A method for preparing camphor fruit peel anthocyanin co-chromogenic copolymer micro / nano particles and its application

Abstract

This invention relates to the field of preparation technology of camphor fruit peel anthocyanin co-coloring copolymer micro / nano particles, and discloses a preparation method and application of camphor fruit peel anthocyanin co-coloring copolymer micro / nano particles. The preparation method is as follows: collect camphor fruit, treat at low temperature, collect the peel, dry at low temperature, pulverize and sieve, defatted, then extract with a high-efficiency solvent, purify by column chromatography to obtain anthocyanin solution, mix with co-coloring substances, then freeze-dry at low temperature to use as the core material of micro / nano particles, add chitosan and casein, stir thoroughly, and freeze-dry to obtain micro / nano particles. The micro / nano particles prepared by this invention can inhibit the inflammatory state caused by crayfish allergy at the Balb / c mouse level, and have potential anti-inflammatory and anti-allergic effects, and can be used as ingredients in anti-inflammatory and anti-allergic products.

Description

Technical Field

[0001] This invention relates to the field of preparation technology of camphor fruit peel anthocyanin co-color copolymer micro / nano particles, and particularly to the preparation of camphor fruit peel anthocyanin co-color copolymer micro / nano particles and the anti-allergic application of camphor fruit peel anthocyanins. Background Technology

[0002] Anthocyanins are water-soluble pigments widely found in natural plants, distributed in the roots, stems, leaves, flowers, fruits, and seeds of various crops. They possess anti-inflammatory and antioxidant properties, improve vision, protect the cardiovascular system, and maintain gut microbiota balance. They significantly enhance and improve human immunity, playing a vital physiological role in human health. Because plants rich in anthocyanins are widely cultivated and easily utilized, they provide a rich and diverse source of raw materials for their development and utilization.

[0003] Camphor trees are excellent for landscaping and afforestation, with large planting volumes and high fruit yields in southern regions. However, due to low utilization, a large amount of camphor fruit resources are not being used rationally, resulting in significant waste. Camphor fruit peel contains abundant anthocyanins. Further efficient utilization can enhance the high-value application of anthocyanins, promote the rational use of camphor tree resources, and improve economic and social benefits. Currently, there is considerable research on anthocyanin extraction methods, but a mature system has not yet been established in terms of extraction efficiency, extraction level, and utilization. The application level of anthocyanins from camphor fruit peel is even lower. Comprehensively improving the preparation level of anthocyanins from camphor fruit peel lays the foundation for increasing extraction efficiency and improving application methods, and also opens up bright prospects for their further application in industries such as biomedicine and health foods.

[0004] The extraction of anthocyanins from plants mainly employs traditional solvent extraction, a method that is relatively simple to operate. The challenge lies in selecting the optimal extraction solvent based on the physicochemical properties and distribution characteristics of different anthocyanins from various crop sources to achieve the best extraction results. Due to the differences in solvent properties, extraction conditions such as temperature, pH, extraction time, and solid-liquid ratio vary. Therefore, optimization based on the properties of the materials, solvents, and extraction conditions is necessary. While this method is time-consuming and consumes a large amount of solvent, it generally yields good extraction results, but it also suffers from low extraction efficiency and may not meet current extraction requirements. Furthermore, with the continuous development of anthocyanin extraction technology, various advanced extraction techniques have emerged, such as microwave extraction, ultrasonic extraction, supercritical fluid extraction, and enzymatic extraction, which are already used in anthocyanin extraction. However, establishing synergistic extraction techniques with different solvents based on specific needs remains a direction worthy of research for all researchers.

[0005] Meanwhile, anthocyanins from camphor fruit peel are rich in disaccharide-substituted structures, which differ from common glycosides. Previous studies have shown that anthocyanins have anti-inflammatory effects, but research on their application in the prevention of crayfish protein allergy in mice is very limited. Moreover, anthocyanins are highly water-soluble, poorly lipid-soluble, and unstable, and are prone to structural changes that cause them to lose their efficacy. Therefore, this invention aims to use the prepared camphor fruit peel anthocyanins for stabilization treatment to prevent crayfish food allergy. This has important research significance and practical value for improving the safety of crayfish food and is of great importance to the safety of crayfish food. Summary of the Invention

[0006] Purpose of the invention: To address the problems existing in the prior art, this invention provides a method for preparing anthocyanin co-coloring copolymer micro / nano particles from camphor fruit peel and its application. The method involves low-temperature drying of camphor fruit peel, pulverizing and sieving, defatting, extraction with a high-efficiency solvent, purification by column chromatography to obtain anthocyanin solution, mixing with co-coloring substances, and then freeze-drying at low temperature to use as the core material for micro / nano particles. Chitosan and casein are then added and thoroughly stirred, followed by freeze-drying to prepare micro / nano particles with sustained-release effects. These particles can be applied to alleviate or relieve inflammation caused by crayfish consumption, thereby improving the safety of crayfish food.

[0007] Technical solution: This invention provides a method for preparing camphor fruit peel anthocyanin co-chromogenic copolymer micro / nano particles, comprising the following steps:

[0008] S1: Add ethyl acetate to camphor fruit bark powder, sonicate, and then evaporate the solvent by rotary evaporation to obtain defatted camphor fruit bark powder;

[0009] S2: The defatted camphor fruit bark powder is mixed with ethanol solution at a certain material-liquid ratio and extracted for a period of time. After filtration of the extraction solvent, the first filtrate and filter residue are obtained by evaporation. The filter residue is mixed with acetone at a certain material-liquid ratio and extracted for a period of time. After filtration of the extraction solution, the second filtrate is obtained by evaporation. The first filtrate and the second filtrate are combined to obtain camphor fruit bark anthocyanins.

[0010] S3: Dissolve the camphor fruit peel anthocyanins in ethanol solution, and then elute them by adsorption and desorption through macroporous resin and dextran gel filtration to obtain pure camphor fruit peel anthocyanins.

[0011] S4: Add the pure anthocyanin from camphor fruit bark to phenolic acid and mix. Adjust the pH and temperature to obtain a stable solution of anthocyanin.

[0012] S5: Chitosan and casein are mixed in a certain proportion, heated to dissolve, cooled and then a stable solution of anthocyanins is added. The mixture is stirred evenly at a fixed temperature and then freeze-dried to obtain camphor fruit peel anthocyanin co-color copolymer micro-nano particles.

[0013] Furthermore, in S4, the molar ratio of the pure anthocyanin from camphor fruit peel to the phenolic acid is 1:30-60;

[0014] And / or, in S5, the mass ratio of chitosan to casein is 1:1 to 1:1.5.

[0015] Furthermore, in S4, the phenolic acid is one or more of chlorogenic acid, cinnamic acid, and rosmarinic acid;

[0016] And / or, in S4, the specific mixing conditions are: adjust the pH value to 1.5 and the temperature to 40°C.

[0017] Furthermore, in S5, the fixed temperature is 35-40°C.

[0018] Further, in S1, the ratio of camphor fruit bark powder to ethyl acetate is 1:5-1:10;

[0019] And / or, in S2, the ratio of the defatted camphor fruit peel powder to the ethanol solution is 1:10~15;

[0020] And / or, in S2, the ratio of the filter residue to acetone is 1:50~100.

[0021] Furthermore, in S1, the specific preparation method of the camphor fruit bark powder is as follows:

[0022] The camphor fruit is washed, frozen at -18℃ for 0.5-2 hours, the peel is collected, dried at 40-45℃ for 6-24 hours, crushed and sieved to obtain camphor fruit peel powder.

[0023] Preferably, in S1, the specific conditions for ultrasonic treatment are: 100W power, 25-35℃ temperature for 2-5 hours.

[0024] Furthermore, in S2, the volume ratio of the ethanol solution is 80%;

[0025] And / or, in S2, the volume ratio of the acetone solution is 80%;

[0026] And / or, in S2, the specific extraction conditions are: room temperature extraction for 5-12 hours;

[0027] And / or, in S2, the specific extraction conditions are: extraction at 50℃-55℃ for 0.5-2h.

[0028] Further, in S3, the macroporous resin is AB-8 or D4020; the eluent for eluting the macroporous resin is an ethanol solution with a pH of 1.5-2.5 and a volume ratio of 85%-95%.

[0029] And / or, in S3, the dextran gel is LH-20.

[0030] The present invention also provides the application of camphor fruit peel anthocyanin co-color copolymer micro / nano particles prepared by any of the above methods in reducing the allergic effects caused by eating crayfish.

[0031] Beneficial Effects: Camphor fruit contains various anthocyanins, but they are mainly found in the pericarp. For purification purposes, the preparation method of the camphor fruit pericarp was optimized, discarding most of the pulp and refining directly from the pericarp. Since the esters and flavonoids in the sample can be removed by ultrasonic extraction, the purity of the sample can be maximized, reducing the influence of impurities. In anthocyanin extraction, ethanol and acetone were used sequentially. The second extraction with acetone solution was performed at near-boiling conditions. At this point, the solvent reached a near-boiling state, and the strong foaming and vigorous stirring significantly improved the anthocyanin extraction efficiency. After separate extraction with ethanol and acetone solutions, the anthocyanin efficiency was greatly improved, as shown in Table 1. Table 1 shows that simple ethanol extraction had poor results, while the extraction efficiency in the comparative example was significantly improved, indicating a superior extraction technique and method. The extracted anthocyanins were further purified with resin to obtain samples with high purity. However, due to the structural and property characteristics of anthocyanins, their stability is poor, making it difficult to exert their anti-inflammatory and other physiological effects. While the stability is greatly improved by preparing them as sustained-release particles, the loss of anthocyanin activity during the micro / nano particle preparation process still cannot be solved. To address this, we selected phenolic acid substances that can form stable complexes with anthocyanins from camphor fruit peel and used them as core materials to further prepare micro / nano particles, thereby improving the stability of the complex. In Balb / c mouse animal experiments, it was found that it can reduce the symptoms of food allergy inflammatory response caused by crayfish intake, thus showing a certain effect in reducing the incidence of food allergies. Therefore, it is necessary to further study to clarify its mechanism of action, which will provide a broader development space for the application of these micro / nano particles in food safety. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the embodiments.

[0033] Implementation method 1:

[0034] Mature camphor fruit with a purplish-black outer skin was collected, washed to remove impurities, air-dried, and rapidly frozen at -18 ℃ for 2 hours. The peel was then removed and the fruit collected. The fruit was dried at 40 ℃ for 8 hours, pulverized, and sieved to obtain camphor fruit peel powder. The camphor fruit peel powder was mixed with ethyl acetate at a ratio of 1:5 and ultrasonically treated at 30 ℃ for 100W for 2 hours to fully defatted it. The defatted camphor fruit peel powder was then thoroughly mixed with 80% ethanol solution at a ratio of 1:12 and extracted at room temperature for 10 hours. After extraction, a first filtrate and a residue were obtained. The residue was thoroughly mixed with 80% acetone solution at a ratio of 1:60 and extracted at 50 ℃ for 2 hours. The extract was filtered and evaporated to dryness to obtain a second filtrate. The second filtrate was combined with the first filtrate obtained above to obtain the crude anthocyanin extract from camphor fruit peel. The crude extract of camphor fruit bark anthocyanins was eluted with AB-8 eluent, which was an ethanol solution with a pH of 1.5 and a volume ratio of 85%. Then, it was eluted with LH-20 dextran gel and dried to obtain pure camphor fruit bark anthocyanins.

[0035] Then, the pure anthocyanin from camphor fruit peel was added to a chlorogenic acid solution with a molar ratio of 1:30 and mixed thoroughly. The pH was adjusted to 1.5 and the temperature to 40℃ to obtain a stable anthocyanin solution. This solution was then added to a chitosan and casein solution that had been mixed in a mass ratio of 1:1 and stirred evenly at 35℃. Finally, the solution was freeze-dried to obtain camphor fruit peel anthocyanin copolymer micro / nano particles.

[0036] Crude allergenic protein was prepared from crayfish meat protein. Balb / c sensitized mice were fed oral gavage, followed by gavage administration of the camphor fruit peel anthocyanin co-chromogenic copolymer micro / nano particles prepared in this application. Serum was collected from allergic mice and mice that had been fed the micro / nano particles prepared in this application after allergy. Changes in serum immunoassay indicators were observed and calculated through immunoassay (specific operation steps and methods can be found in relevant literature of our laboratory: Yang Rongling, et al. Research on the construction of a mouse animal model of allergy to red swamp crayfish [J]. Light Industry Science and Technology, 2019). The absorbance value of the immunoassay was used as an indicator to evaluate the allergenicity of crayfish food.

[0037] Implementation Method 2:

[0038] Mature camphor fruit with a purplish-black outer skin was collected, washed to remove impurities, air-dried, and rapidly frozen at -18 ℃ for 2 hours. The peel was then removed and the fruit collected. The fruit was dried at 40 ℃ for 8 hours, pulverized, and sieved to obtain camphor fruit peel powder. The camphor fruit peel powder was mixed with ethyl acetate at a ratio of 1:8 and ultrasonically treated at 30 ℃ for 90 W for 2.5 hours to fully defatted it. The defatted camphor fruit peel powder was then thoroughly mixed with 80% ethanol solution at a ratio of 1:10 and extracted at room temperature for 10 hours. After extraction, a first filtrate and a residue were obtained. The residue was thoroughly mixed with 80% acetone solution at a ratio of 1:80 and extracted at 50 ℃ for 1.5 hours. The extract was filtered and evaporated to dryness to obtain a second filtrate. The second filtrate was combined with the first filtrate obtained above to obtain the crude anthocyanin extract from camphor fruit peel. The crude extract of camphor fruit bark anthocyanins was eluted with AB-8 eluent, which was an ethanol solution with a pH of 1.5 and a volume ratio of 90%. Then, it was eluted with LH-20 dextran gel and dried to obtain pure camphor fruit bark anthocyanins.

[0039] Then, the pure anthocyanin from camphor fruit peel was added to a chlorogenic acid solution with a molar ratio of 1:45 and mixed thoroughly. The pH was adjusted to 1.5 and the temperature to 40℃ to obtain a stable anthocyanin solution. This solution was then added to a chitosan and casein solution that had been mixed in a mass ratio of 1:1 and stirred evenly at 35℃. Finally, the solution was freeze-dried to obtain camphor fruit peel anthocyanin copolymer micro / nano particles.

[0040] Implementation Method 3:

[0041] Mature camphor fruit with a purplish-black outer skin was collected, washed to remove impurities, air-dried, and rapidly frozen at -18 ℃ for 2 hours. The peel was then removed and the fruit collected. The fruit was dried at 40 ℃ for 8 hours, pulverized, and sieved to obtain camphor fruit peel powder. The camphor fruit peel powder was mixed with ethyl acetate at a ratio of 1:10 and ultrasonically treated at 30 ℃ for 80 W for 4 hours to fully defatted it. The defatted camphor fruit peel powder was then thoroughly mixed with 80% ethanol solution at a ratio of 1:15 and extracted at room temperature for 8 hours. After extraction, a first filtrate and a residue were obtained. The residue was thoroughly mixed with 80% acetone solution at a ratio of 1:90 and extracted at 50 ℃ for 1 hour. The extract was filtered and evaporated to dryness to obtain a second filtrate. The second filtrate was combined with the first filtrate obtained above to obtain the crude anthocyanin extract from camphor fruit peel. The crude extract of camphor fruit bark anthocyanins was eluted with AB-8 eluent, which was an ethanol solution with a pH of 1.5 and a volume ratio of 90%. Then, it was eluted with LH-20 dextran gel and dried to obtain pure camphor fruit bark anthocyanins.

[0042] Then, the pure anthocyanin from camphor fruit peel was added to a chlorogenic acid solution with a molar ratio of 1:55 and mixed thoroughly. The pH was adjusted to 2.0 and the temperature to 40℃ to obtain a stable anthocyanin solution. This solution was then added to a chitosan and casein solution that had been mixed in a mass ratio of 1:1 and stirred evenly at 35℃. Finally, the solution was freeze-dried to obtain camphor fruit peel anthocyanin copolymer micro / nano particles.

[0043] The extraction efficiencies of anthocyanins from camphor fruit using the above-described methods 1 to 3 and conventional 80% ethanol solvent are shown in Table 1.

[0044]

[0045] As can be seen from Table 1, the extraction effect of simple ethanol extraction is poor, while the extraction method provided in this application significantly improves the extraction efficiency.

[0046] The camphor fruit bark anthocyanin co-chromogenic copolymer micro / nano particles obtained in embodiments 1 to 3 were fed to Balb / c mice induced by crayfish food allergy. Serum samples were obtained and compared with unfed mice. Using mouse model serum antibodies as polyclonal antibodies, routine immunoassay procedures were performed on a blank ELISA plate, including coating, blocking, antibody-serum incubation, secondary antibody incubation, and ELISA color development. The absorbance value (450 nm) of each sample well was measured. The results are shown in Table 2.

[0047]

[0048] As shown in Table 2, the absorbance value of the micro-nano particles prepared by the method of this application was significantly reduced after feeding crayfish-allergic mice. This indicates that the prepared camphor fruit peel anthocyanin co-chromogenic copolymer micro-nano particles can slow down or reduce the allergic reaction of Balb / c mice with crayfish-induced food allergy, and therefore have potential anti-allergic effects. They can be used as food ingredients for crayfish-allergic populations.

[0049] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Use of Cinnamomum camphora pericarp anthocyanin co-pigment copolymeric micro-nanoparticles, characterized in that: The camphor fruit peel anthocyanin co-chromogenic copolymer micro / nano particles are used to reduce the allergic effects caused by eating crayfish. The preparation method of the camphor fruit peel anthocyanin co-chromogenic copolymer micro / nano particles includes the following steps: S1: Add ethyl acetate to camphor fruit bark powder, sonicate, and then evaporate the solvent by rotary evaporation to obtain defatted camphor fruit bark powder; S2: The defatted camphor fruit bark powder is mixed with ethanol solution at a certain material-liquid ratio and extracted for a period of time. After filtration of the extraction solvent, the first filtrate and filter residue are obtained by evaporation. The filter residue is mixed with acetone at a certain material-liquid ratio and extracted for a period of time. After filtration of the extraction solution, the second filtrate is obtained by evaporation. The first filtrate and the second filtrate are combined to obtain camphor fruit bark anthocyanins. S3: Dissolve the camphor fruit peel anthocyanins in ethanol solution, and then elute them by adsorption and desorption through macroporous resin and dextran gel filtration to obtain pure camphor fruit peel anthocyanins. S4: Add the pure anthocyanin from camphor fruit bark to phenolic acid and mix. Adjust the pH and temperature to obtain a stable solution of anthocyanin. S5: Chitosan and casein are mixed in a certain proportion, heated to dissolve, cooled and then a stable solution of anthocyanins is added. The mixture is stirred evenly at a fixed temperature and then freeze-dried to obtain camphor fruit peel anthocyanin co-color copolymer micro-nano particles.

2. The use of sambucus nigra L. skin anthocyanins according to claim 1, characterized by the fact that: In S4, the molar ratio of the pure anthocyanin from camphor fruit peel to the phenolic acid is 1:30-60; And / or, in S5, the mass ratio of chitosan to casein is 1:1 to 1:1.

5.

3. The application of the camphor fruit peel anthocyanin co-chromogenic copolymer micro / nano particles according to claim 1, characterized in that: In S4, the phenolic acid is one or more of chlorogenic acid, cinnamic acid, and rosmarinic acid; And / or, in S4, the specific mixing conditions are: adjust the pH value to 1.5 and the temperature to 40°C.

4. The application of the camphor fruit peel anthocyanin co-chromogenic copolymer micro / nano particles according to claim 1, characterized in that: In S5, the fixed temperature is 35-40℃.

5. The application of the camphor fruit peel anthocyanin co-chromogenic copolymer micro / nano particles according to claim 1, characterized in that: In S1, the specific preparation method of the camphor fruit bark powder is as follows: The camphor fruit is washed, frozen at -18℃ for 0.5-2 hours, the peel is collected, dried at 40-45℃ for 6-24 hours, crushed and sieved to obtain camphor fruit peel powder.

6. The application of the camphor fruit peel anthocyanin co-chromogenic copolymer micro / nano particles according to claim 1, characterized in that: In S1, the specific conditions for ultrasonic treatment are: 100W power, 25-35℃ temperature for 2-5 hours.

7. The application of the camphor fruit peel anthocyanin co-chromogenic copolymer micro / nano particles according to claim 1, characterized in that: In S2, the volume ratio of the ethanol solution is 80%; And / or, in S2, the volume ratio of the acetone solution is 80%; And / or, in S2, the specific extraction conditions are: room temperature extraction for 5-12 hours; And / or, in S2, the specific extraction conditions are: extraction at 50℃-55℃ for 0.5-2h.

8. The application of the camphor fruit peel anthocyanin co-chromogenic copolymer micro / nano particles according to claim 1, characterized in that: In S3, the macroporous resin is AB-8 or D4020; the eluent for eluting the macroporous resin is an ethanol solution with a pH of 1.5-2.5 and a volume ratio of 85%-95%. And / or, in S3, the dextran gel is LH-20.

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