g of divinylbenzene and 0.96 g of azobisisoheptanenitrile to the pre-assembled solution, degas by sonication for 20 min, then purge with nitrogen and seal for polymerization and crosslinking reaction at 60 °C for 15 h. Centrifuge and filter, wash with 500 mL of methanol, then elute with a mixed eluent prepared from 850 mL of methanol and 150 mL of acetic acid at 40 °C with shaking for 4 h, then wash with water until neutral, and vacuum dry at 45 °C to obtain capsanthin composite carbon dot molecularly imprinted microspheres. Comparative Example 3

[0032] The difference between this comparative example and Example 7 is that the palmitic acid-β-cyclodextrin complex is replaced with β-cyclodextrin, and the remaining operations are the same as in Example 7. Comparative Example 4

[0033] The difference between this comparative example and Example 7 is that no carbon dots derived from chili waste are added; the remaining operations are the same as in Example 7.

[0034] Performance testing (1) Average particle size Accurately weigh 5.0 mg of molecularly imprinted microspheres and add them to 10.0 mL of water. Disperse the sample suspension by ultrasonication at 200 W for 10 min. Inject 1.0 mL of the suspension into a disposable cuvette, place it in the sample cell of a dynamic light scattering particle size analyzer, and equilibrate at 25°C for 2 min. Measure the scattering angle using a 633 nm helium-neon laser at 173°. Each experiment was repeated three times, and the results are expressed as mean ± standard deviation.

[0035] (2) Imprinting factor Accurately weigh 20.0 mg of molecularly imprinted microspheres and non-imprinted microspheres into two 10 mL centrifuge tubes, respectively. Add 5.0 mL of 0.5 mmol / L acetonitrile solution of the corresponding template molecule to each tube. Incubate at room temperature with shaking for 12 h until equilibrium is reached. Centrifuge at 8000 r / min for 10 min, and collect the supernatant for analysis. Determine the concentration of the remaining template molecule in the supernatant using UV-Vis spectrophotometry, and calculate the equilibrium adsorption capacity Q of the molecularly imprinted microspheres based on the difference between the initial concentration and the equilibrium concentration. MIP The equilibrium adsorption capacity Q of non-imprinted microspheres NIP Where its imprinting factor (IF) = Q MIP / Q NIP Each experiment was repeated three times, and the results are expressed as mean ± standard deviation.

[0036] Table 1. Average particle size and imprinting factor of Examples 1-8 and Comparative Examples 1-4

[0037] As shown in Table 1, the imprinting factors of the capsaicin magnetic composite molecularly imprinted microspheres prepared in Examples 1-4 and the capsaicin composite carbon dot molecularly imprinted microspheres prepared in Examples 5-8 are all greater than 2.5. This indicates that the capsaicin magnetic composite molecularly imprinted microspheres prepared in Examples 1-4 and the capsaicin composite carbon dot molecularly imprinted microspheres prepared in Examples 5-8 have good specific recognition ability for the adsorption of capsaicin or capsaicin.

[0038] Example 9 A highly efficient method for extracting capsanthin includes the following steps: S1. Choline chloride and decanoic acid are mixed in a molar ratio of 1:2 and stirred at 80°C and 300 rpm for 2 hours to obtain a choline chloride-decanoic acid eutectic solvent; S2. Weigh 100.0g of dried chili powder and treat it with low-pressure vacuum plasma at 32℃, discharge power 100W and vacuum degree 20Pa for 10min. Then mix it with 1200mL of choline chloride-decanoic acid deep eutectic solvent and extract it under subcritical conditions at 50℃ and 3.5MPa for 2.5h. Centrifuge the mixture at 4000rpm for 20min and take the upper layer solution to obtain the crude extract of capsanthin. S3. Add 3g of capsaicin magnetic composite molecularly imprinted microspheres prepared in Example 3 and 5g of capsaicin composite carbon dot molecularly imprinted microspheres prepared in Example 7 to 1L of crude capsaicin extract and shake and adsorb at room temperature for 75min. Then place the microspheres in an external magnetic field to remove the capsaicin-adsorbed capsaicin. Filter to obtain capsaicin-adsorbed capsaicin composite carbon dot molecularly imprinted microspheres. Elute with 200mL of anhydrous ethanol. Place the capsaicin-adsorbed capsaicin composite carbon dot molecularly imprinted microspheres back into the filtrate and shake and adsorb for another 30min. Elute a second time with 200mL of anhydrous ethanol. Combine the eluents and dry under vacuum to obtain capsaicin. Example 10

[0039] A highly efficient method for extracting capsanthin includes the following steps: S1. Choline chloride and decanoic acid are mixed in a molar ratio of 1:3 and stirred at 85°C and 300 rpm for 2.5 h to obtain a choline chloride-decanoic acid eutectic solvent; S2. Weigh 100.0g of dried chili powder and treat it with low-pressure vacuum plasma at 40℃, discharge power of 50W and vacuum degree of 30Pa for 15min. Then mix it with 2000mL of choline chloride-decanoic acid deep eutectic solvent and extract it under subcritical conditions at 50℃ and 3.5MPa for 2.5h. Centrifuge the mixture at 4000rpm for 20min and take the upper layer solution to obtain the crude extract of capsanthin. S3. Add 7.5g of capsaicin magnetic composite molecularly imprinted microspheres prepared in Example 3 and 9g of capsaicin composite carbon dot molecularly imprinted microspheres prepared in Example 7 to 1.5L of crude capsaicin extract and shake and adsorb at room temperature for 75min. Then place the capsaicin magnetic composite molecularly imprinted microspheres in an external magnetic field to remove the capsaicin-adsorbed microspheres. Filter to obtain capsaicin-adsorbed capsaicin composite carbon dot molecularly imprinted microspheres. Elute with 200mL of anhydrous ethanol. Put the capsaicin composite carbon dot molecularly imprinted microspheres back into the filtrate and shake and adsorb for another 30min. Elute again with 200mL of anhydrous ethanol. Repeat the cycle once. Combine the eluents and dry under vacuum to obtain capsaicin. Example 11

[0040] A highly efficient method for extracting capsanthin includes the following steps: S1. Choline chloride and decanoic acid are mixed in a molar ratio of 1:2 and stirred at 75°C and 200 rpm for 1.5 h to obtain a choline chloride-decanoic acid eutectic solvent; S2. Weigh 100.0g of dried chili powder and treat it with low-pressure vacuum plasma for 10min at 25℃, discharge power of 50W and vacuum degree of 10Pa. Then mix it with 500mL of choline chloride-decanoic acid deep eutectic solvent and extract it under subcritical conditions at 25℃ and 1.0MPa for 1.5h. Centrifuge the mixture at 4000rpm for 20min and take the upper layer solution to obtain the crude extract of capsanthin. S3. Add 0.4g of capsaicin magnetic composite molecularly imprinted microspheres prepared in Example 3 and 0.8g of capsaicin composite carbon dot molecularly imprinted microspheres prepared in Example 7 to 400mL of crude capsaicin extract and shake and adsorb at room temperature for 60min. Then place the capsaicin magnetic composite molecularly imprinted microspheres in an external magnetic field to remove the capsaicin-adsorbed microspheres. Filter to obtain capsaicin-adsorbed capsaicin composite carbon dot molecularly imprinted microspheres. Elute with 200mL of anhydrous ethanol and then place the capsaicin composite carbon dot molecularly imprinted microspheres back into the filtrate and shake and adsorb for another 30min. Elute a second time with 200mL of anhydrous ethanol, and repeat the cycle once. Combine the eluents and vacuum dry to obtain capsaicin. Example 12

[0041] A highly efficient method for extracting capsanthin includes the following steps: S1. Choline chloride and decanoic acid were mixed in a molar ratio of 1:2.2 and stirred at 75°C and 300 rpm for 1.8 h to obtain a choline chloride-decanoic acid eutectic solvent; S2. Weigh 100.0g of dried chili powder and treat it with low-pressure vacuum plasma at 30℃, discharge power 75W and vacuum degree 15Pa for 10min. Then mix it with 800mL of choline chloride-decanoic acid deep eutectic solvent and extract it under subcritical conditions at 30℃ and 1.5MPa for 2h. Centrifuge the mixture at 4000rpm for 20min and take the upper layer solution to obtain the crude extract of capsanthin. S3. Add 1.4g of capsaicin magnetic composite molecularly imprinted microspheres prepared in Example 3 and 2.45g of capsaicin composite carbon dot molecularly imprinted microspheres prepared in Example 7 to 700mL of crude capsaicin extract and shake to adsorb for 60min at room temperature. Then place the microspheres in an external magnetic field to remove the capsaicin-adsorbed capsaicin. Filter to obtain capsaicin-adsorbed capsaicin composite carbon dot molecularly imprinted microspheres. Elute with 200mL of anhydrous ethanol and then place the microspheres in the filtrate to shake and adsorb for another 30min. Elute a second time with 200mL of anhydrous ethanol. Combine the eluents and dry under vacuum to obtain capsaicin. Example 13

[0042] A highly efficient method for extracting capsanthin includes the following steps: S1. Choline chloride and decanoic acid were mixed in a molar ratio of 1:2.8 and stirred at 85°C and 300 rpm for 2 h to obtain a choline chloride-decanoic acid eutectic solvent. S2. Weigh 100.0g of dried chili powder and treat it with low-pressure vacuum plasma at 35℃, discharge power of 120W and vacuum degree of 25Pa for 10min. Then mix it with 1600mL of choline chloride-decanoic acid deep eutectic solvent and extract it under subcritical conditions at 45℃ and 3.0MPa for 2h. Centrifuge the mixture at 4000rpm for 20min and take the upper layer solution to obtain the crude extract of capsanthin. S3. Add 4g of capsaicin magnetic composite molecularly imprinted microspheres prepared in Example 3 and 6.5g of capsaicin composite carbon dot molecularly imprinted microspheres prepared in Example 7 to 1L of crude capsaicin extract and shake and adsorb at room temperature for 85min. Then place the capsaicin magnetic composite molecularly imprinted microspheres in an external magnetic field to remove the capsaicin-adsorbed microspheres. Filter to obtain capsaicin-adsorbed capsaicin composite carbon dot molecularly imprinted microspheres. Elute with 200mL of anhydrous ethanol. Put the capsaicin composite carbon dot molecularly imprinted microspheres back into the filtrate and shake and adsorb for another 30min. Elute a second time with 200mL of anhydrous ethanol. Combine the eluents and dry under vacuum to obtain capsaicin. Comparative Example 5

[0043] The difference between this comparative example and Example 10 is that the capsaicin magnetic composite molecularly imprinted microspheres prepared in Example 3 are not added; the remaining operations are the same as in Example 10.

[0044] Comparative Example 6 The difference between this comparative example and Example 10 is that the capsanthin composite carbon dot molecularly imprinted microspheres prepared in Example 7 are not added; the remaining operations are the same as in Example 10.

[0045] Comparative Example 7 The difference between this comparative example and Example 10 is that the capsaicin magnetic composite molecularly imprinted microspheres prepared in Comparative Example 1 are used; the remaining operations are the same as in Example 10.

[0046] Comparative Example 8 The difference between this comparative example and Example 10 is that the capsaicin magnetic composite molecularly imprinted microspheres prepared in Comparative Example 2 are used; the remaining operations are the same as in Example 10.

[0047] Comparative Example 9 The difference between this comparative example and Example 10 is that the capsanthin composite carbon dot molecularly imprinted microspheres prepared in Comparative Example 3 are not added; the remaining operations are the same as in Example 10.

[0048] Comparative Example 10 The difference between this comparative example and Example 10 is that the capsanthin composite carbon dot molecularly imprinted microspheres prepared in Comparative Example 4 are not added; the remaining operations are the same as in Example 10.

[0049] Comparative Example 11 The difference between this comparative example and Example 10 is that low-pressure vacuum plasma treatment is not used; the remaining operations are the same as in Example 10.

[0050] Performance testing (1) Capsanthin yield The formula for calculating capsanthin yield is: Capsanthin yield (%) = m1 / m2 × 100%, where m1 represents the mass of capsanthin and m2 represents the mass of dried chili powder. Each experiment was repeated three times, and the results are expressed as mean ± standard deviation.

[0051] (2) Purity of capsanthin The purity of capsanthin samples was determined by high performance liquid chromatography (HPLC) using an ACQUITY UPLC BEH C. 18 A chromatographic column (1.7 μm, 2.1 mm × 50 mm) was used with a gradient elution of 0.05% formic acid aqueous solution-acetonitrile as the mobile phase (0–2.5 min: 80% A; 2.5–7.6 min: 5% A; 7.6–10.0 min: 80% A) at a flow rate of 0.20 mL / min and an injection volume of 2.0 μL. Standard curves were plotted using a series of standard solutions at concentrations of 1.00, 0.40, 0.20, 0.10, and 0.02 mg / mL. Samples were dissolved in methanol and injected directly. The capsanthin content in the samples was calculated based on the capsanthin standard curve, and the ratio of capsanthin content to the capsanthin sample concentration was used to determine the capsanthin purity. Each experiment was repeated three times, and results are expressed as mean ± standard deviation.

[0052] (3) Capsanthin color value Capsanthin color value was determined according to GB 1886.34-2015 "National Food Safety Standard - Food Additives - Capsanthin". Each experiment was repeated three times, and the results are expressed as mean ± standard deviation.

[0053] Table 2. Yields, purity, and color value of capsanthin prepared in Examples 9-13 and Comparative Examples 5-11

[0054] As shown in Table 2, the yield, purity, and color value of capsanthin extracted in Examples 9-10 were all higher than those in Comparative Examples 5-11, indicating that the method described in this invention can achieve efficient extraction and high-quality purification of capsanthin. Example 14

[0055] Animal experiment verification study on the hypoxia tolerance of capsanthin Capsaicin extracted from Examples 10, 5, 6, and 11 was used as samples for hypoxia tolerance animal experiments. Sixty SPF-grade male Kunming mice (weighing 18-22g) were randomly divided into 6 groups (n=10): Example 10 group, Comparative Example 5 group, Comparative Example 6 group, Comparative Example 11 group, blank control group, and positive control group (rhodioloside 100mg / kg). Each group was administered 50mg / kg by gavage daily, while the blank control group was administered an equal volume of water by gavage. The experimental period was 14 days. After the last administration, the mice were fasted for 12 hours but allowed free access to water for normobaric hypoxia testing. The mice were placed alone in an oxygen chamber. Place 10g of soda lime in a 5000mL sealed ground-glass stoppered wide-mouth bottle, and record the respiratory arrest time at room temperature as the hypoxia survival time. Immediately after the hypoxia test, decapitate the animal. Quickly separate the left cerebral cortex on ice, homogenize it on ice, centrifuge and collect the supernatant. ATP content is measured using the luciferase-luciferase method. At the same time, the right lobe of the liver is quick-frozen in liquid nitrogen, and after nuclear protein extraction, the PGC-1α deacetylation level is measured by immunoprecipitation-immunoblotting, and SIRT1 enzyme activity is measured by fluorescent probe method. The left ventricle of the heart is also removed, and mitochondrial DNA copy number is measured by real-time quantitative PCR after genomic DNA extraction.

[0056] Table 3 Evaluation indicators for Example 10, Comparative Examples 5-6, Comparative Example 11, Blank Control Group, and Positive Control Group

[0057] As shown in Table 3, compared with comparative groups 5, 6, and 11, the mice in Example 10 exhibited the best survival time under normobaric hypoxia, comparable to the positive control group. This indicates that high-purity capsanthin possesses excellent hypoxia tolerance activity. Furthermore, the ATP content in the cerebral cortex, the PGC-1α deacetylation level in the liver, the SIRT1 enzyme activity, and the mitochondrial DNA copy number in the heart tissue of Example 10 were all higher than those in the blank control group, comparative groups 5, 6, and 11. This suggests that capsanthin can activate the SIRT1 / PGC-1α pathway to enhance mitochondrial function, thereby prolonging the hypoxia survival time of mice and demonstrating excellent hypoxia tolerance.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A highly efficient extraction method for capsanthin, characterized in that, Includes the following steps: S1. The dried chili powder was subjected to low-pressure vacuum plasma treatment, and then mixed with choline chloride-decanoic acid deep eutectic solvent for subcritical extraction. The extract was obtained by centrifugation and filtration. S2. Add capsaicin magnetic composite molecularly imprinted microspheres and capsaicin composite carbon dot molecularly imprinted microspheres to the crude capsaicin extract for treatment. After adsorption, remove the capsaicin magnetic composite molecularly imprinted microspheres in an external magnetic field, then filter and collect the capsaicin composite carbon dot molecularly imprinted microspheres that have adsorbed capsaicin. After elution and drying, obtain capsaicin.

2. The method for efficient extraction of capsanthin according to claim 1, characterized in that: The addition ratio of dried chili powder and choline chloride-decanoic acid deep eutectic solvent in S1 is 1g:(5-20)mL, wherein the molar ratio of choline chloride to decanoic acid is 1:(2-3); the low-pressure vacuum plasma treatment conditions are discharge power 50-150W, vacuum degree 10-30Pa, treatment temperature 25-40℃, and treatment time 5-15min; the subcritical extraction conditions are extraction temperature 25-50℃, extraction pressure 1-3.5MPa, and extraction time 1-2.5h.

3. The method for efficient extraction of capsanthin according to claim 1, characterized in that: The preparation method of capsaicin magnetic composite molecularly imprinted microspheres in S2 is as follows: first, capsaicin is dissolved in acetonitrile, then 2-vinylpyridine and methacrylic acid are added to prepare a pre-assembled solution, then carboxylated carbon nanotubes and Fe3O4 magnetic nanoparticles are added and ultrasonically mixed, then ethylene glycol dimethacrylate and azobisisobutyronitrile are added, followed by polymerization crosslinking, elution and drying to obtain capsaicin magnetic composite molecularly imprinted microspheres.

4. The method for efficient extraction of capsanthin according to claim 3, characterized in that: The mass ratio of capsaicin, 2-vinylpyridine, and methacrylic acid is 1:(2-5):(2-5); the amount of carboxylated carbon nanotubes added is 5-25%, the amount of Fe3O4 magnetic nanoparticles added is 10-30%, and the amount of azobisisobutyronitrile added is 0.5-2%, all based on the total mass of 2-vinylpyridine and methacrylic acid; the amount of ethylene glycol dimethacrylate added is 1.5-5 times the total mass of 2-vinylpyridine and methacrylic acid.

5. The method for efficient extraction of capsanthin according to claim 3, characterized in that: The conditions for polymerization and crosslinking are a reaction temperature of 60-80℃ and a reaction time of 12-24 h; the elution method is to use a mixed solution of methanol and acetic acid in a volume ratio of (8-9):(1-2) and elute at 30-50℃ for 2-6 h.

6. The method for efficient extraction of capsanthin according to claim 1, characterized in that: The preparation method of the capsanthin composite carbon dot molecularly imprinted microspheres is as follows: capsanthin is first dissolved in acetonitrile, then 2-vinylnaphthalene and N-allyl-2-hydroxybenzamide are added to prepare a pre-assembled solution. Then, palmitic acid-β-cyclodextrin complex and chili waste derivative carbon dots are added and ultrasonically mixed. Subsequently, divinylbenzene and azobisisoheptanenitrile are added for polymerization and cross-linking treatment, followed by elution and drying to obtain capsanthin composite carbon dot molecularly imprinted microspheres. The preparation method of the palmitic acid-β-cyclodextrin complex is as follows: β-cyclodextrin, palmitic acid, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine are mixed at a molar ratio of 1... The mixture of (0.8-1.5): (1.0-1.5): (0.05-0.15) was reacted at a microwave power of 200-350W and 30-40℃ for 2-4 hours. After filtration to remove impurities, purification and drying, a palmitic acid-β-cyclodextrin complex with a degree of substitution of 1-3 was obtained. The preparation method of the carbon dots of the chili waste derivative is as follows: the red chili waste was dried and ground into powder at 50-80℃, and water was added at a material-liquid ratio of 1: (15-30) and stirred to disperse. The mixture was treated at a microwave power of 500-700W and a treatment temperature of 160-220℃ for 1-3 hours, and then microfiltered and dried to obtain the carbon dots of the chili waste derivative.

7. The method for efficient extraction of capsanthin according to claim 6, characterized in that: The mass ratio of capsanthin, 2-vinylnaphthalene, and N-allyl-2-hydroxybenzamide is 1:(3-8):(2-5); the amount of palmitic acid-β-cyclodextrin complex added is 10-25%, the amount of carbon dots from chili waste derivatives added is 10-20%, and the amount of azobisisobutyronitrile added is 0.5-2%, all based on the total mass of 2-vinylnaphthalene and N-allyl-2-hydroxybenzamide; the amount of divinylbenzene added is 1-5 times the total mass of 2-vinylnaphthalene and N-allyl-2-hydroxybenzamide; the polymerization and crosslinking treatment conditions are a treatment temperature of 50-70℃ and a treatment time of 12-24h.

8. The method for efficient extraction of capsanthin according to claim 1, characterized in that: The amount of capsaicin magnetic composite molecularly imprinted microspheres added in S2 is 0.1-0.5%, and the amount of capsanthin composite carbon dot molecularly imprinted microspheres added is 0.2-0.8%, both based on the volume of the crude capsanthin extract.

9. Capsanthin prepared by the method according to any one of claims 1-8.

10. The application of capsanthin according to claim 9 in the preparation of hypoxia-resistant functional foods.

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