A nanoemulsion based on citrus extract and a method for its preparation

Abstract

The application discloses a kind of nanodroplet based on citrus extract and preparation method thereof.The nanodroplet is grafted with gallic acid, ascorbic acid-phosphate and oleic acid in turn with citrus pectin as main chain, and oleic acid is lipophilic, and the rest is hydrophilic, and amphiphilic pectin can form long-term stable storage whitening nanodroplet.The application replaces traditional synthetic ingredients with natural citrus extract, has antioxidant, whitening and anti-inflammatory effects, can reduce the risk of skin sensitivity, and meets the trend of green beauty;At the same time, the hydrophobic block anchors the oil phase, and the hydrophilic segment forms steric hindrance, which significantly improves the stability of the emulsion and prolongs the shelf life to 24 months;The phenolic acid and ascorbic acid contained therein can effectively scavenge free radicals, inhibit tyrosinase activity, reduce melanin production and reduce the release of pro-inflammatory factor IL-6;It is low in cost and environmentally friendly, suitable for cosmetics and pharmaceutical field, and has broad market prospect.

Description

Technical Field

[0001] This invention belongs to the field of nanoemulsion preparation, and particularly relates to a nanoemulsion based on citrus extract and its preparation method. Background Technology

[0002] Citrus extracts have garnered significant attention in recent years across various fields, including medicine, food, cosmetics, and agriculture, due to their rich bioactive components and broad application potential. A team from the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, developed a citrus pectin-based hydrogel loaded with silver nanoparticles derived from trifoliate orange peel, exhibiting both anti-infective and regenerative properties. In a diabetic mouse model, it increased wound healing speed by 2.7 times. Citrus pectin, as a natural thickener and gelling agent, is used in jams, yogurts, and other foods, and its dietary fiber function is utilized for cholesterol reduction and blood sugar management. A citrus flavonoid extract developed by East China University of Science and Technology demonstrates superior inhibition of tyrosinase activity compared to arbutin and has been applied in cosmetics. Citral, as an antifungal agent, can be used for citrus preservation, replacing traditional chemical fungicides. Nanfeng County is converting waste orange peels into pectin for export, addressing both environmental issues and improving economic benefits. Citral from the orange peels is used through nano-encapsulation technology to inhibit citrus blue mold and sour rot, reducing post-harvest losses. The application of citrus extracts is expanding from traditional food additives to high-value-added fields such as medicine, cosmetics and environmental protection, forming an innovative industrial model that combines agriculture and medicine.

[0003] Most skincare products on the market today rely on synthetic substances (such as synthetic fragrances, preservatives, and emulsifiers). While these ingredients are low-cost and highly stable, they may cause skin sensitivity, irritation, and even long-term health risks (such as the controversy surrounding parabens). Furthermore, nanoemulsions in traditional skincare products (such as vitamin E nanoemulsions and sunscreen carriers) are prone to droplet aggregation, layering, or active ingredient deactivation due to Ostwald curing, affecting product stability and shelf life. Some synthetic ingredients (such as silicone oil and plasticizers) are difficult to degrade and may cause endocrine disruption effects through skin absorption. The global trend towards "pure beauty" is driving increased demand for natural ingredients (for example, the EU has restricted the use of 23 synthetic fragrances in cosmetics). Summary of the Invention

[0004] To address the above problems, the present invention aims to provide a citrus extract-based nanoemulsion and its preparation method. To achieve this objective, the following technical solution is adopted:

[0005] A method for preparing a nanoemulsion based on citrus extract includes the following steps:

[0006] 1) Preparation of phenolic acid grafted pectin

[0007] 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 until homogeneous. Ascorbic acid and a 10 mol / L H2O2 solution were then added to initiate the reaction. Nitrogen gas was continuously introduced and the reaction was continued for 1 hour. The mixture was then placed in a dialysis bag and dialyzed in distilled water for 72 hours, followed by freeze-drying for 24 hours. Ungrafted phenolic acids were removed by Soxhlet extraction with ethanol for 8 hours. The resulting copolymer was then dialyzed again with distilled water and freeze-dried to obtain phenolic acid-grafted pectin. The phenolic hydroxyl groups of phenolic acid (gallic acid) and some of the carboxyl groups of citrus pectin molecules underwent esterification polymerization to form phenolic acid polyester-grafted pectin.

[0008] ;

[0009] 2) Preparation of ascorbic acid grafted phosphate

[0010] Add 17-18 mL of ascorbic acid and distilled water to a reactor equipped with a thermometer and pH meter, and stir at room temperature for 10 min. Adjust the pH of the solution to 10.5-11.5 with 10 mol / L sodium hydroxide, slowly add sodium trimetaphosphate, and heat to 55 °C for reaction. Intermittently add 10 mol / L sodium hydroxide to maintain the reaction pH at 10.4-10.7. Monitor the reaction until completion by thin-layer chromatography. Filter the solution and wash the filter cake with a small amount of methanol. After drying, pulverize to obtain a white powder solid, which is ascorbic acid grafted phosphate. Under alkaline conditions, hydrolysis and ring opening generate linear polyphosphate, providing P=O groups to react with the hydroxyl groups (α-position) of ascorbic acid to obtain ascorbic acid (vitamin C) side chains with phosphate ester groups at the ends.

[0011] ;

[0012] 3) Preparation of phosphate ester grafted pectin

[0013] At a temperature of approximately 50°C, phenolic acid-grafted pectin obtained in step 1) and ascorbic acid-grafted phosphate ester obtained in step 2) were added to a reactor. Dicyclohexylcarbodiimide was added for dehydration, and the reaction was continued for 3 hours to obtain phosphate ester-grafted pectin. The terminal phosphate hydroxyl groups of the ascorbic acid-grafted phosphate ester reacted with some of the carboxyl groups of the pectin molecules through an esterification reaction, thereby introducing phenolic acid polyester chains into the pectin backbone and further introducing ascorbic acid-grafted phosphate ester side chains. Both of these side chains are hydrophilic groups.

[0014] ;

[0015] 4) Oleic acid activation treatment

[0016] Oleic acid was added to a device equipped with a stirrer, condenser, and constant-pressure dropping funnel at 60-70℃. Under N2 conditions, excess thionyl chloride was slowly added dropwise through the constant-pressure dropping funnel over 30-60 minutes. The mixture was then refluxed for 2-3 hours to ensure complete reaction. Thin-layer chromatography was used to monitor the reaction until it ended, yielding activated oleic acid. This step is an acyl chloride reaction of the carboxyl group, yielding oleoyl chloride.

[0017] ;

[0018] 5) Hydrophobic linking branch reaction

[0019] The phosphate-grafted pectin obtained in step 3) and the activated oleic acid obtained in step 4) were dissolved in N,N-dimethylformamide in a reactor, stirred evenly, and N2 was continuously introduced. The temperature was controlled at 40-60℃, and the reaction was carried out for 2-3 hours. The mixture was then freeze-dried for 24 hours, and ungrafted oleic acid was removed by Soxhlet extraction with ethanol for 8 hours. Finally, the mixture was placed in a dialysis bag and dialyzed in distilled water for 72 hours. The resulting copolymer was dialyzed again with distilled water and freeze-dried to obtain a multifunctional grafted pectin. Phenolic acid, ascorbic acid, and oleic acid were attached to the side chains of the citrus pectin. Oleyl chloride and hydroxyl groups in the phosphate-grafted pectin underwent an acylation reaction to form a hydrophobic oleic acid segment, thereby forming a hydrophilic and hydrophobic co-chain macromolecular structure.

[0020] ;

[0021] 6) Emulsion preparation

[0022] Using deionized water, glycerol, olive oil, and sodium hyaluronate as the aqueous phase, and citrus pectin grafted with phenolic acid, ascorbic acid, and oleic acid (obtained in step 5) as the oil phase, the aqueous phase was added to the oil phase, followed by the addition of saponins as surfactants. The mixture was stirred at 30°C for 2 hours using a magnetic stirrer to ensure homogeneity. The mixture was then sheared at 14000 r / min for 2 minutes using a high-speed shear mill to obtain a crude emulsion. Finally, the emulsion was homogenized five times under a specific homogenization pressure using a microfluidic homogenizer to obtain a nanoemulsion based on citrus extract, which is the final product. Finally, the multifunctional grafted pectin was mixed with the aqueous phase and, under high-speed shearing conditions, formed an oil-in-water structure. The hydrophobic oleic acid chains dissolved into the internal olive oil phase, while the remaining hydrophilic chains dissolved well in the aqueous phase, resulting in a homogeneous and highly stable emulsion.

[0023] Preferably, the raw materials used in 1) are in the following proportions by weight: 4-6 parts citrus pectin; 8-12 parts gallic acid; 1-2 parts ascorbic acid; and 0.25-0.4 parts 10mol / L H2O2 solution.

[0024] Preferably, the raw materials used in step 2) are: 4-6 parts ascorbic acid; the concentration of the 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.

[0025] Preferably, the raw materials used in 3) are: 4.8-7.2 parts by weight of phenolic acid grafted pectin; 15-18 parts of ascorbic acid grafted phosphate; and 0.3-0.5 parts of dicyclohexylcarbodiimide.

[0026] Preferably, the raw materials used in 4) are: 4.8-7.2 parts by weight of oleic acid and 2.4-3.6 parts by weight of thionyl chloride.

[0027] Preferably, the raw materials used in step 5) are: 19.8-25.2 parts by weight of phosphate-grafted pectin; and 0.4-0.6 parts by weight of activated oleic acid.

[0028] Preferably, the raw materials used in step 6) are in the following proportions by weight: 70-85 parts deionized water; 5-15 parts glycerin; 5 parts olive oil; 0.05-0.5 parts sodium hyaluronate; and 0.5-1.2 parts saponins.

[0029] The beneficial effects of this invention are as follows:

[0030] (1) Graft copolymerization method is used to ensure the stability of nanoemulsion: Unlike traditional nanoemulsions, which suffer from droplet fusion and stratification due to Oswald ripening, affecting product stability, this invention uses an amphiphilic copolymer as a stabilizer. Oleic acid chains act as hydrophobic segments anchored within the oil droplets, reducing oil phase migration. Modified citrus pectin acts as a hydrophilic segment, forming a steric hindrance layer to inhibit droplet aggregation. This copolymer forms a dense network structure (approximately 10 nm thick) at the oil-water interface, effectively blocking the diffusion of small molecules. Copolymer interface engineering extends the shelf life of the nanoemulsion to 24 months (the industry average is 12 months).

[0031] (2) Natural Ingredients: Unlike traditional commercially available skincare products that rely on phenoxyethanol as a preservative and PEG-40 hydrogenated castor oil as an emulsifier, this invention, based on natural citrus extracts, leverages their natural preservative, antibacterial, emulsion stability, and biocompatibility to not only replace synthetic substances and reduce safety risks, but also solve the stability problem of nanoemulsions through interface modification and bio-encapsulation technology. It complies with the EU EC1223 / 2009 regulation's ban on 26 allergenic fragrances and aligns with future developments in green chemistry processes, further promoting the development of natural, functional skincare products.

[0032] (3) The product has whitening, anti-inflammatory and antioxidant effects: Citrus pectin molecules themselves and grafted phenolic acids can effectively scavenge various free radicals such as DPPH and ABTS, and have excellent antioxidant effects. Through esterification, ascorbic acid is grafted onto phosphate esters, and after contact with skin surface cells, ascorbic acid is slowly released, which greatly inhibits the activity of tyrosinase, reduces the production of melanin, and ensures the whitening and anti-inflammatory effects of the product. In addition, in the lipopolysaccharide-induced macrophage experiment, citrus extract can significantly reduce the production of anti-inflammatory factors and reduce the release of the key pro-inflammatory substance IL-6 by 62%, effectively soothing skin redness and irritation.

[0033] (4) Raw materials are cheap and readily available: Citrus is the most produced fruit in my country, with an annual output of over 50 million tons. 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%). Moreover, the required raw materials, such as gallic acid, are present in the citrus peel and can be extracted using low co-solvents. This achieves waste utilization to a certain extent and is in line with the trend of green chemistry. Attached Figure Description

[0034] Figure 1 The 1H NMR spectrum of the phenolic polyester-grafted pectin prepared in Example 3 of this invention;

[0035] Figure 2 The 1H NMR spectrum of the ascorbic acid-grafted phosphate prepared in Example 3 of this invention;

[0036] Figure 3 The 1H NMR spectrum of the phosphate-grafted pectin prepared in Example 3 of this invention;

[0037] Figure 4 The above is the 1H NMR spectrum of oleoyl chloride prepared in Example 3 of this invention;

[0038] Figure 5 The image shows the 1H NMR spectrum of the multifunctional grafted pectin prepared in Example 3 of this invention. Detailed Implementation

[0039] The technical solution and its effects of the present invention will be further described below with reference to the accompanying drawings in the embodiments of the present invention. The following embodiments are only for illustrating the content of the present invention and are not intended to limit the scope of protection of the present invention. Simple modifications made to the present invention based on the concept of the present invention are all within the scope of protection claimed by the present invention.

[0040] The sources of the materials used in the following examples and comparative examples are as follows:

[0041] Citrus pectin: Henan Baizhuo Pectin Biotechnology Co., Ltd.;

[0042] Gallic acid: Shanghai Aladdin Biotechnology Co., Ltd.;

[0043] Acetic acid solution: Shanghai Maclean Biochemical Technology Co., Ltd.

[0044] Sodium tripolyphosphate: Shanghai Aladdin Biotechnology Co., Ltd.;

[0045] Oleic acid: Shanghai Maclean Biochemical Technology Co., Ltd.;

[0046] 10 mol / L H2O2 solution: Shanghai Maclean Biochemical Technology Co., Ltd.;

[0047] N,N-Dimethylformamide: Shanghai Aladdin Biotechnology Co., Ltd.;

[0048] Dicyclohexylcarbodiimide: Shanghai Aladdin Biotechnology Co., Ltd.;

[0049] Sodium hydroxide: Shanghai Maclean Biochemical Technology Co., Ltd.;

[0050] Methanol: Shanghai Aladdin Biotechnology Co., Ltd.;

[0051] Acetone: Shanghai Aladdin Biotechnology Co., Ltd.;

[0052] Ascorbic acid: Tianjin Guangcheng Chemical Technology Co., Ltd.;

[0053] Thionyl chloride: Tianjin Guangcheng Chemical Technology Co., Ltd.;

[0054] Deionized water: homemade;

[0055] Glycerin: Tianjin Guangcheng Chemical Technology Co., Ltd.;

[0056] Sodium hyaluronate: Shanghai Maclean Biochemical Technology Co., Ltd.;

[0057] Saponins: Liaoning Kelong Fine Chemical Co., Ltd.

[0058] Examples 1-5 and Comparative Examples 1-4 all consistently employ the synthesis method of citrus extract-based nanoemulsions as described in the invention.

[0059] Example 1

[0060] 1) At room temperature, 5g of citrus pectin and 150g of 1% acetic acid solution were placed in a container and stirred until completely dissolved. Then, 10g of gallic acid was added and stirred again until homogeneous. Subsequently, 1.5g of ascorbic acid and 0.35g of 10mol / L H2O2 solution were added to start the reaction. Nitrogen gas was continuously introduced and the reaction was continued for 1 hour. The mixture was then placed in a dialysis bag and dialyzed in distilled water for 72 hours. After 24 hours of freeze-drying, ungrafted phenolic acid was removed by Soxhlet extraction with ethanol for 8 hours. The resulting copolymer was then dialyzed again with distilled water and freeze-dried to obtain phenolic acid-grafted pectin.

[0061] 2) Add 5g of ascorbic acid and 17.5g of distilled water to a reactor equipped with a thermometer and pH meter, and stir at room temperature for 10min; adjust the pH of the solution to 10.5-11.5 with 10mol / L sodium hydroxide, slowly add 11.5g of sodium trimetaphosphate, raise the temperature to 55℃ to carry out the reaction, intermittently add 10mol / L sodium hydroxide to maintain the reaction pH at 10.4-10.7, and monitor the reaction until the end of the reaction by thin-layer chromatography; filter, wash the filter cake with a small amount of methanol, dry and pulverize to obtain a white powder solid, which is ascorbic acid grafted phosphate;

[0062] 3) At a temperature of about 50℃, add the phenolic acid grafted pectin obtained in 1) and the ascorbic acid grafted phosphate ester obtained in 2) to the reactor, add 0.4g of dicyclohexylcarbodiimide for dehydration, and continue the reaction for 3h to obtain phosphate ester grafted pectin.

[0063] 4) At 60-70℃, add 6.0g of oleic acid to a device equipped with a stirrer, condenser, and constant pressure dropping funnel. Under N2 conditions, slowly add 3.0g of thionyl chloride dropwise through the constant pressure dropping funnel over 30-60 minutes. Reflux for 2-3 hours to ensure complete reaction. Monitor the reaction by thin-layer chromatography until the end of the reaction to obtain activated oleic acid.

[0064] 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 evenly, and N2 was continuously introduced. The temperature was controlled at 40-60℃ and the reaction was carried out for 2-3 hours. The mixture was then freeze-dried for 24 hours. Ungrafted oleic acid was removed by Soxhlet extraction with ethanol for 8 hours. Finally, the mixture was placed in a dialysis bag and dialyzed in distilled water for 72 hours. The resulting copolymer was dialyzed again with distilled water and freeze-dried to obtain citrus pectin co-grafted with phenolic acid, ascorbic acid and oleic acid.

[0065] 6) Using 78g deionized water, 10g glycerol, 5g olive oil, and 0.01g sodium hyaluronate as the aqueous phase, and the citrus pectin grafted with phenolic acid, ascorbic acid, and oleic acid obtained in 5) as the oil phase, the aqueous phase was added to the oil phase, and 0.7g saponin was added as a surfactant. The mixture was stirred at 30°C for 2 hours with a magnetic stirrer to ensure uniform mixing. The mixture was then sheared at 14000r / min for 2 minutes using a high-speed shear mill to obtain a crude emulsion. Finally, the mixture was homogenized 5 times using a microfluidic homogenizer under a certain homogenization pressure to obtain a nanoemulsion based on citrus extract, which is the final product.

[0066] Example 2

[0067] 1) At room temperature, 4g of citrus pectin and 100g of 1% acetic acid solution were placed in a container and stirred until completely dissolved. Then, 8g of gallic acid was added and stirred again until homogeneous. Subsequently, 1.0g of ascorbic acid and 0.25g of 10mol / L H2O2 solution were added to start the reaction. Nitrogen gas was continuously introduced and the reaction was continued for 1 hour. The mixture was then placed in a dialysis bag and dialyzed in distilled water for 72 hours. After 24 hours of freeze-drying, ungrafted phenolic acid was removed by Soxhlet extraction with ethanol for 8 hours. The resulting copolymer was then dialyzed again with distilled water and freeze-dried to obtain phenolic acid-grafted pectin.

[0068] 2) Add 6g of ascorbic acid and 18g of distilled water to a reactor equipped with a thermometer and pH meter, and stir at room temperature for 10min; adjust the pH of the solution to 10.5-11.5 with 10mol / L sodium hydroxide, slowly add 11.0g of sodium trimetaphosphate, raise the temperature to 55℃ to carry out the reaction, intermittently add 10mol / L sodium hydroxide to maintain the reaction pH at 10.4-10.7, and monitor the reaction until the end of the reaction by thin-layer chromatography; filter, wash the filter cake with a small amount of methanol, dry and pulverize to obtain a white powder solid, which is ascorbic acid grafted phosphate;

[0069] 3) At a temperature of about 50℃, add the phenolic acid grafted pectin obtained in 1) and the ascorbic acid grafted phosphate ester obtained in 2) to the reactor, add 0.3g of dicyclohexylcarbodiimide for dehydration, and continue the reaction for 3h to obtain phosphate ester grafted pectin.

[0070] 4) At 60-70℃, add 4.8g of oleic acid to a device equipped with a stirrer, condenser, and constant pressure dropping funnel. Under N2 conditions, slowly add 2.4g of thionyl chloride dropwise through the constant pressure dropping funnel over 30-60 minutes. Reflux for 2-3 hours to ensure complete reaction. Monitor the reaction by thin-layer chromatography until it ends to obtain activated oleic acid.

[0071] 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 evenly, and N2 was continuously introduced. The temperature was controlled at 40-60℃ and the reaction was carried out for 2-3 hours. The mixture was then freeze-dried for 24 hours. Ungrafted oleic acid was removed by Soxhlet extraction with ethanol for 8 hours. Finally, the mixture was placed in a dialysis bag and dialyzed in distilled water for 72 hours. The resulting copolymer was dialyzed again with distilled water and freeze-dried to obtain citrus pectin co-grafted with phenolic acid, ascorbic acid and oleic acid.

[0072] 6) Using 70g deionized water, 5g glycerol, 5g olive oil, and 0.5g sodium hyaluronate as the aqueous phase, and the citrus pectin grafted with phenolic acid, ascorbic acid, and oleic acid obtained in 5) as the oil phase, the aqueous phase was added to the oil phase, and 1.2g saponin was added as a surfactant. The mixture was stirred at 30°C for 2 hours with a magnetic stirrer to ensure uniform mixing. The mixture was then sheared at 14000r / min for 2 minutes using a high-speed shear mill to obtain a crude emulsion. Finally, the mixture was homogenized 5 times using a microfluidic homogenizer under a certain homogenization pressure to obtain a nanoemulsion based on citrus extract, which is the final product.

[0073] Example 3

[0074] 1) At room temperature, 6g of citrus pectin and 200g of 1% acetic acid solution were placed in a container and stirred until completely dissolved. Then, 12g of gallic acid was added and stirred again until homogeneous. Subsequently, 2.0g of ascorbic acid and 0.4g of 10mol / L H2O2 solution were added to initiate the reaction. Nitrogen gas was continuously introduced for 1 hour, and the mixture was then placed in a dialysis bag and dialyzed in distilled water for 72 hours. After 24 hours of freeze-drying, ungrafted phenolic acids were removed by Soxhlet extraction with ethanol for 8 hours. The resulting copolymer was then dialyzed again with distilled water and freeze-dried to obtain phenolic acid-grafted pectin, as shown below. Figure 1 As shown, the product has a distinct characteristic peak of hydrogen phenolate at δ9.7;

[0075] 2) Add 6g of ascorbic acid and 18.0g of distilled water to a reactor equipped with a thermometer and pH meter, and stir at room temperature for 10min. Adjust the pH of the solution to 10.5-11.5 with 10mol / L sodium hydroxide, slowly add 11g of sodium trimetaphosphate, and heat to 55℃ to carry out the reaction. Intermittently add 10mol / L sodium hydroxide to maintain the reaction pH at 10.4-10.7. Monitor the reaction until completion using thin-layer chromatography. Filter the solution, wash the filter cake with a small amount of methanol, dry it, and pulverize it to obtain a white powdery solid, which is ascorbic acid grafted phosphate. Figure 2 As shown, the product has a distinct characteristic hydrogen peak of phosphate ester at δ4.0;

[0076] 3) At approximately 50℃, add the phenolic acid-grafted pectin obtained in 1) and the ascorbic acid-grafted phosphate ester obtained in 2) to the reactor, add 0.5g of dicyclohexylcarbodiimide for dehydration, and continue the reaction for 3 hours to obtain phosphate ester-grafted pectin. Figure 3 As shown, the hydrogen phenolate and hydrogen phosphate esters change relative to 1) and 2);

[0077] 4) Under conditions of 60-70℃, add 7.2g of oleic acid to a device equipped with a stirrer, condenser, and constant-pressure dropping funnel. Under N2 conditions, slowly add 3.6g of thionyl chloride dropwise through the constant-pressure dropping funnel over 30-60 minutes. Reflux for 2-3 hours to ensure complete reaction. Monitor the reaction until it ends using thin-layer chromatography to obtain activated oleic acid. Figure 4 The oleoyl chloride shown;

[0078] 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 evenly, and N2 was continuously introduced. The temperature was controlled at 40-60℃, and the reaction was carried out for 2-3 hours. The mixture was then freeze-dried for 24 hours, and ungrafted oleic acid was removed by Soxhlet extraction with ethanol for 8 hours. Finally, the mixture was placed in a dialysis bag and dialyzed in distilled water for 72 hours. The resulting copolymer was dialyzed again with distilled water and freeze-dried to obtain citrus pectin co-grafted with phenolic acid, ascorbic acid, and oleic acid, as shown in the figure. Figure 5 As shown, the active hydrogen at δ9.7 is not displayed;

[0079] 6) Using 85g deionized water, 15g glycerol, 5g olive oil, and 0.5g sodium hyaluronate as the aqueous phase, and the citrus pectin grafted with phenolic acid, ascorbic acid, and oleic acid obtained in 5) as the oil phase, the aqueous phase was added to the oil phase, and then 1.2g saponin was added as a surfactant. The mixture was stirred at 30℃ for 2 hours with a magnetic stirrer to ensure uniform mixing. The mixture was then sheared at 14000r / min for 2 minutes using a high-speed shear mill to obtain a crude emulsion. Finally, the mixture was homogenized 5 times using a microfluidic homogenizer under a certain homogenization pressure to obtain a nanoemulsion based on citrus extract, which is the final product.

[0080] Example 4

[0081] Compared with Examples 1-3, the difference in Example 4 is that the proportion of raw materials is changed to: 4g of citrus pectin, 200g of 10% acetic acid solution, 8g of gallic acid, 1g of ascorbic acid in 1), 0.25g of 10mol / L H2O2, 6g of ascorbic acid in 2), 11g of sodium trimetaphosphate, 0.3g of dicyclohexylcarbodiimide, 7.2g of oleic acid, 2.4g of thionyl chloride, 85g of deionized water, 5g of glycerol, 5g of olive oil, 0.05g of sodium hyaluronate, and 1.2g of saponins.

[0082] Example 5

[0083] Compared with Examples 1-3, the difference in Example 5 is that the proportion of raw materials is changed to: 6g of citrus pectin, 100g of 10% acetic acid solution, 12g of gallic acid, 2g of ascorbic acid in 1), 0.40g of 10mol / L H2O2, 4g of ascorbic acid in 2), 12g of sodium trimetaphosphate, 0.5g of dicyclohexylcarbodiimide, 4.8g of oleic acid, 3.6g of thionyl chloride, 70g of deionized water, 15g of glycerol, 5g of olive oil, 0.5g of sodium hyaluronate, and 0.5g of saponins.

[0084] Comparative Example 1

[0085] It is basically the same as Example 1, except that phenolic acid (gallic acid) is not used for grafting.

[0086] Comparative Example 2

[0087] It is basically the same as Example 1, except that ascorbic acid is not grafted with phosphate ester.

[0088] Comparative Example 3

[0089] It is basically the same as Example 1, except that oleic acid chains are not grafted onto the pectin.

[0090] Comparative Example 4

[0091] Chennuo Nanobiotechnology Co., Ltd. sells whitening and anti-inflammatory nanoemulsions.

[0092] Examples 1-5 and Comparative Examples 1-4 were all subjected to the following tests:

[0093] 1. Stability Testing

[0094] (1) Dynamic light scattering (DLS) measurement

[0095] Test method: A dynamic light scattering (DLS) instrument (Malvern Zetasizer Nano ZS) was used at a test temperature of 25℃. The nanoemulsion was diluted to a suitable concentration (to avoid multiple scattering interference) and filtered through a 0.45μm filter before injection. The DLS instrument detected changes in the intensity of scattered light, and the time scale of these fluctuations was analyzed using the autocorrelation function to calculate the diffusion coefficient (D) of the particles. The diffusion coefficient was then converted into the hydrodynamic diameter (d) using the Stokes-Einstein equation. H ):

[0096] d H= =

[0097] (k) B Boltzmann constant; T: absolute temperature (K); D: Solvent viscosity; D: Diffusion coefficient

[0098] Test objective: To determine its hydrodynamic diameter (d) H The polydispersity index (PDI) and long-term storage stability were used to verify its resistance to aggregation and Ostwald ripening. The polydispersity index (PDI) reflects the uniformity of particle size.

[0099] 2) Zeta potential monitoring

[0100] Test Method: After applying an electric field using a DLS instrument, the electrophoretic mobility is calculated by detecting the Doppler frequency shift of particle motion and then converted into a Zeta potential. The sample is diluted with a low-conductivity buffer (e.g., 1 mM KCl or pure water) to avoid high ionic strength shielding of surface charges; impurities are removed using a 0.45 μm filter membrane (crucial for nanoemulsions). Platinum or carbon electrodes are used (to avoid interference from electrolytic reactions), typically 5–20 V / cm (too high may cause Joule heating, too low a signal), and the temperature is maintained at 25 ± 0.1 °C (temperature affects solvent viscosity and dielectric constant). The Zeta potential mode is selected, and the instrument automatically applies positive and negative electric fields alternately. Each measurement lasts approximately 30–60 seconds, repeated three times and the average value is taken. The instrument software directly outputs the Zeta potential (unit: mV).

[0101] Test objective: Zeta potential is a key parameter for characterizing the surface charge properties of nanoparticles and directly affects the stability of colloidal systems.

[0102] 2. Efficacy Test

[0103] (1) Free radical scavenging test (antioxidant performance)

[0104] Determination of hydroxyl radical scavenging capacity: 2 mL of 1.0 mg / mL citrus pectin graft copolymer was added, along with an equal volume of 6 mmol / L ferric sulfate. The mixture was stirred thoroughly and allowed to stand in the dark for 10 min. Then, an equal concentration and volume of salicylic acid were added, and the mixture was stirred thoroughly and allowed to stabilize in a light-free environment for 30 min. The reaction was repeated three times at a UV wavelength of 510 nm. The hydroxyl radical scavenging rate was calculated as follows:

[0105] Hydroxyl radical scavenging rate

[0106] In the formula: A y : Absorbance value of the 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.

[0107] (2) Determination of DPPH free radical scavenging capacity: 2 mL of sample solution was mixed with an equal volume of DPPH-anhydrous ethanol solution (0.1 mmol / L), stabilized in the dark for 30 min, and repeated three times at a UV wavelength of 517 nm. The DPPH free radical scavenging rate was calculated as follows:

[0108] DPPH free radical scavenging rate

[0109] In the formula: A y : Absorbance value of the sample solution; A h : Absorbance value of anhydrous ethanol instead of DPPH anhydrous ethanol solution; A j Anhydrous ethanol is used to replace the absorbance value of the sample solution.

[0110] 2) Tyrosinase activity test (whitening performance)

[0111] Test method: Human epidermal melanocytes were seeded into 96-well plates (1×10⁻⁶ cells / well). 4 / well), cultured for 24 hours, with the blank group without tyrosinase but with the same concentration of citrus nanoemulsion, and the control group without sample; incubated at 37℃ for 30 min, and immediately measured the absorbance at 475 nm wavelength (characteristic absorption peak of dopaquinone) to calculate the inhibition rate:

[0112] Inhibition rate (%) = (1- ) × 100%

[0113] Test principle: Tyrosinase catalyzes the oxidation of the substrate (L-DOPA) to generate dopaquinone (a melanin precursor). The enzyme activity inhibition rate is assessed by measuring the change in absorbance of the reaction product.

[0114] 3) Macrophage model test (anti-inflammatory properties)

[0115] RAW264.7 macrophages were cultured in cell culture flasks until the cell surface area reached 80%-90%. The cells were then digested, and the culture flasks and medium were replaced for passage. Once the cells reached the logarithmic growth phase, the normally growing cells were digested, centrifuged, resuspended, and reseeded into 96-well plates. The cells were then cultured in an incubator at 37°C with 5% carbon dioxide. The experimental groups were: a blank group (DMEM complete medium without other reagents), sample groups (cultured with high, medium, and low doses of sample solution), and a positive control group (cultured with lipopolysaccharide).

[0116] After group culture, 20 μL of 5 mg / mL LMT solution was added to each well and incubated for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well, followed by shaking for 10 min. The microplate reader was set to 490 nm, and cell viability was calculated.

[0117] Cell viability (%) = ×100%

[0118] 3. Practical application simulation test

[0119] Under conditions of 40±2℃ and 75%-80% humidity, physical and chemical stability were tested monthly, and the remaining content of active ingredients was detected by high performance liquid chromatography; the tests were conducted continuously for 4 months.

[0120] All the above performance tests were performed on Examples 1-5 and the control group. The test results are shown in Table 1.

[0121] Table 1 Performance tests conducted in Examples 1-5

[0122]

[0123] It should be noted that the four data points in Table 1 representing the retention of active ingredients in the emulsion after one, two, three, and four months respectively.

[0124] The data in the table show that the citrus extract-based nanoemulsions demonstrated in Examples 1-5 exhibited good performance with minimal differences. Example 4 showed the largest hydrodynamic diameter, significantly reducing the likelihood of Ostwald ripening. The core reason for this is that in this invention, the oleic acid chain, as a hydrophobic segment, can anchor within the oil droplet. Its hydrophobic interaction with the oil phase tightly binds the oil phase molecules, significantly reducing their migration rate. This effectively encapsulates the oil phase within the droplet, preventing the oil from diffusing from small droplets to larger droplets, thus cutting off the inducing factor of droplet size unevenness at the starting point of ripening and avoiding subsequent fusion and stratification. Examples 3-5 have larger particle sizes, potentially better suited for long-term use. Examples 2-3 showed slightly higher free radical scavenging rates, possibly related to the grafting of phenolic acid components. Example 5 exhibited the most prominent tyrosinase inhibition effect, potentially making it suitable as a product primarily targeting whitening effects. Cell viability in all examples was greater than 80%, meeting the requirements for biomaterials. Monthly testing results showed that even in the fourth month, the remaining content of active ingredients reached 85% or higher, indicating excellent shelf life.

[0125] Table 2 compares the performance of Examples 1-4 with Example 1.

[0126]

[0127] As shown in Table 2, the performance of Comparative Examples 1-4 was weaker than that of Example 1, with Comparative Example 4 showing the most significant performance difference compared to Example 1. Specifically, Comparative Example 1 exhibited a significant decrease in hydroxyl radical and DPPH radical scavenging abilities compared to the other comparative examples, demonstrating the indispensable role of phenolic acids in antioxidation and free radical scavenging. Comparative Example 2 showed the most significant difference in tyrosinase inhibition rate, highlighting the irreplaceable role of ascorbic acid in whitening. Comparative Example 3 showed significant differences in hydrodynamic diameter, polydispersity index, and Zeta potential, indicating that the polymer's dispersion function had failed. The hydrophobicity of the citrus pectin polymer, lacking oleic acid, decreased significantly, making the emulsion prone to Ostwald polymerization and greatly shortening the product's shelf life. This highlights the importance of oleic acid molecules as hydrophobic linkages.

[0128] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a nanoemulsion based on citrus extract, characterized in that, Includes the following steps: 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 until homogeneous. Ascorbic acid and a 10 mol / L H2O2 solution were then added to initiate the reaction. Nitrogen gas was continuously introduced and the reaction was continued for 1 hour. The mixture was then placed in a dialysis bag and dialyzed in distilled water for 72 hours, followed by freeze-drying for 24 hours. Ungrafted phenolic acids were removed by Soxhlet extraction with ethanol for 8 hours. The resulting copolymer was then dialyzed again with 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 pH meter, and stir at room temperature for 10 min. Adjust the pH of the solution to 10.5-11.5 with 10 mol / L sodium hydroxide, slowly add sodium trimetaphosphate, and heat to 55 °C to carry out the reaction. Intermittently add 10 mol / L sodium hydroxide to maintain the reaction pH at 10.4-10.

7. Monitor the reaction until it is complete using thin-layer chromatography. Filter the solution, wash the filter cake with a small amount of methanol, dry it, and pulverize it to obtain a white powdery solid, which is ascorbic acid grafted phosphate. 3) Preparation of phosphate ester grafted pectin At a temperature of about 50℃, phenolic acid grafted pectin obtained in 1) and ascorbic acid grafted phosphate ester obtained in 2) were added to the reactor, and dicyclohexylcarbodiimide was added for dehydration. The reaction was continued for 3 hours to obtain phosphate ester grafted pectin. 4) Oleic acid activation treatment Oleic acid was added to a device equipped with a stirrer, condenser, and constant pressure dropping funnel at 60-70℃. Under N2 conditions, excess thionyl chloride was slowly added dropwise through the constant pressure dropping funnel over 30-60 minutes. The mixture was then refluxed for 2-3 hours to ensure complete reaction. Thin-layer chromatography was used to monitor the reaction until it ended, yielding activated oleic acid. 5) Hydrophobic linking branch reaction 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 evenly, and N2 was continuously introduced. The temperature was controlled at 40-60℃ and the reaction was carried out for 2-3 hours. The mixture was then freeze-dried for 24 hours. Ungrafted oleic acid was removed by Soxhlet extraction with ethanol for 8 hours. Finally, the mixture was placed in a dialysis bag and dialyzed in distilled water for 72 hours. The resulting copolymer was dialyzed again with distilled water and freeze-dried to obtain a multifunctional grafted pectin with phenolic acid, ascorbic acid and oleic acid attached to the side chains of citrus pectin. 6) Emulsion preparation Using deionized water, glycerol, olive oil, and sodium hyaluronate as the aqueous phase, and citrus pectin grafted with phenolic acid, ascorbic acid, and oleic acid (5) as the oil phase, the aqueous phase was added to the oil phase, followed by the addition of saponins as surfactants. The mixture was stirred at 30°C for 2 hours with a magnetic stirrer to ensure homogeneity. The mixture was then sheared at 14000 r / min for 2 minutes using a high-speed shear mill to obtain a crude emulsion. Finally, the mixture was homogenized 5 times using a microfluidic homogenizer under a certain homogenization pressure to obtain a nanoemulsion based on citrus extract, which is the final product.

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

3. The method for preparing the citrus extract-based nanoemulsion according to claim 1, characterized in that, The raw material molar ratio used in 2) is as follows: 4-6 parts 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 the citrus extract-based nanoemulsion according to claim 1, characterized in that, The raw materials used in 3) are as follows: 4.8-7.2 parts by weight of phenolic acid grafted pectin; 15-18 parts of ascorbic acid grafted phosphate; and 0.3-0.5 parts of dicyclohexylcarbodiimide.

5. The method for preparing a citrus extract-based nanoemulsion according to claim 1, characterized in that, The raw materials used in 4) are: 4.8-7.2 parts by weight of oleic acid and 2.4-3.6 parts by weight of thionyl chloride.

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

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

8. A nanoemulsion based on citrus extract, characterized in that, It is prepared by any one of the preparation methods described in claims 1-7.

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