Nano-emulsion based on citrus extract and preparation method of nano-emulsion

Abstract

The invention discloses a nanometer emulsion based on a citrus extract and a preparation method of the nanometer emulsion. The nano-emulsion takes citrus pectin as a main chain, gallic acid, ascorbic acid-phosphate and oleic acid are sequentially grafted, the oleic acid is lipophilic, the rest is hydrophilic, and the amphiphilic pectin can form the whitening nano-emulsion which can be stably stored for a long time. The natural citrus extract is used for replacing traditional synthetic components, has the anti-oxidation, whitening and anti-inflammatory effects, can reduce the skin sensitivity risk, and conforms to the green beauty makeup trend; meanwhile, the oil phase is anchored by a hydrophobic block, steric hindrance is formed by a hydrophilic block, the stability of the emulsion is remarkably improved, and the shelf life is prolonged to 24 months; phenolic acid and ascorbic acid contained in the traditional Chinese medicine composition can effectively scavenge free radicals, inhibit tyrosinase activity, reduce melanogenesis and reduce release of a proinflammatory factor IL-6; the composition is low in cost, environment-friendly, suitable for the fields of cosmetics and medicines and wide in market prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanoemulsion preparation, and particularly relates to a nanoemulsion based on citrus extract and a preparation method thereof. BACKGROUND

[0002] Citrus extract has attracted high attention in the fields of medicine, food, cosmetics and agriculture in recent years due to its rich bioactive components and wide application potential. A citrus pectin-based hydrogel loaded with nimbicin-derived silver nanoparticles developed by the team of Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, has both anti-infection and pro-regeneration functions, and can increase the wound healing rate of diabetic mouse models by 2.7 times. As a natural thickening agent and gelling agent, citrus pectin is used in food such as jam and yogurt, and is used for cholesterol and blood sugar management due to its dietary fiber function. The citrus flavonoid extract developed by East China University of Technology is superior to arbutin in inhibiting tyrosinase activity, and has been applied to cosmetics. Lemon aldehyde, as an antifungal agent, can be used for citrus preservation and replace traditional chemical fungicides. Nanfeng County transforms waste orange peel into pectin for export, which not only solves environmental problems but also improves economic benefits. Lemon aldehyde in orange peel is used to inhibit citrus penicilliosis and sour rot through nano-encapsulation technology to reduce postharvest losses. The application of citrus extract is expanding from traditional food additives to high-value medical, cosmetic and environmental protection fields, forming an innovative industrial model of "combination of agriculture and medicine".

[0003] Most of the skin care products on the market rely on synthetic substances (such as synthetic fragrances, preservatives, emulsifiers, etc.). Although these ingredients have low cost and high stability, they may cause skin sensitivity, irritation, and even long-term health risks (such as controversy over parabens). In addition, nanoemulsions in traditional skin care products (such as vitamin E nanoemulsion, sunscreen carrier, etc.) are prone to Ostwald ripening, which leads to droplet aggregation, delamination or inactivation of active ingredients, affecting product stability and shelf life. Some synthetic ingredients (such as silicone oil, plasticizer) are difficult to degrade and may be absorbed through the skin to produce endocrine disrupting effects. Under the global "pure makeup trend, the demand for natural ingredients is increasing (such as the European Union has limited the use of 23 synthetic fragrances in cosmetics). SUMMARY

[0004] In view of the above problems, the present application aims to provide a nanoemulsion based on citrus extract and a preparation method thereof. To achieve the purpose of the present application, the following technical solutions are adopted: A preparation method of a nanoemulsion based on citrus extract, comprising the following steps: 1) Preparation of phenolic acid grafted pectin At room temperature, the citrus pectin and 1% acetic acid solution were put into a container, stirred until completely dissolved, then added gallic acid, stirred again, then added ascorbic acid and 10 mol / L H2O2 solution to start the reaction; continuously filled with nitrogen for 1 h, loaded into dialysis bag and dialyzed in distilled water for 72 h, freeze-dried for 24 h; the ungrafted phenolic acid was removed by Soxhlet extraction with ethanol for 8 h, and the obtained copolymer was again dialyzed in distilled water and freeze-dried to obtain phenolic acid grafted pectin; the phenolic hydroxyl group of phenolic acid (gallic acid) and part of the carboxyl group of the citrus pectin molecule form phenolic acid polyester grafted pectin through esterification polymerization reaction; ; 2) Preparation of ascorbic acid grafted phosphate ester Into a reactor equipped with a thermometer and a pH meter, ascorbic acid and distilled water 17-18 mL were added, stirred at room temperature for 10 min; the solution pH was adjusted to 10.5-11.5 with 10 mol / L sodium hydroxide, and sodium trimetaphosphate was slowly added, and the temperature was raised to 55°C for reaction, 10 mol / L sodium hydroxide was intermittently added to keep the reaction pH at 10.4-10.7, and thin layer chromatography was used for tracking detection until the reaction was completed; suction filtration, and the filter cake was washed with a small amount of methanol, dried and crushed to obtain a white powder solid, ascorbic acid grafted phosphate ester was obtained; under alkaline conditions, hydrolysis ring opening generates linear polyphosphate, providing P=O group to react with the hydroxyl group (α position) of ascorbic acid, obtaining ascorbic acid (vitamin C) side chain with phosphate ester group at the end; ; 3) Preparation of phosphate ester grafted pectin At a temperature of about 50°C, the phenolic acid grafted pectin obtained in 1) and the ascorbic acid grafted phosphate ester obtained in 2) were added to the reactor, and dicyclohexyl carbodiimide was added to dehydrate, and the reaction was continued for 3 h to obtain phosphate ester grafted pectin; the terminal phosphate ester hydroxyl group of ascorbic acid grafted phosphate ester reacts with part of the carboxyl group of the pectin molecule through esterification, so that after the pectin main chain introduces the phenolic acid polyester chain, the ascorbic acid grafted phosphate ester side chain is continuously introduced, and the two side chains are both hydrophilic groups; ; 4) Oil acid activation treatment Under the condition of 60-70°C, oil acid was added to a device equipped with stirring function, condenser tube and constant pressure dropping funnel, and then excess thionyl chloride was slowly dropped through the constant pressure dropping funnel under N2 condition, and the dropping was completed in 30-60 min, and the reaction was completely guaranteed by refluxing for 2-3 h, and thin layer chromatography was used for tracking detection until the reaction was completed, and the activated oil acid was obtained; this step is the acyl chloride reaction of carboxyl group, and oil acid chloride is obtained; ; 5) Hydrophobic linkage grafting reaction The phosphonate grafted pectin obtained in 3) and the activated oleic acid obtained in 4) are dissolved in N, N-dimethylformamide in a reactor, stirred uniformly, continuously charged with N2, the temperature is controlled at 40-60℃, the reaction is carried out for 2-3h, frozen and dried for 24h, the ungrafted oleic acid is removed by Soxhlet extraction with ethanol for 8h, finally, the obtained copolymer is put into a dialysis bag and dialyzed in distilled water for 72h, the obtained copolymer is again dialyzed in distilled water and frozen and dried, thus obtaining the multifunctional group grafted pectin, which has phenolic acid, ascorbic acid and oleic acid on the side chain of citrus pectin; the acylation reaction of oleoyl chloride and the hydroxyl group in the phosphonate grafted pectin is carried out, thus forming the hydrophobic oleic acid segment, thereby forming the hydrophilic and hydrophobic co-chain macromolecular structure; ; 6) Emulsion preparation The deionized water, glycerol, olive oil and sodium hyaluronate are used as the water phase, the phenolic acid, ascorbic acid and oleic acid co-grafted citrus pectin obtained in 5) is used as the oil phase, the water phase is added into the oil phase, the saponin is added as the surfactant, a magnetic stirrer is used to stir at 30℃ for 2h, the high-speed shearing machine is used to shear at 14000r / min for 2min, thus obtaining the coarse emulsion; finally, the microfluidizer homogenizer is used to homogenize for 5 times under a certain homogenization pressure, thus obtaining the citrus extract-based nanoemulsion, i.e. the final product. Finally, the multifunctional group grafted pectin is mixed with the water phase, the oil-in-water structure is formed under the high-speed shearing condition, the hydrophobic oleic acid chain is dissolved into the internal olive oil phase, and the remaining hydrophilic chain part is well dissolved in the water phase, thus finally obtaining the homogeneous and high-stability emulsion.

[0005] Preferably, the raw materials used in 1) are as follows: citrus pectin 4-6 parts; gallic acid 8-12 parts; ascorbic acid 1-2 parts; 10mol / L H2O2 solution 0.25-0.4 parts.

[0006] Preferably, the raw materials used in 2) are as follows: ascorbic acid 4-6 parts; the concentration of the ascorbic acid solution is 1.5-2.0mol / L; 10mol / L H2O2 solution 0.25-0.4 parts; sodium trimetaphosphate 11-12 parts.

[0007] Preferably, the raw materials used in 3) are as follows: phenolic acid grafted pectin 4.8-7.2 parts; ascorbic acid grafted phosphonate 15-18 parts; dicyclohexyl carbodiimide 0.3-0.5 parts.

[0008] Preferably, the raw materials used in 4) are as follows: oleic acid 4.8-7.2 parts; thionyl chloride 2.4-3.6 parts.

[0009] Preferably, the raw materials used in 5) are as follows: phosphonate grafted pectin 19.8-25.2 parts; activated oleic acid 0.4-0.6 parts.

[0010] Preferably, the raw material quality parts used in 6) are: deionized water 70-85 parts; glycerol 5-15 parts; olive oil: 5 parts; sodium hyaluronate 0.05-0.5 parts; saponin 0.5-1.2 parts.

[0011] The beneficial effects of the present application are: (1) The graft copolymerization method ensures the stability of the nanoemulsion: Unlike traditional nanoemulsions, which cause Osswald ripening leading to droplet fusion, delamination, and affecting product stability. The present application uses an amphiphilic copolymer as a stabilizer, in which the oleic acid chain is anchored in the oil droplet as a hydrophobic segment, reducing the oil phase migration rate, and the modified citrus pectin forms a steric hindrance layer as a hydrophilic segment, inhibiting droplet aggregation. The copolymer forms a dense network structure (thickness about 10 nm) at the oil-water interface, effectively blocking the diffusion of small molecules. The interface engineering of the copolymer makes the shelf life of the nanoemulsion up to 24 months (industry average 12 months).

[0012] (2) Natural ingredients: Unlike traditional commercially available skin care products that rely on phenoxyethanol as a preservative and PEG-40 hydrogenated castor oil as an emulsifier, these ingredients, although low in cost and high in stability, may cause skin sensitivity, irritation, and even long-term health risks. The present application is based on natural ingredients from citrus extracts, which have natural preservative, antibacterial, emulsifying stability, and biocompatibility. Not only can it replace synthetic substances to reduce safety risks, but also can solve the stability problem of nanoemulsion through interface modification and biological wrapping technology. It meets the EU EC1223 / 2009 regulation on the prohibition of 26 kinds of sensitizing fragrances, and will further promote the development of natural functional skin care products by combining green chemical processes in the future.

[0013] (3) The product has the functions of whitening, anti-inflammatory and antioxidant: The citrus pectin molecule itself and the grafted phenolic acid can effectively scavenge various free radicals such as DPPH and ABTS, and have excellent antioxidant effect. Through esterification reaction, ascorbic acid is grafted onto the phosphate, which slowly releases ascorbic acid after contacting with the skin surface cells, greatly inhibits the activity of tyrosinase, reduces the generation of melanin, and ensures the whitening and anti-inflammatory effect of the product. In addition, in the experiment of lipopolysaccharide-induced macrophages, citrus extract can significantly reduce the production of anti-inflammatory factors, and the release of key pro-inflammatory substance IL-6 is reduced by 62%, effectively relieving skin redness and irritation.

[0014] (4) Raw materials are cheap and easy to obtain: Citrus is the highest yield fruit in China, with an annual output of over 500 million tons. The processing by-products (peel accounting for 20%-30%) are rich in active ingredients such as gallic acid (0.5%-1.2%) and pectin (15%-30%). The required raw materials such as gallic acid exist in the citrus peel, which can be extracted by low common solvent. To some extent, it realizes waste utilization and meets the green chemical trend. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 NMR spectrum of the phenolic acid polyester grafted pectin prepared in Example 3 of the present application; Figure 2 NMR spectrum of the ascorbic acid grafted phosphate prepared in Example 3 of the present application; Figure 3 NMR spectrum of the phosphate grafted pectin prepared in Example 3 of the present application; Figure 4 NMR spectrum of the oleoyl chloride prepared in Example 3 of the present application; Figure 5 NMR spectrum of the multifunctional group grafted pectin prepared in Example 3 of the present application. DETAILED DESCRIPTION

[0016] The technical solutions of the present application and their effects will be further described below in combination with the drawings in the embodiments of the present application. The following examples are only used to illustrate the content of the present application and are not used to limit the protection scope of the present application. Simple changes made by applying the concept of the present application to the present application are within the scope of protection claimed by the present application.

[0017] The sources of the materials in the following examples and comparative examples are as follows: Citrus pectin: Henan Bai Zhuo Pectin Biotechnology Co., Ltd.; Gallic acid: Shanghai Aladdin Bio-Science Co., Ltd.; Acetic acid solution: Shanghai Macklin Biochemical Technology Co., Ltd. Sodium trimetaphosphate: Shanghai Aladdin Bio-Science Co., Ltd.; Oleic acid: Shanghai Macklin Biochemical Technology Co., Ltd.; 10 mol / L H2O2 solution: Shanghai Macklin Biochemical Technology Co., Ltd.; N,N-dimethylformamide: Shanghai Aladdin Bio-Science Co., Ltd.; Dicyclohexyl carbodiimide: Shanghai Aladdin Bio-Science Co., Ltd.; Sodium hydroxide: Shanghai Macklin Biochemical Technology Co., Ltd.; Methanol: Shanghai Aladdin Bio-Science Co., Ltd.; Acetone: Shanghai Aladdin Bio-Science Co., Ltd.; Ascorbic acid: Tianjin Guangcheng Chemical Co., Ltd.; Thionyl chloride: Tianjin Guangcheng Chemical Co., Ltd.; Deionized water: self-made; Glycerol: Tianjin Guangcheng Chemical Co., Ltd.; Sodium hyaluronate: Shanghai Macklin Biochemical Technology Co., Ltd.; Saponin: Liaoning Kelong Fine Chemical Co., Ltd.

[0018] Examples 1-5, Comparative Examples 1-4 all use the same synthesis method of the nanoemulsion based on citrus extract in the invention content.

[0019] Example 1 1) At room temperature, 5 g of citrus pectin and 150 g of 1% acetic acid solution were placed in a container, stirred until completely dissolved, then 10 g of gallic acid was added, and stirred again to be uniform, then 1.5 g of ascorbic acid and 0.35 g of 10 mol / L H2O2 solution were added to start the reaction; continuously charge nitrogen for 1 h, load into a dialysis bag and dialyze in distilled water for 72 h, freeze-dry for 24 h; use ethanol soxhlet extraction for 8 h to remove ungrafted phenolic acid, and the obtained copolymer is again dialyzed in distilled water and freeze-dried to obtain phenolic acid grafted pectin; 2) Into a reactor equipped with a thermometer and pH meter, 5 g of ascorbic acid and 17.5 g of distilled water were added, stirred at room temperature for 10 min; adjust the solution pH to 10.5-11.5 with 10 mol / L sodium hydroxide, slowly add 11.5 g of sodium trimetaphosphate, heat to 55°C for reaction, intermittently add 10 mol / L sodium hydroxide to keep the reaction pH at 10.4-10.7, and track the detection by thin layer chromatography until the reaction is completed; suction filtration, and the filter cake is washed with a small amount of methanol, dried and crushed to obtain a white powder solid, obtaining ascorbic acid grafted phosphate; 3) At a temperature of about 50°C, the phenolic acid grafted pectin obtained in 1) and the ascorbic acid grafted phosphate obtained in 2) were added to the reactor, and 0.4 g of dicyclohexyl carbodiimide was added to dehydrate, and the reaction was continued for 3 h to obtain phosphate grafted pectin; 4) Under the condition of 60-70°C, 6.0 g of oleic acid was added to a device equipped with stirring function, condenser tube and constant pressure dropping funnel, and 3.0 g of chlorosulfoxide was slowly added through the constant pressure dropping funnel under N2 condition, and the dropping was completed in 30-60 min. Reflux for 2-3 h to ensure complete reaction, and track the detection by thin layer chromatography until the reaction is completed, to obtain activated oleic acid; 5) The phosphate grafted pectin obtained in 3) and the activated oleic acid obtained in 4) were dissolved in N,N-dimethylformamide in a reactor, stirred uniformly, continuously charged with N2, and the temperature was controlled at 40-60°C, the reaction was carried out for 2-3 h, freeze-dried for 24 h, and then ethanol soxhlet extraction was used for 8 h to remove ungrafted oleic acid, finally loaded into a dialysis bag and dialyzed in distilled water for 72 h, and the obtained copolymer was again dialyzed in distilled water and freeze-dried to obtain citrus pectin grafted with phenolic acid, ascorbic acid and oleic acid; 6) with 78 g of deionized water, 10 g of glycerol, 5 g of olive oil and 0.01 g of sodium hyaluronate as the water phase, 5) the obtained phenolic acid, ascorbic acid and oleic acid co-grafted citrus pectin as the oil phase, the water phase was added to the oil phase, and 0.7 saponin was added as a surfactant, and stirred at 30°C for 2h with a magnetic stirrer to make it uniform, and sheared at 14000r / min for 2min with a high-speed shearing machine to obtain a coarse emulsion; finally, under a certain homogenization pressure, the microfluidizer homogenizer was used for 5 times to obtain a citrus extract-based nanoemulsion, i.e. the final product.

[0020] Example 2 1) At room temperature, 4 g of citrus pectin was placed in a container with 100 g of 1% acetic acid solution, stirred until completely dissolved, then 8 g of gallic acid was added, and stirred again to make it uniform, then 1.0 g of ascorbic acid and 0.25 g of 10 mol / L H2O2 solution were added to start the reaction; continuously fill in nitrogen for 1h, put it in a dialysis bag and dialyze in distilled water for 72h, freeze-dry for 24h; use ethanol soxhlet extraction for 8h to remove ungrafted phenolic acid, and the obtained copolymer is again subjected to distilled water dialysis and freeze-drying to obtain phenolic acid grafted pectin; 2) Into a reactor equipped with a thermometer and pH meter, add 6 g of ascorbic acid and 18 g of distilled water, stir at room temperature for 10 min; adjust the solution pH to 10.5-11.5 with 10 mol / L sodium hydroxide, slowly add 11.0 g of sodium trimetaphosphate, heat to 55°C for reaction, intermittently add 10 mol / L sodium hydroxide to maintain the reaction pH at 10.4-10.7, and track the reaction to completion by thin layer chromatography; suction filtration, and wash the filter cake with a small amount of methanol, dry and crush to obtain a white powder solid, to obtain ascorbic acid grafted phosphate; 3) At a temperature of about 50°C, add the phenolic acid grafted pectin obtained in 1) and the ascorbic acid grafted phosphate obtained in 2) to the reactor, add 0.3 g of dicyclohexyl carbodiimide to dehydrate, and continue to react for 3h to obtain phosphate grafted pectin; 4) Under the condition of 60-70°C, add 4.8 g of oleic acid to the device equipped with stirring function, condenser tube and constant pressure dropping funnel, and then slowly drop 2.4 g of chlorosulfoxide through the constant pressure dropping funnel under N2 condition, drop for 30-60 min, reflux for 2-3h to ensure the reaction is complete, and track the reaction to completion by thin layer chromatography, to obtain activated oleic acid; 5) The phosphonate grafted pectin obtained in 3) and the activated oleic acid obtained in 4) were dissolved in N,N-dimethylformamide in a reactor, stirred uniformly, continuously filled with N2, the temperature was controlled at 40-60℃, and reacted for 2-3h, then freeze-dried for 24h, and the ungrafted oleic acid was removed by ethanol Soxhlet extraction for 8h, and finally the product was put into a dialysis bag and dialyzed in distilled water for 72h to obtain the copolymer, which was then dialyzed in distilled water again, freeze-dried to obtain the citrus pectin grafted with phenolic acid, ascorbic acid and oleic acid; 6) The water phase was prepared by using 70g deionized water, 5g glycerol, 5g olive oil and 0.5g sodium hyaluronate, the oil phase was prepared by using the citrus pectin grafted with phenolic acid, ascorbic acid and oleic acid obtained in 5), the water phase was added into the oil phase, 1.2 saponins were added as surfactants, and the mixture was stirred at 30℃ for 2h by using a magnetic stirrer, then sheared at 14000r / min for 2min by using a high-speed shearing machine to obtain a coarse emulsion, and finally the nanoemulsion based on citrus extract, i.e. the final product, was obtained by homogenizing the coarse emulsion for 5 times by using a microfluidic homogenizer under a certain homogenization pressure.

[0021] Example 3 1) At room temperature, 6g of citrus pectin was put into a container with 200g of 1% acetic acid solution, stirred until completely dissolved, then 12g of gallic acid was added, stirred again, followed by the addition of 2.0g of ascorbic acid and 0.4g of 10mol / L H2O2 solution to start the reaction, continuously filled with nitrogen for 1h, put into a dialysis bag and dialyzed in distilled water for 72h, and freeze-dried for 24h, then the ungrafted phenolic acid was removed by ethanol Soxhlet extraction for 8h, and the obtained copolymer was dialyzed in distilled water again, freeze-dried to obtain the phenolic acid grafted pectin, as shown in Figure 1 , the product has obvious hydrogen characteristic peaks of phenolic acid δ9.7; 2) A reactor equipped with a thermometer and a pH meter was used, 6g of ascorbic acid and 18.0g of distilled water were added, stirred at room temperature for 10min, the pH of the solution was adjusted to 10.5-11.5 by using 10mol / L sodium hydroxide, 11g of sodium trimetaphosphate was slowly added, the temperature was raised to 55℃ for reaction, 10mol / L sodium hydroxide was intermittently added to keep the reaction pH at 10.4-10.7, and thin layer chromatography was used for tracking detection until the reaction was completed, then the mixture was suction filtered, the filter cake was washed with a small amount of methanol, dried and crushed to obtain a white powder solid, and the ascorbic acid grafted phosphate was obtained, as shown in Figure 2 , the product has obvious hydrogen characteristic peaks of phosphate δ4.0; 3) At a temperature of about 50℃, the phenolic acid grafted pectin obtained in 1) and the ascorbic acid grafted phosphate obtained in 2) were added into a reactor, 0.5g of dicyclohexyl carbodiimide was added to dehydrate, and the reaction was continued for 3h to obtain the phosphonate grafted pectin, as shown in Figure 3 , the hydrogen of phenolic acid and the hydrogen of phosphate have changed compared with 1) and 2). 4) In the device equipped with stirring function, condenser, constant pressure dropping funnel, 7.2 g of oleic acid was added at 60-70 °C, 3.6 g of chlorosulfoxide was slowly added through the constant pressure dropping funnel under the condition of N2, and the dropping was completed in 30-60 min, and the reflux was carried out for 2-3 h to ensure the completion of the reaction, and the thin layer chromatography was used for tracking detection until the reaction was completed, and the activated oleic acid, i.e. oleoyl chloride, was obtained. Figure 4 5) The phosphonate grafted pectin obtained in 3) and the activated oleic acid obtained in 4) were dissolved in N,N-dimethylformamide in a reactor, stirred uniformly, continuously filled with N2, and the temperature was controlled at 40-60 °C, and the reaction was carried out for 2-3 h, and the freeze-drying was carried out for 24 h, and the non-grafted oleic acid was removed by using ethanol soxhlet extraction for 8 h, and finally the dialysis bag was loaded and dialyzed in distilled water for 72 h, and the obtained copolymer was again subjected to dialysis in distilled water, freeze-dried, and the pectin grafted with gallic acid, ascorbic acid and oleic acid was obtained, as shown in Figure 5 6) The water phase was prepared by using 85 g of deionized water, 15 g of glycerol, 5 g of olive oil and 0.5 g of sodium hyaluronate, the oil phase was prepared by using the pectin grafted with gallic acid, ascorbic acid and oleic acid obtained in 5), the water phase was added to the oil phase, 1.2 g of saponin was added as a surfactant, a magnetic stirrer was used for stirring at 30 °C for 2 h to make the mixture uniform, a high-speed shearing machine was used for shearing at 14000 r / min for 2 min to obtain a coarse emulsion, and finally a micro-jet homogenizer was used for homogenization under a certain homogenization pressure for 5 times to obtain a nanoemulsion based on citrus extract, i.e. the final product.

[0022] Example 4 Compared with examples 1-3, the difference of example 4 is that the proportion of raw materials is changed as follows: citrus pectin 4 g, 10% acetic acid solution 200 g, gallic acid 8 g, ascorbic acid in 1) 1 g, 10 mol / L H2O2 0.25 g, ascorbic acid in 2) 6 g, sodium trimetaphosphate 11 g, dicyclohexyl carbodiimide 0.3 g, oleic acid 7.2 g, chlorosulfoxide 2.4 g, deionized water 85 g, glycerol 5 g, olive oil 5 g, sodium hyaluronate 0.05 g, saponin 1.2 g.

[0023] Example 5 Compared with examples 1-3, the difference of example 5 is that the proportion of raw materials is changed as follows: citrus pectin 6 g, 10% acetic acid solution 100 g, gallic acid 12 g, ascorbic acid in 1) 2 g, 10 mol / L H2O2 0.40 g, ascorbic acid in 2) 4 g, sodium trimetaphosphate 12 g, dicyclohexyl carbodiimide 0.5 g, oleic acid 4.8 g, chlorosulfoxide 3.6 g, deionized water 70 g, glycerol 15 g, olive oil 5 g, sodium hyaluronate 0.5 g, saponin 0.5 g. ​​

[0024] Comparative Example 1 The same as Example 1, except that no phenolic acid (gallic acid) was grafted.

[0025] Comparative Example 2 The same as Example 1, except that no phosphate ester was grafted to ascorbic acid.

[0026] Comparative Example 3 The same as Example 1, except that no oleic acid chain was grafted on pectin.

[0027] Comparative Example 4 A whitening and anti-inflammatory nanoemulsion commercially available from the company Dianonano Biotech Co., Ltd.

[0028] Example 1-5 and Comparative Examples 1-4 were all tested as follows: 1. Stability test (1) Dynamic light scattering (DLS) determination Test method: Dynamic light scattering instrument (Malvern Zetasizer Nano ZS) was used, and the test temperature was 25°C. The nanoemulsion was diluted to an appropriate concentration (to avoid multiple scattering interference), filtered through a 0.45 μm filter membrane, and then injected. The DLS instrument detects the change in scattering light intensity, and through autocorrelation function analysis of the time scale of these fluctuations, the diffusion coefficient (D) of the particles is calculated, and the diffusion coefficient is converted to the hydrodynamic diameter (d H ) using the Stokes-Einstein equation: d H= =

[0029] (k B : Boltzmann constant; T: absolute temperature (K); : solvent viscosity; D: diffusion coefficient) Test purpose: To determine the hydrodynamic diameter (d H ), polydispersity index (PDI), and long-term storage stability, and to verify the anti-aggregation and anti-Ostwald ripening ability. The polydispersity index (PDI) reflects the uniformity of the particle size.

[0030] 2) Zeta potential monitoring Test method: Zeta potential is calculated from the Doppler shift of the scattered light as the particles move in an applied electric field. The sample is diluted with a low conductivity buffer (e.g. 1 mM KCI or pure water) to avoid screening of the surface charges by high ionic strength; 0.45 pm filter is used to remove impurities (important for nanoemulsions). Platinum or carbon electrodes are used (to avoid interference from electrolytic reactions), typically 5-20 V / cm (too high can cause Joule heating, too low can give weak signals), and the temperature is kept constant at 25 ± 0.1 °C (temperature affects the viscosity and dielectric constant of the solvent). The instrument is set to Zeta potential mode, and the instrument automatically alternates between positive and negative electric fields. A single measurement takes about 30-60 seconds, and the average of three measurements is taken. The instrument software directly outputs the Zeta potential (unit: mV).

[0031] Test purpose: Zeta potential is a key parameter to characterize the surface charge property of nanoparticles, and directly affects the stability of colloidal systems.

[0032] 2. Efficacy test (1) Free radical scavenging test (antioxidant performance) Determination of hydroxyl radical scavenging ability: 1.0 mg / mL of citrus pectin graft copolymer 2 mL, add 6 mmol / L equal volume of ferric sulfate, stir evenly, stand in the dark for 10 min, then add the same concentration and volume of salicylic acid, stir evenly, and stabilize in the dark environment for 30 min. Set the ultraviolet wavelength to 510 nm and repeat three times. The hydroxyl radical scavenging rate is calculated as follows: Hydroxyl radical scavenging rate

[0033] In the formula: A y : absorbance value of sample solution; A h : absorbance value of deionized water instead of salicylic acid solution; A j : absorbance value of deionized water instead of sample solution.

[0034] (2) Determination of DPPH free radical scavenging ability, 2 mL of sample solution is mixed with equal volume of DPPH-anhydrous ethanol solution (0.1 mmol / L), and is stabilized in the dark environment for 30 min. The ultraviolet wavelength is set to 517 nm and repeated three times. The DPPH free radical scavenging rate is calculated as follows: DPPH free radical scavenging rate

[0035] In the formula: A y : absorbance value of sample solution; A h : absorbance value of anhydrous ethanol instead of DPPH anhydrous ethanol solution; A j : absorbance value of anhydrous ethanol instead of sample solution.

[0036] 2) Tyrosinase activity test (whitening performance) Test method: human epidermal melanocytes were inoculated in 96-well plates (1 x 10 4 / well) and cultured for 24 hours. The blank group was not added with tyrosinase, and the same concentration of citrus nanoemulsion was added. The control group was not added with sample. Incubation was carried out at 37°C for 30 min, and the absorbance at 475 nm wavelength (characteristic absorption peak of dopaquinone) was immediately determined, and the inhibition rate was calculated: Inhibition rate (%) = (1- ) x 100% Test principle: tyrosinase catalyzes the oxidation of substrate (L-dopa) to generate dopaquinone (melanin precursor), and the inhibition rate of enzyme activity is evaluated by determining the absorbance change of the reaction product.

[0037] 3) Macrophage model test (anti-inflammatory performance) RAW264.7 macrophages were cultured in cell bottles, and when the area of cell growth accounted for 80%-90%, the cells were digested and replaced with culture bottles and medium for subculture. When the cells were observed to be in the logarithmic phase, the normally growing cells were digested, centrifuged, resuspended and inoculated in 96-well plates. Continue to culture in a constant temperature incubator at 37°C with a carbon dioxide content of 5%. The grouping of this experiment is as follows: blank group (DMEM complete medium without other reagents), sample group (add high, medium and low dose sample solution for culture), positive control group (add lipopolysaccharide for culture).

[0038] After grouping and culturing, 20 μL of 5 mg / mL MTT solution was added to each well and incubated for 4 h. The supernatant was discarded, 150 μL of DMSO was added to each well, and oscillation was carried out for 10 min. The wavelength of the enzyme marker was set to 490 nm, and the cell survival rate was calculated: Cell survival rate (%) = x 100% 3. Actual application simulation test Under the conditions of temperature 40±2°C and humidity 75%-80%, the physical and chemical stability was detected once a month, and the residual content of active ingredients was detected by high performance liquid chromatography; continuous detection for 4 months.

[0039] The above performance tests were carried out on examples 1-5 and the control group, and the test results are shown in table 1 Table 1 Performance test of examples 1-5

[0040] It should be noted that the four data in the residual content of active ingredients in table 1 are the retention of active ingredients after the emulsion is prepared for the first, second, third and fourth months, respectively.

[0041] The data in the table shows that the citrus extract-based nanoemulsions of Examples 1-5 perform well and have little difference, with the largest hydrodynamic diameter of Example 4 greatly reducing the possibility of Ostwald ripening, the core reason being that the oleic acid chain as the hydrophobic segment can be anchored in the oil droplet interior, which can tightly bind the oil phase molecules through hydrophobic interaction, significantly reducing the migration rate of the oil phase molecules, equivalent to wrapping the oil phase in the oil droplet interior, preventing the oil in the small droplets from diffusing to the large droplets, cutting off the cause of uneven droplet size from the beginning of ripening, and avoiding subsequent fusion and stratification. The particle sizes of Examples 3-5 are larger, which may be more suitable for long-term needs; the free radical scavenging rates of Examples 2-3 perform slightly higher, which may be related to the grafting of phenolic acid components; the amylase inhibitory effect of Example 5 is the most outstanding, which may be used as a product focusing on whitening effect; the cell survival rates of all examples are greater than 80%, which meet the requirements of biological materials; every other month test results show that even in the fourth month, the remaining content of active ingredients reaches 85% and above, and the product has excellent shelf life.

[0042] Table 2, Comparative Examples 1-4 and Example 1 were tested for performance

[0043] As can be seen from Table 2, the performance of each data of Comparative Examples 1-4 is weaker than that of Example 1, and the performance gap of Comparative Example 4 compared with Example 1 is the most significant. The hydroxyl radical and DPPH radical scavenging capacity of Comparative Example 1 decreases significantly compared with other comparative examples, which reflects the indispensable role of phenolic acid in antioxidant and free radical scavenging; the difference in amylase inhibition rate of Comparative Example 2 is the most obvious, which reflects the irreplaceable role of ascorbic acid in whitening; Comparative Example 3 has obvious differences in hydrodynamic diameter, polydispersity index, and Zeta potential, indicating that the dispersion function of the polymer has failed, the hydrophobicity of the citrus pectin polymer without oleic acid has decreased significantly, and the emulsion is prone to Ostwald polymerization, greatly shortening the shelf life of the product, thus showing the importance of the oleic acid molecule as the hydrophobic segment.

[0044] The above examples are only illustrative of the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. A method for preparing a nanoemulsion based on citrus extract, characterized in that: The following steps are involved: 1) Preparation of phenolic acid grafted pectin At room temperature, citrus pectin and a 1% acetic acid solution were placed in a container and stirred until completely dissolved. Gallic acid was then added and stirred again. Ascorbic acid and a 10 mol / L H2O2 solution were then added to initiate the reaction. Nitrogen was continuously aerated and the reaction continued for 1 hour. The mixture was then placed in a dialysis bag and dialyzed in distilled water for 72 hours and freeze-dried for 24 hours. Ungrafted phenolic acids were removed by Soxhlet extraction with ethanol for 8 hours. The resulting copolymer was then dialyzed against distilled water and freeze-dried to obtain phenolic acid-grafted pectin. 2) Preparation of ascorbic acid grafted phosphate Add ascorbic acid and 17-18 mL of distilled water to a reactor equipped with a thermometer and a pH meter, and stir at room temperature for 10 minutes. Adjust the pH of the solution to 10.5-11.5 with 10 mol / L sodium hydroxide, slowly add sodium trimetaphosphate, raise the temperature to 55°C for reaction, intermittently add 10 mol / L sodium hydroxide to maintain the reaction pH at 10.4-10.7, and monitor the reaction by thin-layer chromatography until the reaction is complete. Filter with suction, wash the filter cake with a small amount of methanol, dry, and then pulverize to obtain a white powdery solid to obtain ascorbic acid grafted phosphate. 3) Preparation of phosphate grafted pectin At a temperature of about 50° C., the phenolic acid grafted pectin obtained in 1) and the ascorbic acid grafted phosphate obtained in 2) were added to a reactor, dicyclohexylcarbodiimide was added for dehydration, and the reaction was continued for 3 hours to obtain phosphate grafted pectin; 4) Oleic acid activation treatment At 60-70°C, oleic acid is added to a device equipped with a stirring function, a condenser, and a constant pressure dropping funnel. Under nitrogen, an excess amount of thionyl chloride is slowly added dropwise through the constant pressure dropping funnel over 30-60 minutes. The mixture is refluxed for 2-3 hours to ensure complete reaction. Thin layer chromatography is used to monitor the reaction until completion, thereby obtaining activated oleic acid. 5) Hydrophobic chain grafting reaction The phosphate-grafted pectin obtained in 3) and the activated oleic acid obtained in 4) are dissolved in N,N-dimethylformamide in a reactor, stirred evenly, continuously filled with nitrogen, and the temperature is controlled at 40-60°C. The reaction is carried out for 2-3 hours, freeze-dried for 24 hours, and subjected to Soxhlet extraction with ethanol for 8 hours to remove ungrafted oleic acid. Finally, the mixture is placed in a dialysis bag and dialyzed in distilled water for 72 hours. The resulting copolymer is further dialyzed in distilled water and freeze-dried to obtain a multifunctional grafted pectin having phenolic acid, ascorbic acid, and oleic acid attached to the side chains of citrus pectin. 6) Emulsion preparation Deionized water, glycerin, olive oil and sodium hyaluronate are used as the aqueous phase, and the citrus pectin grafted with phenolic acid, ascorbic acid and oleic acid obtained in step 5 is used as the oil phase. The aqueous phase is added to the oil phase, and then saponin is added as a surfactant. The mixture is stirred at 30°C for 2 hours with a magnetic stirrer to mix evenly. The mixture is sheared at 14,000 r / min for 2 minutes with a high-speed shearing machine to obtain a coarse emulsion. Finally, the mixture is homogenized 5 times with a microfluidizer at a certain homogenizing pressure to obtain a nanoemulsion based on citrus extract, which is the final product.

2. The method for preparing a nanoemulsion based on citrus extract according to claim 1, characterized in that: The raw materials used in the above 1) are as follows: 4-6 parts by weight of citrus pectin; 8-12 parts by weight of gallic acid; 1-2 parts by weight of ascorbic acid; and 0.25-0.4 parts by weight of a 10 mol / L H2O2 solution.

3. The method for preparing a nanoemulsion based on citrus extract according to claim 1, characterized in that: The molar ratio of the raw materials used in the above 2) is: 4-6 parts of ascorbic acid; the concentration of ascorbic acid solution is 1.5-2.0 mol / L; 0.25-0.4 parts of 10 mol / L H2O2 solution; and 11-12 parts of sodium trimetaphosphate.

4. The method for preparing a nanoemulsion based on citrus extract according to claim 1, wherein: The raw materials used in the above 3) are as follows: 4.8-7.2 parts by weight of phenolic acid grafted pectin; 15-18 parts by weight of ascorbic acid grafted phosphate; and 0.3-0.5 parts by weight of dicyclohexylcarbodiimide.

5. The method for preparing a nanoemulsion based on citrus extract according to claim 1, wherein: The raw materials used in the above 4) are as follows: 4.8-7.2 parts by weight of oleic acid; 2.4-3.6 parts by weight of thionyl chloride.

6. The method for preparing a nanoemulsion based on citrus extract according to claim 1, characterized in that: The raw materials used in the above 5) are as follows: 19.8-25.2 parts by weight of phosphate-grafted pectin; 0.4-0.6 parts by weight of activated oleic acid.

7. The method for preparing a nanoemulsion based on citrus extract according to claim 1, characterized in that: The raw materials used in the above 6) are as follows: 70-85 parts of deionized water; 5-15 parts of glycerol; 5 parts of olive oil; 0.05-0.5 parts of sodium hyaluronate; and 0.5-1.2 parts of saponin.

8. A nanoemulsion based on citrus extract, characterized in that The invention is prepared by the preparation method according to any one of claims 1 to 7.

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