Camellia oleifera flavonoid nanoparticles with whitening effect and preparation method and application thereof

Abstract

The present invention belongs to the technical field of cosmetics, and specifically relates to a camellia flavonoid nanoparticle with whitening effect, and a preparation method and application thereof. The camellia flavonoid nanoparticle of the present invention is based on the electrostatic interaction between a polysaccharide compound and a metal ion crosslinking agent, and is cross-linked to form nanoparticles with a polymer network, and an olefin succinic anhydride ester is used as a surfactant to improve the stability of the nanoparticles. The camellia flavonoid compound is wrapped in the nanoparticles through the action of hydrogen bonds and hydrophobic bonds, which can improve the water solubility of the camellia flavonoid, reduce the degradation problem of the camellia flavonoid caused by the environment, and then significantly improve its stability, and finally achieve the effect of significantly improving the activity and bioavailability of the camellia flavonoid.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and more specifically, relates to a camellia oleifera flavonoid nanoparticle with whitening effect, and a preparation method and application thereof. Background Art

[0002] Camellia oleifera is a unique oil crop in China. It blooms from October to March of the following year. Its flowers are rich in nutrients and bioactive ingredients such as protein, amino acids, reducing sugars, polysaccharides, polyphenols, flavonoids, anthocyanins and vitamins. It has antioxidant, antibacterial, antiviral, antitumor and anti-aging biological activities, and has broad application prospects in food, medicine, cosmetics and other fields. However, there is little research on Camellia oleifera at present, and its development and utilization is limited to directly using it as a scented tea beverage or adding it to facial masks as a conditioning agent. There is no in-depth study on its functional activity, and there is no reasonable development and utilization of Camellia oleifera resources.

[0003] Among the active ingredients of Camellia oleifera, flavonoids are the most abundant. Flavonoids can inhibit the catalytic activity of enzymes (tyrosinase) related to melanin synthesis, reduce melanin production from the source, prevent pigment deposition, and reduce the formation of skin aging spots, wrinkles, chloasma, etc., to achieve the effect of whitening. It is a green and safe natural whitening agent. Camellia oleifera flavonoids also have an antioxidant effect and can reduce melanin intermediates such as quinone, DHI, and DHICA. It is another whitening method to inhibit melanin production. For example, Chinese patent application CN112263625A discloses a Camellia oleifera concentrate, which is obtained by heating and extracting Camellia oleifera, Camellia oleifera branches and leaves, and Camellia oleifera with an aqueous solution of propylene glycol, glycerol or butanediol, and has good antioxidant properties. However, it is found in the actual application process that Camellia oleifera flavonoids are unstable in the environment. Not only are they easily degraded when exposed to acid, alkali, light, high temperature, etc., but they are also unstable in room temperature, resulting in a significant reduction in their biological activity, which greatly limits their application. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing camellia flavonoids with poor stability and to provide a camellia flavonoids nanoparticle with whitening effect and significantly improved stability.

[0005] The purpose of the present invention is to provide a method for preparing the camellia oleifera flavonoid nanoparticles with whitening effect.

[0006] Another object of the present invention is to provide the application of the Camellia oleifera flavonoids nanoparticles having whitening effect.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] The invention discloses camellia oleifera flavonoid nanoparticles with whitening effect, which are mainly made of camellia oleifera flavonoids, polysaccharide compounds, metal ion crosslinking agents and alkenyl succinic anhydride esters.

[0009] The nanoparticles of the present invention are based on the electrostatic interaction between polysaccharide compounds and metal ion crosslinking agents, and crosslink to form nanoparticles with polymer networks, and have the advantages of being nontoxic, free of organic solvents, convenient, and controllable. The oil camellia flavonoid compounds are wrapped in nanoparticles through hydrogen bonds and hydrophobic bonds and interact with flavonoid substances, and the degradation problem of oil camellia flavonoids caused by the external environment can be reduced, and then its stability can be significantly improved. At the same time, adding surfactant alkenyl succinic anhydride ester can further improve the water solubility of oil camellia flavonoids and the stability of nanoparticles, and finally reach the effect of significantly improving the activity and bioavailability of oil camellia flavonoids. After oil camellia flavonoids are made into nanoparticles, their particle diameter is usually less than 500nm, and the solubility and stability of oil camellia flavonoids can be improved, and the stability of nanoparticles can be further improved by adding alkenyl succinic anhydride ester.

[0010] Furthermore, the mass ratio of the camellia flavonoids extract, the polysaccharide compound, and the metal ion cross-linking agent is (3-16):10:(5-1):(10-1).

[0011] Furthermore, the polysaccharide compound is selected from one or more of chitosan, pectin, sodium alginate and xanthan gum.

[0012] Furthermore, the metal ion cross-linking agent is selected from one or more of sodium polyphosphate, calcium chloride and magnesium chloride.

[0013] Furthermore, the preparation of the camellia oleifera flavonoids extract comprises the following steps:

[0014] The camellia oleifera is added into an ethanol solution, subjected to complete microwave extraction, and subjected to post-treatment to obtain the camellia oleifera flavonoid extract.

[0015] Further, the volume concentration of the ethanol solution is 30% to 70%. Preferably, the volume concentration of the ethanol solution is 40% to 60%; more preferably, the volume concentration of the ethanol solution is 50%.

[0016] Furthermore, the solid-liquid ratio of the camellia oleifera added to the ethanol solution is 1:(15-35) g / mL. Preferably, the solid-liquid ratio of the camellia oleifera added to the ethanol solution is 1:(25-35) g / mL; more preferably, the solid-liquid ratio of the camellia oleifera added to the ethanol solution is 1:30 g / mL.

[0017] Further, the power of the microwave extraction is 80-720 W. Preferably, the power of the microwave extraction is 80-400 W; more preferably, the power of the microwave extraction is 240 W.

[0018] Furthermore, the microwave extraction time is (30-150) s. Preferably, the microwave extraction time is (60-120) s; more preferably, the microwave extraction time is 90 s.

[0019] Furthermore, the camellia oleifera is dried before extraction, crushed, sieved with a 100-mesh sieve, and then extracted.

[0020] Preferably, the post-treatment is filtration, concentration of the filtrate under reduced pressure, and freeze-drying.

[0021] In addition, the present invention also provides a method for preparing the Camellia oleifera flavonoid nanoparticles, which specifically comprises the following steps:

[0022] Add alkenyl succinic anhydride ethanol solution to the polysaccharide compound solution, mix evenly to obtain a mixed solution; mix the camellia flavonoids extract solution and the metal ion crosslinking agent solution, add them to the mixed solution, complete the crosslinking reaction, obtain a camellia flavonoids nanoparticle suspension, and vacuum dry to obtain camellia flavonoids nanoparticles.

[0023] Furthermore, the concentration of the polysaccharide compound solution is 1-5 mg / mL. Preferably, the concentration of the polysaccharide compound solution is 1-3 mg / mL; more preferably, the concentration of the polysaccharide compound solution is 1 mg / mL.

[0024] Furthermore, the concentration of the metal ion crosslinking agent solution is 0.5-4.5 mg / mL. Preferably, the concentration of the metal ion crosslinking agent solution is 1.5-3.5 mg / mL; more preferably, the concentration of the metal ion crosslinking agent solution is 2.5 mg / mL.

[0025] Further, the concentration of the camellia flavonoids extract solution is 2-10 mg / mL. Preferably, the concentration of the camellia flavonoids extract solution is 2-6 mg / mL; more preferably, the concentration of the camellia flavonoids extract solution is 4 mg / mL.

[0026] Furthermore, the mass ratio of the polysaccharide compound to the metal ion crosslinking agent is (2-6): 1. Preferably, the mass ratio of the polysaccharide compound to the metal ion crosslinking agent is (2-4): 1; more preferably, the mass ratio of the polysaccharide compound to the metal ion crosslinking agent is 3:1.

[0027] Furthermore, the mass ratio of the polysaccharide compound to the camellia flavonoids extract is 10:(3-16).

[0028] Furthermore, the mass ratio of the polysaccharide compound to the alkenyl succinic anhydride ester is (1-9): 1. Preferably, the mass ratio of the polysaccharide compound to the alkenyl succinic anhydride ester is (3-7): 1; more preferably, the mass ratio of the polysaccharide compound to the alkenyl succinic anhydride ester is 5:1.

[0029] Preferably, the temperature for uniform mixing is (40-60)°C and the time is (2-10) hours.

[0030] Preferably, the cross-linking reaction is carried out at room temperature for (30 to 60) minutes.

[0031] The present invention proves through experiments that after the camellia flavonoids are made into camellia flavonoids nanoparticles, the antioxidant and tyrosinase inhibition activities are significantly improved, and the nanoparticles have a good whitening effect.

[0032] Therefore, the present invention also claims to protect the application of the Camellia oleifera flavonoid nanoparticles in whitening products. The products may be cosmetics, health products, and the like.

[0033] The present invention has the following beneficial effects:

[0034] The camellia flavonoids nanoparticles of the present invention are based on the electrostatic interaction between polysaccharide compounds and metal ion crosslinking agents to form nanoparticles with a polymer network through crosslinking. Alkenyl succinic anhydride ester is used as a surfactant to improve the stability of the nanoparticles. The camellia flavonoids compounds are wrapped in the nanoparticles through the action of hydrogen bonds and hydrophobic bonds, which can improve the water solubility of the camellia flavonoids, reduce the degradation problem of the camellia flavonoids caused by the environment, and then significantly improve the stability, ultimately achieving the effect of significantly improving the activity and bioavailability of the camellia flavonoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a statistical graph of the research data on the effect of the volume fraction of ethanol solution on the extraction of flavonoids from Camellia oleifera in Example 1 of the present invention.

[0036] Figure 2 This is a statistical graph of the research data on the effect of the material-liquid ratio on the extraction of flavonoids from Camellia oleifera in Example 1 of the present invention.

[0037] Figure 3 This is a statistical graph of the research data on the effect of microwave power on the extraction of flavonoids from Camellia oleifera in Example 1 of the present invention.

[0038] Figure 4 This is a statistical graph of the research data on the effect of microwave time on the extraction of flavonoids from Camellia oleifera in Example 1 of the present invention.

[0039] Figure 5 This is the chromatogram detected by liquid chromatography-mass spectrometry in Experimental Example 1 of the present invention.

[0040] Figure 6 This is a statistical graph of the measurement data of the antioxidant activity DPPH free radical scavenging ability of Experimental Example 2 of the present invention.

[0041] Figure 7 This is a statistical diagram of the measurement data of the antioxidant activity ABTS free radical scavenging ability of Experimental Example 2 of the present invention.

[0042] Figure 8 This is a statistical graph of the data of the tyrosinase inhibition activity determination of Camellia flavonoids nanoparticles in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0044] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0045] Example 1 Screening of conditions for extracting flavonoids from Camellia oleifera

[0046] 1. Effect of ethanol solution volume fraction on the extraction of flavonoids from Camellia oleifera

[0047] Taking 1g of camellia oleifera pollen dried through a 100-mesh sieve as raw material, the volume of ethanol solution was set as a single factor variable, and ethanol solutions with volume concentrations of 30%, 40%, 50%, 60%, and 70% were used respectively to make a solid-liquid ratio of 1:20g / mL. Under a microwave power of 400W, microwave extraction was performed for 120s, and the extract was filtered. The filtrate was concentrated under reduced pressure and freeze-dried to obtain the camellia oleifera flavonoid extract.

[0048] Determine the flavonoid content in the camellia extract and calculate the flavonoid content of the camellia. The results are shown in Figure 1 As can be seen from the figure, the flavonoids content extracted under the conditions of 30% to 70% volume concentration of ethanol solution is relatively high, among which the extraction amount of 50% volume concentration of ethanol solution is the largest, which can be as high as 91.44 mg / g.

[0049] 2. Effect of solid-liquid ratio on the extraction of flavonoids from Camellia oleifera

[0050] 1 g of camellia oleifera pollen dried through a 100-mesh sieve was used as raw material, and a 50% volume concentration of ethanol solution was added. The solid-liquid ratio was set as a single factor variable, and the solid-liquid ratio was 1:15 g / mL, 1:20 g / mL, 1:25 g / mL, 1:30 g / mL, and 1:35 g / mL, respectively. Under a microwave power of 400 W, microwave extraction was performed for 120 s, and the extract was filtered. The filtrate was concentrated under reduced pressure and freeze-dried to obtain the camellia oleifera flavonoid extract.

[0051] Determine the flavonoid content in the camellia extract and calculate the flavonoid content of the camellia. The results are shown in Figure 2 As can be seen from the figure, the flavonoid content extracted under the conditions of a solid-liquid ratio of 1:(15-35) g / mL is relatively high, among which the extraction amount at a solid-liquid ratio of 1:30 g / mL is the largest, reaching up to 93.92 mg / g.

[0052] 3. Effect of microwave power on the extraction of flavonoids from Camellia oleifera

[0053] 1 g of camellia oleifera pollen dried through a 100-mesh sieve was used as raw material, and 50% volume concentration of ethanol solution was added to make the solid-liquid ratio of 1:20 g / mL. The microwave power was set as a single factor variable. Microwave extraction was performed for 120 s under microwave power conditions of 80 W, 240 W, 400 W, 560 W, and 720 W, respectively. The extract was filtered, the filtrate was concentrated under reduced pressure, and freeze-dried to obtain the camellia oleifera flavonoid extract.

[0054] Determine the flavonoid content in the camellia extract and calculate the flavonoid content of the camellia. The results are shown in Figure 3 As can be seen from the figure, the flavonoids content extracted under the microwave extraction conditions of 80-720W power is relatively high, among which the extraction amount is the largest when the microwave power is 240W, which can be as high as 90.39mg / g.

[0055] 4. Effect of microwave extraction time on the extraction of flavonoids from Camellia oleifera

[0056] 1 g of camellia oleifera pollen dried through a 100-mesh sieve was used as raw material, and 50% volume concentration of ethanol solution was added to make the solid-liquid ratio of 1:20 g / mL. Under a microwave power of 400 W, the microwave extraction time was set as a single factor variable, and microwave extraction was performed for 30 s, 60 s, 90 s, 120 s, and 150 s respectively. The filtrate was filtered, concentrated under reduced pressure, and freeze-dried to obtain the camellia oleifera flavonoid extract.

[0057] Determine the flavonoid content in the camellia extract and calculate the flavonoid content of the camellia. The results are shown in Figure 4 As can be seen from the figure, the flavonoids content extracted with microwave extraction time (30-150) s is relatively high, among which the extraction amount is the largest when microwave extraction is 90 s, which can be as high as 94.64 mg / g.

[0058] Example 2 Extraction of Camellia oleifera flavonoids

[0059] The extraction of camellia oleifera flavonoids specifically comprises the following steps:

[0060] Camellia oleifera pollen dried through a 100-mesh sieve was used as raw material, and a 50% volume concentration ethanol solution was added to make a solid-liquid ratio of 1:30 g / mL. Under a microwave power of 240 W, microwave extraction was performed for 90 s, and the filtrate was filtered, concentrated under reduced pressure, and freeze-dried to obtain the camellia oleifera flavonoid extract.

[0061] Example 3 Screening of conditions for preparing Camellia oleifera flavonoid nanoparticles

[0062] 1. Effect of the mass ratio of chitosan (CS) to sodium polyphosphate (TPP) on the particle size, PDI and encapsulation efficiency of nanoparticles

[0063] By using the ion gel method, a chitosan solution with a mass concentration of 3 mg / mL and an ethanol solution of alkenyl succinic anhydride ester with a mass concentration of 1 mg / mL were mixed (the mass ratio of chitosan to alkenyl succinic anhydride ester was 5:1), and the mixture was stirred on a magnetic stirrer at 40°C at a speed of 550 rpm for 2 hours to obtain a mixed solution; 1 mL of a 2 mg / mL camellia flavonoid extract solution (the mass ratio of chitosan to camellia flavonoid extract was 3:1) and a 2.5 mg / mL sodium polyphosphate solution were mixed evenly, filtered through a 0.45 μm microporous filter membrane, and added dropwise to the above-mentioned mixed solution at a speed of 1 drop / second through a microinjection pump; wherein the mass ratio of chitosan to TPP was set as a single factor variable, which were 2:1, 3:1, 4:1, 5:1, and 6:1, respectively, and stirring was continued for 30 minutes to obtain a suspension of camellia flavonoid nanoparticles, which was vacuum dried at -20°C to obtain camellia flavonoid nanoparticles. The particle size, PDI and encapsulation efficiency of the obtained Camellia oleifera flavonoids nanoparticles were measured. The results are shown in Table 1.

[0064] Table 1 Effect of CS to TPP mass ratio on nanoparticle size, PDI and encapsulation efficiency

[0065] Serial number CS:TPP Particle size / nm PDI Encapsulation rate / % 1 2:1 388.1 0.548 74.44 2 3:1 443 0.591 78.60 3 4:1 625.3 0.698 71.02 4 5:1 665.3 0.730 68.97 5 6:1 518.2 0.603 68.12

[0066] As can be seen from the table, the mass ratio of chitosan to TPP can achieve good particle size, PDI, and encapsulation efficiency in the range of (2-6):1. The data in the table were statistically analyzed using the grey correlation coefficient method. It can be seen that the comprehensive evaluation values ​​of the nanoparticles prepared by different CS to TPP mass ratios are: Z1=1.50913, Z2=1.534273, Z3=0.348054, Z4=0.042569, Z5=0.76138 (the larger the comprehensive evaluation value, the better the comparison evaluation object); it can be concluded that when the mass ratio of CS to TPP is 3:1, the comprehensive score of the nanoparticles obtained is the highest.

[0067] 2. Effect of chitosan (CS) concentration on nanoparticle size, PDI and encapsulation efficiency

[0068] The ion gel method was used to set the concentration of chitosan as a single factor variable. Chitosan solutions with mass concentrations of 1, 2, 3, 4, and 5 mg / mL and alkenyl succinic anhydride ester ethanol solution with a mass ratio of 1 mg / mL were mixed (the mass ratio of chitosan to alkenyl succinic anhydride ester was 5:1), and the mixture was stirred on a magnetic stirrer at 40°C and 550 rpm for 2 h to obtain a mixed solution; 1 mL of a 2 mg / mL camellia flavonoids extract solution (the mass ratio of chitosan to camellia flavonoids extract was 5:1) was added to the mixture. The mixture was uniformly mixed with a sodium polyphosphate solution having a mass ratio of 0.5:1, 1:1, 1.5:1, 2:1, and 2.5:1, and a mass concentration of 2.5 mg / mL, filtered through a 0.45 μm microporous filter membrane, and added to the mixed solution at a speed of 1 drop / second by a microinjection pump, wherein the mass ratio of chitosan to TPP was 3:1, stirred on a magnetic stirrer at a speed of 550 rpm for 30 min, and a suspension of flavonoid nanoparticles of oil camellia was obtained, which was vacuum dried at -20°C to obtain flavonoid nanoparticles of oil camellia. The particle size, PDI, and encapsulation efficiency of the obtained flavonoid nanoparticles of oil camellia were measured, and the results are shown in Table 2.

[0069] Table 2 Effect of CS concentration on nanoparticle size, PDI and encapsulation efficiency

[0070] Serial number <![CDATA[CS concentration / mg·mL -1 > Particle size / nm PDI Encapsulation rate / % 1 1 307.8 0.457 69.81 2 2 423.9 0.583 71.37 3 3 434.8 0.584 73.66 4 4 445 0.632 73.92 5 5 452 0.703 76.88

[0071] As can be seen from the table, chitosan concentrations in the range of (1-5) mg / mL can achieve good particle size, PDI, and encapsulation efficiency. The data in the table were statistically analyzed using the grey correlation coefficient method, and it can be seen that the comprehensive evaluation values ​​of nanoparticles prepared with different CS concentrations are: Z1 = 1.030985, Z2 = 0.48364, Z3 = 0.626423, Z4 = 0.50563, Z5 = 0.558353 (the larger the comprehensive evaluation value, the better the comparison evaluation object); it can be concluded that when the CS concentration is 1 mg / mL, the comprehensive score of the nanoparticles obtained is the highest.

[0072] 3. Effect of sodium polyphosphate (TPP) concentration on nanoparticle size, PDI and encapsulation efficiency

[0073] By using the ion gel method, a chitosan solution with a mass concentration of 3 mg / mL and an ethanol solution of alkenyl succinic anhydride ester with a mass concentration of 1 mg / mL were mixed (the mass ratio of chitosan to alkenyl succinic anhydride ester was 5:1), and the mixture was stirred on a magnetic stirrer at 40°C and a speed of 550 rpm for 2 hours to obtain a mixed solution. 1 mL of 2 mg / mL camellia flavonoids extract solution (the mass ratio of chitosan to camellia flavonoids extract is 3:1) and sodium polyphosphate solution were mixed evenly and filtered through a 0.45 μm microporous membrane. The mixture was added dropwise to the mixture at a speed of 1 drop / second by a microinjection pump, and stirred on a magnetic stirrer at a speed of 550 rpm for 30 min. The concentration of TPP was set as a single factor variable, and sodium polyphosphate solutions with mass concentrations of 0.5, 1.5, 2.5, 3.5, and 4.5 mg / mL were added dropwise to the mixture, respectively, wherein the mass ratio of chitosan to TPP was 3:1. The mixture was stirred for 30 min to obtain a suspension of camellia flavonoids nanoparticles, which was vacuum dried at -20 °C to obtain camellia flavonoids nanoparticles. The particle size, PDI, and encapsulation efficiency of the obtained camellia flavonoids nanoparticles were measured, and the results are shown in Table 3.

[0074] Table 3 Effect of TPP concentration on nanoparticle size, PDI and encapsulation efficiency

[0075] Serial number <![CDATA[TPP concentration / mg·mL -1 > Particle size / nm PDI Encapsulation rate / % 1 0.5 267.5 0.513 66.6 2 1.5 366.9 0.606 68.95 3 2.5 435.8 0.703 76.5 4 3.5 760.7 0.812 74.68 5 4.5 821.7 0.984 69.47

[0076] As can be seen from the table, TPP concentrations in the range of (0.5-4.5) mg / mL can achieve good particle size, PDI, and encapsulation efficiency. The data in the table were statistically analyzed using the grey correlation coefficient method, and it can be seen that the comprehensive evaluation values ​​of the nanoparticles prepared with different TPP concentrations are: Z1=1.219311, Z2=1.125678, Z3=1.363619, Z4=0.75965, Z5=0.166659 (the larger the comprehensive evaluation value, the better the comparative evaluation object); it can be concluded that when the concentration of TPP is 2.5 mg / mL, the nanoparticles obtained have the highest comprehensive score.

[0077] 4. Effect of Camellia oleifera flavonoids extract solution concentration on nanoparticle size, PDI and encapsulation efficiency

[0078] Using the ion gel method, a chitosan solution with a mass concentration of 3 mg / mL and an ethanol solution of alkenyl succinic anhydride ester with a mass concentration of 1 mg / mL were mixed (the mass ratio of chitosan to alkenyl succinic anhydride ester was 5:1), and the mixture was stirred on a magnetic stirrer at 40°C at a speed of 550 rpm for 2 hours to obtain a mixed solution; 1 mL of the camellia flavonoid extract solution and the sodium polyphosphate solution were mixed evenly and filtered through a 0.45 μm microporous filter membrane, and then added dropwise to the mixed solution of polysaccharide compounds and alkenyl succinic anhydride esters at a speed of 1 drop / second through a microinjection pump. The concentration of the camellia flavonoid extract of the camellia flavonoid extract solution was set as a single factor variable, and the concentrations were 2, 4, 6, 8, and 10 mg / mL (the mass ratio of chitosan to the camellia flavonoid extract was 0.6:1, 0.75:1, 1:1, 1.5:1, and 3:1), wherein the mass ratio of chitosan to TPP was 3:1, and the mixture was stirred on a magnetic stirrer at a speed of 550 rpm for 30 min, and then stirred for 30 min to obtain a camellia flavonoid nanoparticle suspension, which was vacuum dried at -20°C to obtain camellia flavonoid nanoparticles. The particle size, PDI, and encapsulation efficiency of the obtained camellia flavonoid nanoparticles were measured, and the results are shown in Table 4.

[0079] Table 4 Effect of Camellia oleifera flavonoids extract solution concentration on nanoparticle size, PDI and encapsulation efficiency

[0080] Serial number <![CDATA[Flavone concentration / mg·mL -1 > Particle size / nm PDI Encapsulation rate / % 1 2 310.2 0.461 69.81 2 4 247.5 0.358 66.10 3 6 284.3 0.383 63.24 4 8 312.3 0.434 59.97 5 10 404.5 0.536 59.24

[0081] As can be seen from the table, the concentration of the camellia flavonoids extract solution in the range of (2-10) mg / mL can achieve good particle size, PDI, and encapsulation efficiency. The data in the table were statistically analyzed using the grey correlation coefficient method, and it can be seen that the comprehensive evaluation values ​​of the nanoparticles prepared with different TPP concentrations are: Z1=1.156168, Z2=1.576864, Z3=1.202377, Z4=0.756767, Z5=0 (the larger the comprehensive evaluation value, the better the comparative evaluation object); it can be concluded that when the concentration of the camellia flavonoids extract is 4 mg / mL, the nanoparticles prepared have the highest comprehensive score.

[0082] Example 4 Preparation of Camellia oleifera flavonoids nanoparticles

[0083] The preparation of the Camellia oleifera flavonoid nanoparticles specifically comprises the following steps:

[0084] By using the ion gel method, a chitosan solution with a mass concentration of 1 mg / mL and an alkenyl succinic anhydride ester ethanol solution with a mass concentration of 1 mg / mL were mixed (the mass ratio of chitosan to alkenyl succinic anhydride ester was 5:1), and the mixture was continuously stirred on a magnetic stirrer at 40°C at a speed of 550 rpm for 2 hours to obtain a mixed solution; 1 mL of a 4 mg / mL camellia flavonoid extract solution (the mass ratio of chitosan to the camellia flavonoid extract was 3:2) and a 2.5 mg / mL sodium polyphosphate solution were mixed evenly, filtered through a 0.45 μm microporous filter membrane, and added dropwise to the above-mentioned mixed solution at a speed of 1 drop / second through a microinjection pump, wherein the mass ratio of chitosan to TPP was 3:1; the mixture was stirred on a magnetic stirrer at a speed of 550 rpm for 30 minutes to obtain a camellia flavonoid nanoparticle suspension, which was vacuum dried at -20°C to obtain camellia flavonoid nanoparticles.

[0085] The results showed that the particle size of the obtained Camellia flavonoids nanoparticles was 329.5 nm, the PDI was 0.451, and the zeta potential was 35.3 mV.

[0086] Experimental Example 1 Qualitative Analysis of Camellia oleifera Flavonoid Extract

[0087] The camellia oleifera flavonoids extract prepared in Example 2 was qualitatively analyzed by liquid chromatography-mass spectrometry, and the specific detection conditions were as follows:

[0088] 1. Chromatographic conditions

[0089] Using ACQUITY UHPLC BEH C 18 Chromatographic column (2.1×150mm, 1.8μm); injection volume 1.0μL; flow rate: 0.2mL / min; column temperature: 30℃. Gradient elution was performed with acetonitrile (A) and ultrapure water (B) as mobile phases, elution program: 0~10min, 80% B; 11~20min, 10% B; 21~25min, 95% B.

[0090] 2. Mass spectrometry conditions

[0091] Drying gas flow (N2) at 8.0 L / min; drying gas temperature: 300°C; nebulizing pressure: 35 psig; capillary voltage: 3.5 kV; collision voltage: 100 V; cone voltage: 65 V. Electrospray ionization was operated in negative ionization mode with a scan range of 100 to 1700 m / z and a collision energy of 20 to 40 eV.

[0092] Results Figure 5, combined with the figure and existing literature, a total of 6 flavonoid compounds were identified in the Camellia oleifera flavonoid extract prepared in Example 2, specifically catechin, glucogallocatechin, kaempferol-3-O-α-L-rhamnoside, quercetin, myricetin and epicatechin.

[0093] Among them, the exact molecular formula of component 1 is C 13 H 16 O 10 , primary mass spectrometry [MH] - m / z 331; the main fragment of secondary mass spectrum is [MH] - m / z 169 and 271, component 1 was identified as glucogallocin. (Rodriguez-Perez, C., Quirantes-Pine, R., Amessis-Ouchemoukh, N., Madani, K., Segura-Carretero, A., Fernandez-Gutierrez, A. (2013) A metabolite-profiling approach allows the identification of new compounds from Pistacia lentiscus leaves. J Pharm Biomed Anal 77, 167-174.)

[0094] Component 2 and component 3 have different retention times, but both have the same molecular formula of C 15 H 14 O6 and have the same primary mass spectrum and secondary mass spectrum fragments, so the two are isomers. According to the secondary fragment ion [MH] - m / z109 and 203 indicate that component 2 and component 3 are catechin and epicatechin, respectively. (Sun, Y., Qin, Y., Li, H., Peng, H., Chen, H., Xie, H.-r., Deng, Z. (2015) Rapid characterization of chemical constituents in Radix Tetrastigma, a functional herbal mixture, before and after metabolism and their antioxidant / antiproliferative activities. Journal of Functional Foods 18, 300-318.)

[0095] The exact molecular formula of component 4 calculated by mass spectrometry software is C 21H 20 O 12 , [MH] - m / z 463 determines its molecular weight as 463, and the secondary ion fragment is [MH] - m / z 361, so it is inferred that component 4 is myricetin-3-O-rhamnoside, i.e. myricetin. (Yan, S., Zhang, X., Wen, X., Lv, Q., Xu, C., Sun, C., Li, X. (2016) Purification of Flavonoids from Chinese Bayberry (Morella rubra Sieb. et Zucc.) Fruit Extracts and α-Glucosidase Inhibitory Activities of Different Fractionations. Molecules 21.)

[0096] Component 5, primary scan in negative ion mode produces ion fragments [MH] - m / z 447, fragmented in MS / MS to produce [MH] - The fragment ion of m / z 301, so component 5 is quercetin. (Song Jianping, Xu Hu, Chen Fei, Liu Xunhong, Zhang Fengsu, Hou Ya, Ma Yang. UPLC-Q-TOF-MS analysis of flavonoids in Apocynum venetum leaves[J]. Chinese Medicinal Materials, 2014, 37(07):1199-1204.)

[0097] The molecular formula of component 6 accurately calculated by mass spectrometry software is C 21 H 20 O 10 The molecular ion peak produced by the primary mass spectrometry is [MH] - m / z 431, the fragment ion produced by secondary mass spectrometry is [MH] - m / z 285, so it is inferred that component 6 is kaempferol-3-O-α-L-rhamnoside. (Kerhoas, L., Aouak, D., Cingoz, A., Routaboul, JM, Lepiniec, L., Einhorn, J., Birlirakis, N. (2006) Structural characterization of the majorflavonoid glycosides from Arabidopsis thaliana seeds. J Agr Food Chem 54, 6603-6612.)

[0098] Experimental Example 2 Activity determination of different carrier nanoparticles

[0099] Referring to Example 4, chitosan and sodium polyphosphate were replaced, and different carriers (sodium alginate CaCl2, sodium alginate MgCl2, pectin-CaCl2, xanthan gum-CaCl2, zein, soy protein isolate) were used to load Camellia oleifera flavonoid extract to prepare nanoparticles. The ability of the nanoparticles to scavenge DPPH and ABTS free radicals and inhibit tyrosinase activity at a concentration of 25 μg / mL was determined, and the results are shown in Table 5.

[0100] Table 5 Activity determination of different carrier nanoparticles

[0101] Nanoparticles DPPH removal rate / % ABTS clearance rate / % Tyrosinase inhibition rate / % Chitosan-sodium polyphosphate 47.67 99.47 94.53 <![CDATA[Sodium alginate - CaCl2]]> 45.62 98.83 90.75 <![CDATA[Sodium alginate - MgCl2]]> 44.95 98.39 89.57 <![CDATA[Pectin - CaCl2]]> 42.59 96.63 87.78 <![CDATA[Xanthan gum - CaCl2]]> 41.57 95.63 86.14 Zein 32.46 89.59 74.45 Soy Protein Isolate 31.62 89.37 72.61

[0102] As can be seen from the table, encapsulating Camellia flavonoids extract in nanoparticle carriers can improve its biological activity. The biological activity of nanoparticles with polysaccharides as carriers is higher than that of nanoparticles with proteins as carriers. Among the five nanoparticles with polysaccharides as carriers, chitosan nanoparticles have the highest biological activity and their whitening effect is significantly better than that of nanoparticles with other carriers.

[0103] Experimental Example 3 Comparison of different surfactants and alkenyl succinic anhydride esters

[0104] Referring to Example 4, different surfactants (sodium dodecyl sulfate, Tween 80, polyethylene glycol and glycidyl trimethylammonium chloride) were used instead of alkenyl succinic anhydride to prepare nanoparticles. The nanoparticles were stored in a constant temperature incubator at 25°C, and samples were taken out after 30 days to measure the changes in particle size and water solubility (the amount of flavonoids dissolved in water).

[0105] Table 6 Comparison of different surfactants and alkenyl succinic anhydride esters

[0106]

[0107] Comparison of the effects of alkenyl succinic anhydride esters and other surfactants (sodium dodecyl sulfate, Tween 80, polyethylene glycol and glycidyl trimethylammonium chloride) on the stability and water solubility of the prepared nanoparticles showed that the nanoparticles with alkenyl succinic anhydride esters as surfactants showed excellent storage stability and water solubility. After 30 days of storage, the particle size only increased by 87.7nm, and the water solubility was 880 times that of free Camellia oleifera flavonoids, and much higher than that of nanoparticles with other surfactants added. It can be seen that alkenyl succinic anhydride esters can significantly improve the stability of nanoparticles and the water solubility of active substances.

[0108] Experimental Example 4 Effect of the Addition Amount of Alkenyl Succinic Anhydride Ester on the Storage Stability of Nanoparticles

[0109] The ion gel method was used to mix a chitosan solution with a mass concentration of 3 mg / mL and an ethanol solution of alkenyl succinic anhydride ester with a mass concentration of 1 mg / mL. The mass ratio of chitosan to alkenyl succinic anhydride ester was set as a single factor variable, which were 1:1, 3:1, 5:1, 7:1, and 9:1, respectively. The mixture was stirred on a magnetic stirrer at 40 °C and 550 rpm for 2 h to obtain a mixed solution. 1 mL of a 2 mg / mL Camellia oleifera flavonoids extract solution (chitosan and alkenyl succinic anhydride ester) was added. The mass ratio of camellia flavonoids extract is 3:1) and a sodium polyphosphate solution with a mass concentration of 2.5 mg / mL is mixed evenly, filtered through a 0.45 μm microporous filter membrane, and added to the above mixed solution at a speed of 1 drop / second through a microinjection pump, wherein the mass ratio of chitosan to TPP is 3:1, and stirred on a magnetic stirrer at a speed of 550 rpm for 30 minutes to obtain a camellia flavonoids nanoparticle suspension, which is vacuum dried at -20°C to obtain camellia flavonoids nanoparticles.

[0110] The nanoparticles with different amounts of alkenyl succinic anhydride ester were placed at room temperature for 7, 14, 21, 28, and 35 days, and the changes in particle size were measured. The results are shown in Table 7.

[0111] Table 7 Effect of the mass ratio of chitosan to alkenyl succinic anhydride ester on the particle size change of nanoparticles during storage

[0112]

[0113] As shown in the table, the stability of nanoparticles increased with the addition of surfactant alkenyl succinic anhydride ester. The greater the amount of alkenyl succinic anhydride ester added, the more stable the nanoparticles. However, when the amount added is too large, flocculation will occur, resulting in an increase in the particle size of the nanoparticles. Particles with a size less than 500 nm are considered nanoparticles. Therefore, the mass ratio of chitosan to alkenyl succinic anhydride ester is 5:1, which is the optimal amount of alkenyl succinic anhydride ester added.

[0114] Experimental Example 5 Determination of Antioxidant Activity of Camellia oleifera Flavonoid Extract and Nanoparticles

[0115] The antioxidant properties of camellia flavonoids (COAF) and camellia flavonoids nanoparticles (COAF-NPs) were compared using DPPH and ABTS methods respectively:

[0116] 1. Determination of DPPH free radical scavenging ability: 0.5 mL of sample (concentrations of 5, 10, 15, 20, and 25 μg / mL) was mixed with 2.5 mL of 0.1 mmol / L DPPH solution, and reacted in the dark for 30 min. The sample was adjusted to zero with anhydrous ethanol, and vitamin C (Vc) was used as a positive control. The OD value at 517 nm was measured. The results are shown in Figure 6 .

[0117] As can be seen from the figure, within the concentration range of 5 to 25 μg / mL, the maximum DPPH radical scavenging abilities of camellia flavonoids and nanoparticles were 29.92% and 47.67%, respectively, that is, the DPPH radical scavenging ability of camellia flavonoids was increased by 1.59 times after being embedded in nanoparticles.

[0118] 2. Determination of ABTS free radical scavenging ability: 0.5 mL of sample (concentrations of 5, 10, 15, 20, 25 μg / mL) was mixed with 2.5 mL of ABTS solution and reacted in the dark for 5 min. Vitamin C (Vc) was used as a positive control and the OD value at 734 nm was measured. The results are shown in Figure 7 .

[0119] As can be seen from the figure, within the concentration range of 5 to 25 μg / mL, the maximum ABTS free radical scavenging abilities of camellia flavonoids and nanoparticles were 89.16% and 99.47%, respectively, that is, the ABTS free radical scavenging ability of camellia flavonoids was increased by 1.12 times after being embedded in the nanoparticles.

[0120] Experimental Example 6 Determination of Tyrosinase Inhibitory Activity of Camellia oleifera Flavonoid Nanoparticles

[0121] 40 μL of sample (concentrations were 22.5, 32.5, 42.5, 52.5, 62.5 μg / mL, respectively) was mixed with 40 μL of 124 U / mL tyrosinase solution and 80 μL of phosphate buffer (0.1 mol / L, pH 6.8), and reacted at 37°C in the dark for 10 min. Then 40 μL of 5 mmol / L substrate L-DOPA (L-DOPA) was added, and the reaction was continued at 37°C for another 30 min. The OD value at 475 nm was measured using an enzyme reader. The results are shown in Figure 8 .

[0122] As can be seen from the figure, within the concentration range of 22.5-62.5 μg / mL, the maximum tyrosinase inhibition activity of camellia flavonoids and nanoparticles were 70.12% and 94.53%, respectively, that is, the DPPH free radical scavenging ability of camellia flavonoids was increased by 1.35 times after being embedded in nanoparticles.

[0123] Experimental Example 7 Study on the Stability of Camellia oleifera Flavonoids-Loaded Nanoparticles

[0124] 1. Storage stability study

[0125] The camellia flavonoids extract solution and the nanoparticles loaded with camellia flavonoids in Example 4 were stored in a constant temperature incubator at 25°C. Samples were taken out every 7 days to measure their tyrosinase inhibition activity and antioxidant capacity. The results are shown in Table 8.

[0126] Table 8 Storage stability test results

[0127]

[0128] It can be seen from the above table that compared with the camellia flavonoids extract solution, the camellia flavonoids loaded in nanoparticles have a smaller trend of inhibiting tyrosinase activity and antioxidant activity with increasing storage time, proving that nanoparticles can improve the storage stability of camellia flavonoids.

[0129] 2. Thermal stability study

[0130] The camellia flavonoids extract solution and the camellia flavonoids nanoparticles loaded in Example 4 were incubated in a constant temperature water bath at 20°C, 40°C, 60°C or 80°C for 5 min, respectively, and the changes in their tyrosinase inhibition activity and antioxidant capacity were measured. The results are shown in Table 9.

[0131] Table 9 Thermal stability test results

[0132]

[0133] It can be seen from the above table that compared with the camellia flavonoids extract solution, the temperature of loading camellia flavonoids in nanoparticles has less effect on its inhibition of tyrosinase activity and antioxidant activity, which proves that nanoparticles can improve the thermal stability of camellia flavonoids.

[0134] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A nanoparticle of camellia flavonoids with whitening effect, characterized in that: The camellia oleifera flavonoid nanoparticles are made of camellia oleifera flavonoid extract, polysaccharide compounds, metal ion crosslinking agents and alkenyl succinic anhydride esters; The mass ratio of the camellia oleifera flavonoids extract, the polysaccharide compound, the metal ion crosslinking agent and the alkenyl succinic anhydride ester is (3-16): 10: (5-1): (10-1); The polysaccharide compound is selected from one or more of chitosan, pectin, sodium alginate and xanthan gum; The metal ion cross-linking agent is selected from one or more of sodium polyphosphate, calcium chloride, and magnesium chloride; The method for preparing the camellia oleifera flavonoid nanoparticles specifically comprises the following steps: Adding alkenyl succinic anhydride ethanol solution to the polysaccharide compound solution, mixing evenly to obtain a mixed solution; mixing the camellia flavonoids extract solution and the metal ion crosslinking agent solution, adding the mixture to the mixed solution, and completing the crosslinking reaction to obtain a camellia flavonoids nanoparticle suspension, vacuum drying to obtain camellia flavonoids nanoparticles; The concentration of the camellia flavonoids extract solution is 2-10 mg / mL; The concentration of the polysaccharide compound solution is 1-5 mg / mL; The concentration of the metal ion crosslinking agent solution is 0.5-4.5 mg / mL.

2. The camellia oleifera flavonoid nanoparticles according to claim 1, characterized in that: The preparation of the camellia oleifera flavonoids extract comprises the following steps: The camellia oleifera is added into an ethanol solution, subjected to complete microwave extraction, and subjected to post-treatment to obtain the camellia oleifera flavonoid extract.

3. The camellia oleifera flavonoid nanoparticles according to claim 2, characterized in that: The camellia oleifera is added into an ethanol solution with a volume concentration of 30% to 70%.

4. The camellia oleifera flavonoid nanoparticles according to claim 2 or 3, characterized in that: The solid-liquid ratio of the camellia oleifera added to the ethanol solution is 1 g: (15-35) mL.

5. Use of the camellia oleifera flavonoid nanoparticles according to any one of claims 1 to 4 in the preparation of cosmetics with whitening effect.

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