Paeonol-hyaluronic acid-fullerene compound as well as preparation method and application thereof

Abstract

The invention provides a paeonol-hyaluronic acid-fullerene compound as well as a preparation method and application thereof, and belongs to the technical field of compounds. The paeonol-hyaluronic acid-fullerene compound provided by the invention comprises fullerene and an ester compound coated on the surface of the fullerene, and the ester compound is an ester compound of paeonol and hyaluronic acid. According to the compound provided by the invention, the fullerene is coated with the ester of the paeonol and the hyaluronic acid by utilizing the hydrophilicity of the chemical structure of the hyaluronic acid, so that the problem of poor water solubility of the paeonol and the fullerene is solved, and the ester of the paeonol and the hyaluronic acid does not have biotoxicity, so that the compound has a good application prospect. Therefore, the paeonol-hyaluronic acid-fullerene compound has excellent water solubility and biological safety.

Description

Technical Field

[0001] The present invention relates to the technical field of composites, and in particular to a paeonol-hyaluronic acid-fullerene composite and a preparation method and application thereof. Background Art

[0002] Fullerene is a special allotrope in carbon nanomaterials. It is a hollow molecule composed of carbon atoms, usually in the shape of a sphere, ellipse, column or tube. Fullerene has excellent ability to scavenge free radicals and is widely used in the fields of cosmetics, biomedicine and the like. However, since fullerene is insoluble in water, its application in the fields of cosmetics, biomedicine and the like is severely limited. In order to improve the water solubility of fullerene, it is usually embedded with water-soluble molecules, such as xanthan gum molecules. However, studies have found that the potential biological toxicity of the fullerene derivatives prepared in this way is not conducive to their application in the fields of cosmetics, biomedicine and the like. Another method for modifying fullerene is to couple sodium hyaluronate with fullerene to obtain a hyaluronic acid-fullerene conjugate. This method uses an azidation reagent and an organic solvent in the preparation process, which increases the residue of toxic and harmful reagents, and the process is complicated, which is limited in practical applications.

[0003] Paeonol is a natural compound extracted from plants and a natural non-toxic cosmetic raw material. It has the functions of scavenging free radicals, inhibiting melanin, reducing and fading pigments deposited in the skin, removing stasis and spots, reducing inflammation, reducing swelling and relieving pain, anti-allergy, and anti-virus. However, paeonol is slightly soluble in water, which affects its biological activity in practical applications.

[0004] Paeonol and fullerene are both ingredients in the catalog of cosmetic raw materials. They both have good biological activity, but poor water solubility. Improving their water solubility and biosafety is of great significance for the application of paeonol and fullerene. Summary of the invention

[0005] The object of the present invention is to provide a paeonol-hyaluronic acid-fullerene complex and a preparation method and application thereof. The paeonol-hyaluronic acid-fullerene complex provided by the present invention has excellent water solubility and biological safety, and is conducive to application in cosmetics.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a paeonol-hyaluronic acid-fullerene complex, comprising fullerene and an esterified product coated on the surface of the fullerene, wherein the esterified product is an esterified product of paeonol and hyaluronic acid.

[0008] Preferably, the mass ratio of the fullerene to the ester is 1:(1-50).

[0009] The present invention also provides a method for preparing the paeonol-hyaluronic acid-fullerene complex described in the above technical solution, comprising the following steps:

[0010] 1) mixing a paeonol aqueous solution and a sodium hyaluronate aqueous solution to perform an esterification reaction to obtain an esterified aqueous solution; the temperature of the paeonol aqueous solution is 60 to 80° C.; the pH value of the sodium hyaluronate aqueous solution is 4 to 6;

[0011] 2) The esterified aqueous solution obtained in step 1) is mixed with fullerene and then intermittently ball-milled to obtain a paeonol-hyaluronic acid-fullerene complex; the temperature of the intermittent ball-milling is 15-20°C.

[0012] Preferably, in the step 1), the mass ratio of paeonol in the paeonol aqueous solution to sodium hyaluronate in the sodium hyaluronate aqueous solution is (0.5-5):(20-60).

[0013] Preferably, the temperature of the esterification reaction in step 1) is 60-80°C.

[0014] Preferably, the molecular weight of sodium hyaluronate in step 1) is 42.5-145 kD.

[0015] Preferably, the intermittent ball milling in step 2) is performed by stopping the ball mill for 20 to 40 minutes after running the ball mill for 1 to 20 minutes.

[0016] Preferably, the rotation speed of the intermittent ball milling in step 2) is 500 to 800 rpm, and the time of the intermittent ball milling is 6 to 12 hours.

[0017] Preferably, the medium of the intermittent ball milling in step 2) is 1,3-butanediol, and the volume ratio of the ball milling medium to the ball milling material in the intermittent ball milling is (2-4):1.

[0018] The present invention also provides the use of the paeonol-hyaluronic acid-fullerene complex described in the above technical solution in the preparation of cosmetics.

[0019] The present invention provides a paeonol-hyaluronic acid-fullerene complex, including fullerene and an ester coated on the surface of the fullerene, wherein the ester is an ester of paeonol and hyaluronic acid. The complex provided by the present invention utilizes the hydrophilicity of the chemical structure of hyaluronic acid, and coats the fullerene with the ester of paeonol and hyaluronic acid, thereby solving the problem of poor water solubility of paeonol and fullerene, and the ester of paeonol and hyaluronic acid has no biological toxicity, so that the paeonol-hyaluronic acid-fullerene complex has excellent water solubility and biological safety. The results of the embodiment show that the paeonol-hyaluronic acid-fullerene complex provided by the present invention can be prepared into a 10g / L aqueous solution, which has good water solubility; it has no cytotoxicity within the concentration range of 0.05 to 1mg / mL, and can promote cell growth; using a 10mg / mL paeonol-hyaluronic acid-fullerene complex aqueous solution as a moisturizing water, 10 people used it for 8 weeks without any adverse skin reactions, and has good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a macroscopic state diagram of the paeonol-hyaluronic acid-fullerene complex prepared in Example 1;

[0021] Figure 2 The infrared spectra of the paeonol-hyaluronic acid-fullerene complex prepared in Example 1 and the mixture of paeonol, sodium hyaluronate and fullerene;

[0022] Figure 3 This is a macroscopic state diagram of the aqueous solution of the paeonol-hyaluronic acid-fullerene complex prepared in Example 2 after standing for 24 hours. DETAILED DESCRIPTION

[0023] All raw materials of the present invention have no particular limitation on their sources and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0024] There is no particular limitation on the purity of all raw materials in the present invention. The present invention preferably uses analytically pure materials or materials with conventional purity in the field of complexes.

[0025] The invention provides a paeonol-hyaluronic acid-fullerene complex, comprising fullerene and an esterified product coated on the surface of the fullerene, wherein the esterified product is an esterified product of paeonol and hyaluronic acid.

[0026] The paeonol-hyaluronic acid-fullerene complex provided by the present invention has good water solubility, stability and biosafety; has no cytotoxicity and can promote cell growth; has a good scavenging effect on DPPH and ABTS free radicals; has an inhibitory effect on tyrosinase, hyaluronidase and elastase; can increase the water content of the skin, and the effect is better than that of commercially available lotions; the paeonol-hyaluronic acid-fullerene complex is used to prepare lotions, which has an obvious whitening effect.

[0027] In the present invention, the mass ratio of the fullerene to the ester is preferably 1:(1-50); as an embodiment of the present invention, the mass ratio of the fullerene to the ester can be 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45. When the mass ratio of fullerene to ester is within the above range, the ester can effectively encapsulate the fullerene, which is beneficial to improve the biological activity of the complex.

[0028] The present invention also provides a method for preparing the paeonol-hyaluronic acid-fullerene complex described in the above technical solution, comprising the following steps:

[0029] 1) mixing a paeonol aqueous solution and a sodium hyaluronate aqueous solution to perform an esterification reaction to obtain an esterified aqueous solution; the temperature of the paeonol aqueous solution is 60 to 80° C.; the pH value of the sodium hyaluronate aqueous solution is 4 to 6;

[0030] 2) The esterified aqueous solution obtained in step 1) is mixed with fullerene and then intermittently ball-milled to obtain a paeonol-hyaluronic acid-fullerene complex; the temperature of the intermittent ball-milling is 15-20°C.

[0031] The invention mixes a paeonol aqueous solution and a sodium hyaluronate aqueous solution, performs an esterification reaction, and obtains an esterified product aqueous solution.

[0032] In the present invention, the temperature of the paeonol solution is 60-80°C. The present invention uses water as a solvent to avoid the use of organic solvents to cause the residue of toxic and harmful reagents. At room temperature, paeonol is slightly soluble in water, and the solubility is too low, and the prepared product cannot fully exert the biological activity of paeonol; increasing the temperature can increase the solubility of paeonol, thereby increasing the content of paeonol in the product and giving full play to its biological activity.

[0033] As an embodiment of the present invention, the concentration of the paeonol aqueous solution can be 1-5 g / L, or 2-4 g / L; in an embodiment of the present invention, the concentration of the paeonol aqueous solution is 1 g / L and 5 g / L.

[0034] In the present invention, the pH value of the sodium hyaluronate aqueous solution is 4 to 6. The present invention preferably adjusts the pH value of the sodium hyaluronate aqueous solution by adding an inorganic acid to the mixture of sodium hyaluronate and water, and the inorganic acid is preferably 4 to 8% dilute hydrochloric acid. There are a large number of sodium carboxylates in the sodium hyaluronate molecule, which can undergo an esterification reaction with the phenolic hydroxyl group of paeonol under acidic conditions, grafting paeonol onto the hyaluronic acid molecular chain, thereby solving the problem of poor water solubility of paeonol.

[0035] In the present invention, the molecular weight of sodium hyaluronate in the sodium hyaluronate aqueous solution is preferably 42.5 to 145 kD. The molecular weight of sodium hyaluronate directly affects its performance. The larger the molecular weight, the longer the molecular chain and the greater the viscosity after dissolution. If the molecular weight is too small, the sodium hyaluronate molecules will quickly enter the skin, affecting the moisture balance of the skin and causing the skin to dry out. If the molecular weight is too large, the viscosity after dissolution will be too high and the use experience will be poor. When the molecular weight of sodium hyaluronate is within the above range, the use experience is better and it will not quickly enter the skin and destroy the moisture balance of the skin.

[0036] As an embodiment of the present invention, the concentration of the sodium hyaluronate aqueous solution can be 10-30 g / L, or 15-25 g / L; in an embodiment of the present invention, the concentration of the sodium hyaluronate aqueous solution is 10 g / L and 30 g / L.

[0037] In the present invention, the mass ratio of paeonol in the paeonol aqueous solution to sodium hyaluronate in the sodium hyaluronate aqueous solution is preferably (0.5-5): (20-60). As an embodiment of the present invention, the mass ratio of paeonol in the paeonol aqueous solution to sodium hyaluronate in the sodium hyaluronate aqueous solution can be 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:120. If the mass of paeonol is too little, its biological activity cannot be exerted, and if it is too much, it cannot be completely grafted onto the hyaluronic acid molecular chain. The present invention limits the mass ratio of paeonol to sodium hyaluronate within the above range, so that paeonol can be grafted onto the hyaluronic acid molecular chain and exert the biological activity of paeonol.

[0038] The present invention has no particular requirement on the mixing method of the paeonol aqueous solution and the sodium hyaluronate aqueous solution, as long as paeonol and sodium hyaluronate can be fully contacted. Stirring is preferably used in the present invention.

[0039] In the present invention, the temperature of the esterification reaction is preferably 60-80°C. As an embodiment of the present invention, the temperature of the esterification reaction can be 60°C, 65°C, 70°C, 75°C, 80°C. The temperature of the esterification reaction affects the reaction rate. If the temperature is too low, the reaction rate is too slow. If the temperature is too high, side reactions will occur and energy consumption will increase. When the temperature of the esterification reaction is within the above range, the reaction rate can be increased without side reactions and energy consumption will not be too high.

[0040] After obtaining the esterified aqueous solution, the present invention mixes the esterified aqueous solution with fullerene and then performs intermittent ball milling to obtain a paeonol-hyaluronic acid-fullerene complex.

[0041] As an embodiment of the present invention, the fullerene can be C60 or C70; in the embodiment of the present invention, the fullerene used is C60. In the present invention, the mass ratio of the esterified product to the fullerene in the esterified product aqueous solution is preferably (1-50):1.

[0042] In the present invention, the temperature of the intermittent ball milling is 15-20° C. The ester of paeonol and hyaluronic acid will hydrolyze at high temperature. The present invention prevents the hydrolysis of the ester by controlling the temperature of the intermittent ball milling.

[0043] In the present invention, it is preferred to add ball milling media for intermittent ball milling. The ball milling media is preferably 1,3-butanediol. The volume ratio of the ball milling media to the ball milled material is preferably (2-4):1. As an embodiment of the present invention, the volume ratio of the ball milling media to the ball milled material can be 2:1, 3:1, or 4:1. 1,3-Butanediol is used as a medium for intermittent ball milling to improve the effect of intermittent ball milling.

[0044] In the present invention, the intermittent ball milling is preferably performed by stopping the ball milling machine for 20 to 40 minutes after each operation of the ball milling machine for 1 to 20 minutes. The present invention adopts intermittent ball milling to prevent the molecular structure of fullerene from being damaged by continuous long-term ball milling. Moreover, when the ball milling machine stops operating, the heat generated during the ball milling process can also be dissipated, which is conducive to controlling the ball milling temperature.

[0045] In the present invention, the speed of the intermittent ball mill is preferably 500 to 800 rpm. As an embodiment of the present invention, the speed of the intermittent ball mill can be 500 rpm, 600 rpm, 700 rpm, or 800 rpm. When the speed of the intermittent ball mill is within the above range, the ester of paeonol and hyaluronic acid can be coated on the surface of fullerene, while preventing the raw material structure from being damaged by excessive speed.

[0046] In the present invention, the intermittent ball milling time is preferably 6 to 12 hours. As an embodiment of the present invention, the intermittent ball milling time can be 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, and 12 hours. When the intermittent ball milling time is within the above range, the ester of paeonol and hyaluronic acid can be coated on the fullerene surface, and the raw material structure can be prevented from being destroyed.

[0047] The present invention has no special requirements for the ball milling equipment, as long as intermittent ball milling can be achieved and the ball milling temperature can be controlled. The present invention preferably uses a low-temperature ball mill.

[0048] After the intermittent ball milling is completed, the present invention preferably sequentially performs solid-liquid separation and drying on the obtained material to obtain the paeonol-hyaluronic acid-fullerene complex.

[0049] As an embodiment of the present invention, the solid-liquid separation may be centrifugation; the rotation speed of the centrifugation is preferably 800 to 1200 rpm, and the time of the centrifugation is preferably 20 to 40 min.

[0050] As an embodiment of the present invention, the drying may be freeze drying; the freeze drying temperature is preferably -30 to -60°C, and the freeze drying time is preferably 20 to 30 hours.

[0051] The present invention utilizes esterification reaction between paeonol and sodium hyaluronate in an acidic aqueous solution to graft paeonol onto a hyaluronic acid molecular chain, thereby solving the problem of poor water solubility of paeonol; the obtained ester of paeonol-hyaluronic acid is coated on the surface of fullerene by intermittent ball milling, thereby overcoming the water solubility problem of fullerene; meanwhile, the ester of paeonol and hyaluronic acid has no biological toxicity and no organic solvent is used, thereby making the paeonol-hyaluronic acid-fullerene complex have excellent water solubility and biological safety.

[0052] The present invention also provides the use of the paeonol-hyaluronic acid-fullerene complex described in the above technical solution in the preparation of cosmetics.

[0053] The present invention has no special requirements for the application method, and the application method commonly used in the art can be adopted.

[0054] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Example 1

[0056] A method for preparing a paeonol-hyaluronic acid-fullerene complex, comprising the following specific steps:

[0057] With H at 80℃ 2 O dissolves paeonol and prepares 10 mL of a paeonol aqueous solution with a concentration of 2 g / L; prepares 20 mL of a sodium hyaluronate (molecular weight 100 kD) aqueous solution with a concentration of 10 g / L, and adjusts its pH to 4 with 5% diluted HCl; at 80° C., mix the sodium hyaluronate aqueous solution and the paeonol aqueous solution, and stir for 2 hours to obtain an ester aqueous solution.

[0058] 0.015 g of fullerene was added to the esterified aqueous solution, and then 120 mL of 1,3-butanediol was added, and intermittent ball milling was performed in a low-temperature ball mill at a speed of 800 rpm for 6 hours. After the ball mill was operated for 15 minutes, it was stopped for 40 minutes, and the ball milling temperature was 20°C; after ball milling, centrifugation was carried out at 1000 rpm for 30 minutes, and the upper layer solution obtained was the paeonol-hyaluronic acid-fullerene complex solution. After freeze-drying at -40°C for 24 hours, a powdered paeonol-hyaluronic acid-fullerene complex was obtained, and the macroscopic photo is shown as follows: Figure 1 shown.

[0059] The infrared absorption of the paeonol-hyaluronic acid-fullerene complex prepared in Example 1 was measured using an infrared spectrometer. At the same time, a mixture of paeonol, sodium hyaluronate and fullerene (using the same raw materials as in Example 1, with a mass ratio of 4:20:3) was used as a comparative sample to obtain an infrared spectrum. Figure 2 shown.

[0060] from Figure 2 It can be seen that hyaluronic acid is at 1616cm -1 The characteristic peak of carboxylate carbonyl group at 1645 cm -1 , increased by 29cm -1 This is because the carbonyl peak moves toward the high frequency direction after paeonol forms an ester bond with hyaluronic acid, indicating that paeonol has been successfully grafted onto the hyaluronic acid molecular chain.

[0061] Example 2

[0062] A method for preparing a paeonol-hyaluronic acid-fullerene complex, comprising the following specific steps:

[0063] With H at 70℃ 2 O to dissolve paeonol and prepare 5 mL of a paeonol aqueous solution with a concentration of 1 g / L; prepare 20 mL of a sodium hyaluronate (molecular weight 80 kD) aqueous solution with a concentration of 30 g / L, and adjust its pH to 6 with 6% dilute HCl; at 60° C., mix the sodium hyaluronate aqueous solution and the paeonol aqueous solution and stir for 2 hours to obtain an esterified aqueous solution.

[0064] 0.25 g of fullerene was added to the esterified aqueous solution, and then 60 mL of 1,3-butanediol was added, and intermittent ball milling was carried out in a low-temperature ball mill at a rotation speed of 500 rpm and a ball milling time of 12 h. After the ball mill was operated for 20 min, it was stopped for 40 min, and the ball milling temperature was 15° C. After ball milling, centrifugation was performed at 1000 rpm for 30 min, and the obtained upper layer solution was the paeonol-hyaluronic acid-fullerene complex solution. After freeze-drying at -40° C. for 24 h, a powdered paeonol-hyaluronic acid-fullerene complex was obtained.

[0065] The paeonol-hyaluronic acid-fullerene complex prepared in Example 2 was prepared into an aqueous solution with a concentration of 10 g / L and allowed to stand for 24 hours. Figure 3 As shown in the figure, it can be seen that the composite prepared in Example 2 has good water solubility and is a clear transparent liquid with no precipitation in the solution, showing good stability.

[0066] Test Example 1 Stability test in physiological solution

[0067] The paeonol-hyaluronic acid-fullerene complex prepared in Example 1 was diluted in physiological saline and phosphate buffer at a ratio of 50 mg / 10 mL, respectively, and then centrifuged at 6000 rpm for 10 min. No precipitation was produced in the diluted solutions. This shows that the paeonol-hyaluronic acid-fullerene complex prepared in the present invention exhibits high stability in these two physiological solutions, which is conducive to effective interaction with human cells.

[0068] Test Example 2 Detection of the ability to scavenge DPPH free radicals

[0069] Experimental group 1 is the paeonol-hyaluronic acid-fullerene complex prepared in Example 1 of the present invention;

[0070] Experimental group 2 was paeonol;

[0071] Experimental group 3 was commercially available water-soluble fullerene (Xiamen Funa New Materials Technology Co., Ltd.).

[0072] Use deionized water to prepare different concentrations (0.02mg / mL, 0.04mg / mL, 0.06mg / mL, 0.08mg / mL and 1.0mg / mL) of samples from different experimental groups. Take 2mL of sample solutions of different concentrations in test tubes, add 2mL of prepared DPPH solution (0.004g DPPH powder is added to a 50mL volumetric flask, dilute with 95% ethanol, and store at 4°C away from light), mix well, and place at room temperature away from light for 30 minutes. After placing, centrifuge each sample at a speed of 10000rpm for 10 minutes, take the supernatant and measure the absorbance at a wavelength of 517nm, which is recorded as A j , 2 mL of different samples were mixed with 2 mL of 95% ethanol to obtain a mixed solution, and the absorbance of the mixed solution was measured at a wavelength of 517 nm, which was recorded as A i , 2 mL of DPPH solution and 2 mL of distilled water were mixed to obtain a mixed solution, and the absorbance of the mixed solution was measured at a wavelength of 517 nm, which was recorded as A 0 The scavenging rate of each sample on DPPH free radicals was calculated according to the following formula.

[0073] DPPH free radical scavenging rate (%) = [A 0 -(A j -A i )] / A 0 ×100%.

[0074] The scavenging rate of DPPH free radicals by different samples is shown in Table 1.

[0075] Table 1 Results of scavenging rate of DPPH free radicals by different samples

[0076]

[0077] It can be seen from Table 1 that the paeonol-hyaluronic acid-fullerene complex prepared by the present invention has a strong DPPH free radical scavenging ability, and its DPPH free radical scavenging ability is higher than that of water-soluble fullerene.

[0078] Test Example 3: Detection of the ability to scavenge ABTS free radicals

[0079] The sample grouping of this test example is the same as that of test example 2.

[0080] Weigh 0.384 g ABTS and add to 40 mL dHO 2 O to obtain solution A, weigh 0.066 g potassium persulfate (K 2 S 2 O 8 ), add to 40mL dH 2 O to obtain solution B, mix solution A and solution B, and add dH 2O was diluted to 100 mL to obtain a 7 mmol / L ABTS stock solution. The ABTS stock solution was allowed to stand at room temperature and in the dark for 12 h, and then diluted with 95% ethanol solution until the absorbance of the diluted solution at a wavelength of 734 nm was 0.70 ± 0.02 A, thereby obtaining an ABTS free radical stock solution. 0.9 mL of each experimental group sample solution of different concentrations (0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL and 1.0 mg / mL) was mixed with 2 mL of ABTS free radical stock solution, and allowed to stand at room temperature for 6 min. The mixture was then centrifuged at 10,000 rpm for 10 min, and the absorbance of the mixture was measured at a wavelength of 734 nm using the supernatant, which was recorded as A. j 0.9 mL of different samples were mixed with 2 mL of 95% ethanol to obtain a mixed solution, and the absorbance of the mixed solution was measured at a wavelength of 734 nm, which was recorded as A i , 2 mL of ABTS free radical stock solution and 2 mL of distilled water were mixed to obtain a mixed solution, and the absorbance of the mixed solution was measured at a wavelength of 734 nm, which was recorded as A 0 The scavenging rate of each sample on ABTS free radicals was calculated according to the following formula.

[0081] ABTS free radical scavenging rate (%) = [A 0 -(A j -A i )] / A 0 ×100%;

[0082] The scavenging rate of ABTS free radicals by different samples is shown in Table 2.

[0083] Table 2 Results of scavenging rate of ABTS free radicals by different samples

[0084]

[0085] It can be seen from Table 2 that the paeonol-hyaluronic acid-fullerene complex prepared in the present invention has a strong ABTS free radical scavenging ability, and its ABTS free radical scavenging ability is higher than that of water-soluble fullerene.

[0086] Test Example 4 Biological Toxicity Test

[0087] The sample grouping of this test example is the same as that of test example 2, and a control group is added, which is composed of normally growing cells.

[0088] The MTT method was used to observe the proliferation or inhibitory effects of different samples on human epithelial cells. The MTT method is based on the metabolic reduction of tetrazolium blue (MTT). There are NADP-related dehydrogenases in the mitochondria of living cells, which can reduce the yellow MTT to insoluble blue-purple formazan. However, this enzyme disappears after cell death, resulting in the failure of MTT to be reduced. After dissolving formazan with dimethyl sulfoxide (DMSO), the absorbance is detected at a wavelength of 570nm using an enzyme reader.

[0089] The samples of different experimental groups with different concentrations were mixed with 4.5×10 4 100 μL of HaCaT cells in the logarithmic growth phase were prepared into 200 μL of culture medium (GSGPs were prepared to different concentrations in DMEM (high glucose) medium containing 10% fetal bovine serum) and incubated in 5% CO 2 After culturing at 37°C in an incubator for 72 hours, 20 μL of 5g / L MTT solution was added and cultured for another 4 hours, centrifuged, and the culture medium was discarded. 180 μL DMSO was added to each experimental group, and after oscillating on a constant temperature oscillator for 60 seconds, the absorbance was detected at a wavelength of 570 nm using an enzyme marker. Three parallel samples were made for each concentration sample. The inhibition rate was calculated using the following formula based on the measured absorbance value.

[0090] Inhibition rate = (absorbance value of control group - absorbance value of experimental group) / absorbance value of control group × 100%;

[0091] The inhibition results of different samples on human epithelial cells are shown in Table 3.

[0092] Table 3 Inhibition results of different samples on human epithelial cells

[0093]

[0094] As can be seen from Table 3, after 72 hours of culture, the paeonol-hyaluronic acid-fullerene complex prepared by the present invention has no inhibitory effect on epithelial cell (HaCaT cell) mitosis, but promotes cell mitosis, and the promotion effect is higher than that of other experimental groups, indicating that the paeonol-hyaluronic acid-fullerene complex prepared by the present invention has a good mitogenic effect. At the same time, the water-soluble fullerenes of the experimental group 3 began to show cytotoxicity to HaCaT cells when their concentration was above 0.05 mg / mL. This is similar to the results reported in the literature (Chen Chuan et al., Effect of water-soluble fullerenes on the activity of human skin fibroblasts [J]. Chemistry and Bioengineering, 2019, 36 (10): 27-30, 34). The above experimental results show that the paeonol-hyaluronic acid-fullerene complex prepared by the present invention greatly reduces the toxicity of water-soluble fullerenes to cells.

[0095] Test Example 5 Tyrosinase Inhibition Test

[0096] The test sample grouping of this test example is the same as that of test example 2.

[0097] To determine the tyrosinase inhibition rate, prepare the test reagents for the tyrosinase inhibition rate of the samples in the experimental group 1 according to Table 4. The steps are as follows: first, add phosphate buffer, the samples in the experimental group 1 and L-tyrosine solution in four test tubes according to the compositions of A1 to A4 in Table 4, then keep the four test tubes in a water bath at 30°C for 10 minutes at the same time, and after the insulation is completed, add 0.5 mL of L-tyrosinase solution with a concentration of 100 U / mL to the four test tubes according to Table 4, shake well and react for 10 minutes, and measure the absorbance values ​​of the solutions in the four test tubes at a wavelength of 475 nm, which are recorded as A and B respectively. 1 , A 2 , A 3 , A 4 The same experimental group samples were tested for inhibition rates of tyrosinase at three different concentrations, namely 0.05 mg / mL, 0.1 mg / mL and 0.2 mg / mL. When testing the samples of Experimental Group 2 and Experimental Group 3, the samples of Experimental Group 1 in the above steps were replaced with the samples of Experimental Group 2 and Experimental Group 3 respectively.

[0098] Table 4 Tyrosinase inhibition test reagent composition

[0099]

[0100] The tyrosinase inhibition rate of the experimental group samples was calculated according to the following formula:

[0101] Tyrosinase inhibition rate = [(A 3 -A 4 ) / (A 1 -A 2 )]×100%;

[0102] The inhibitory effects of samples in different experimental groups on tyrosinase were calculated according to the above formula. The results of the inhibitory effects of samples in different experimental groups on tyrosinase are shown in Table 5.

[0103] Table 5 Results of tyrosinase inhibition by samples from different experimental groups

[0104]

[0105]

[0106] It can be seen from Table 5 that the paeonol-hyaluronic acid-fullerene complex prepared by the present invention has a higher inhibitory effect on tyrosinase, and its inhibitory effect is better than that of water-soluble fullerene, can reduce the production of melanin in the human body, and has a better whitening effect.

[0107] Test Example 6 Hyaluronidase Inhibition Test

[0108] The paeonol-hyaluronic acid-fullerene complex prepared in Example 1 was prepared into a 5 mg / mL solution, and then deionized water was added for gradient dilution (5, 4, 3, 2, 1 mg / mL) for standby use. Group A1 added 50 μL of paeonol-hyaluronic acid-fullerene complex solution and 50 μL of hyaluronidase solution (pH 5.4, 0.2 mol / L acetic acid buffer prepared to 500 U / mL), reacted at 37°C for 20 min, and added 10 μL of 2.5 mol / L CaCl 2 React at 37°C for 20 minutes, add 50μL 0.5mg / mL potassium hyaluronate at 37°C for 40 minutes, then react at room temperature for 10 minutes, add 50μL acetylacetone solution (3.5mL acetylacetone dissolved in 50mL 0.1mol / L sodium carbonate solution, freshly prepared), 10μL 5mol / L NaOH, 50μL deionized water, react in a boiling water bath for 15 minutes, cool to room temperature and react in a 4°C cold water bath for 10 minutes. Add 100μL P-DAB color developer (0.8g DBMA mixed with 15mL concentrated hydrochloric acid and 15mL anhydrous ethanol) and shake thoroughly, then add 350μL anhydrous ethanol, color at room temperature for 30 minutes, measure OD530, recorded as A 1 ; Group A2 replaced the hyaluronidase solution and potassium hyaluronate solution with an equal volume of acetic acid buffer, and the test results were recorded as A 2 In group B1, the paeonol-hyaluronic acid-fullerene complex solution was replaced with an equal volume of deionized water, and the test results were recorded as B 1 In group B2, the paeonol-hyaluronic acid-fullerene complex solution was replaced with an equal volume of deionized water, and the enzyme solution and potassium hyaluronate solution were replaced with an equal volume of acetic acid buffer. The test results were recorded as B 2 . Calculate the hyaluronidase inhibition rate:

[0109] Hyaluronidase inhibition rate (%) = [1-(A 1 -A 2 ) / (B 1 -B 2 )]×100%.

[0110] The inhibition rates of samples of different concentrations on hyaluronidase are shown in Table 6.

[0111] Table 6 Results of inhibition of hyaluronidase by samples of different concentrations

[0112] Sample concentration (mg / mL) 1 2 3 4 5 Hyaluronidase inhibition rate (%) 29.76 58.56 68.981 74.67 76.32

[0113] It can be seen from Table 6 that the paeonol-hyaluronic acid-fullerene complex prepared by the present invention has an inhibitory effect on hyaluronidase and is concentration-dependent. When the concentration of the paeonol-hyaluronic acid-fullerene complex solution is 5 mg / ml, the hyaluronidase inhibition rate is 76.32%.

[0114] Test Example 7 Elastase Inhibition Test

[0115] After the paeonol-hyaluronic acid-fullerene complex prepared in Example 1 was prepared into a 50 mg / mL solution, deionized water was added in proportion for gradient dilution (50, 40, 30, 20, 10 mg / mL) for standby use. Group A1 added 50 μL of paeonol-hyaluronic acid-fullerene complex solution, 50 μL of 0.5 U / mL elastase, and 50 μL of 0.05 mol / L pH 8.5 Tris-HCl buffer, and reacted at 37°C for 15 min; then added 50 μL of 1 mmol / L N-succinyl-Ala-Ala-Val-Ala p-nitroaniline solution and reacted at 37°C for 20 min, and OD410 was measured, which was recorded as A. 1 ; Group A2 replaced the enzyme solution and substrate solution with equal volumes of buffer solution, and the test results were recorded as A 2 ; Group B1 replaced the paeonol-hyaluronic acid-fullerene complex solution with an equal volume of buffer solution, and the test results were recorded as B 1 In group B2, the paeonol-hyaluronic acid-fullerene complex solution and substrate solution were replaced with an equal volume of buffer solution, and the test results were recorded as B 2 . Calculate the elastase inhibition rate:

[0116] Elastase inhibition rate (%) = [1-(A 1 -A 2 ) / (B 1 -B 2 )]×100%.

[0117] The inhibition rates of samples with different concentrations on elastase are shown in Table 7.

[0118] Table 7 Results of inhibition of elastase by samples of different concentrations

[0119] Sample concentration (mg / mL) 10 20 30 40 50 Elastase inhibition rate (%) 22.31 37.79 63.21 70.83 81.35

[0120] As can be seen from Table 7, the paeonol-hyaluronic acid-fullerene complex prepared by the present invention has an inhibitory effect on elastase, which is concentration-dependent, and the inhibition rate is 81.35% at a concentration of 50 mg / mL. Therefore, the paeonol-hyaluronic acid-fullerene complex has the effects of controlling inflammatory reactions and allergic reactions, promoting skin barrier repair, and having an anti-aging effect.

[0121] Test Example 8: Preventive and repairing effects on UVB-induced damage to human fibroblasts

[0122] Human skin fibroblasts (HFF-1, purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO 2 The cells in the logarithmic growth phase were cultured in an environment and digested with trypsin EDTA to make a cell density of 5×10 5 The suspension of cells / mL was inoculated into a 96-well plate at 100 μL / well and cultured for 24 hours. The paeonol-hyaluronic acid-fullerene complex prepared in Example 1 was dissolved in a DMEM basal culture medium without fetal bovine serum, and the cells in the 96-well plate were divided into groups, with 6 replicate wells in each group.

[0123] Human skin fibroblasts incubated for 24 hours were irradiated with UVB at an energy density of 300 mL / cm 2 , height 10cm, after irradiation for 24h, change culture medium and culture for 24h. The group of paeonol-hyaluronic acid-fullerene complex provided in Example 1 was used as the experimental group, and 100 μL of paeonol-hyaluronic acid-fullerene complex solution was added to human skin fibroblasts after irradiation, so that the final concentrations were 0.125mg / mL, 0.25mg / mL, 0.5mg / mL, 1.0mg / mL, and 2.0mg / mL, respectively, and the cells were incubated for 24h; a blank control group (containing only normal saline) was set, with 5 replicates in each group, and the absorbance was measured at 570nm by MTT method after 24h, and the relative viability of the cells was calculated according to the following formula, and the test results are shown in Table 8.

[0124] Relative survival rate = (A n -A o ) / (A x -A o )×100%

[0125] A n : absorbance value of experimental group;

[0126] A x : absorbance value of negative control group (cell group not irradiated by ultraviolet light, i.e., normal control group in Table 8);

[0127] A o : Absorbance value of blank control group.

[0128] Table 8 Cell activity after UVB irradiation and treatment with different drugs (%)

[0129]

[0130] As shown in Table 8, the relative survival rate of cells after UVB irradiation is 62.37%. The addition of the paeonol-hyaluronic acid-fullerene complex improves the survival rate of fibroblasts after UVB irradiation. When the concentration of the complex is 2.0 mg / ml, the cell survival rate reaches 90.44%. Therefore, the paeonol-hyaluronic acid-fullerene complex provided by the present invention has a repairing effect on cell damage caused by UVB irradiation.

[0131] Test Example 9: Skin Moisture Improvement Test

[0132] Twenty women aged 30 to 40 were divided into two groups, 10 in each group, with a commercially available moisturizing water (Touzhen Huanyan Soothing Moisturizing Softening Toner) as the control group, and a moisturizing water containing 10 mg / mL of paeonol-hyaluronic acid-fullerene was applied to the face twice a day, morning and evening, for 8 weeks, and the skin moisture content was measured using a stratum corneum moisture detector (CorneometerCM825). The skin moisture content is shown in Table 9.

[0133] Table 9 Effects of different moisturizing water on skin moisture content (%)

[0134] Group 0 Weeks 8 weeks Comparison group 20.5 24.5 Experimental Group 20.7 39.6

[0135] It can be seen from Table 9 that the paeonol-hyaluronic acid-fullerene provided by the present invention can effectively increase the water content in the skin, and the improvement effect is significantly better than that of commercially available products.

[0136] Test Case 10 Safety Evaluation Results

[0137] The safety of the present invention on human skin was observed by human trial experiment of Test Example 9. When the subjects were visited, they were carefully questioned, checked and recorded any adverse events that occurred during the use of the samples. The results showed that the 10 subjects in Test Example 9 did not have any adverse skin reactions during the 8-week human experimental study, indicating that the samples in the experimental group and the test group had good safety.

[0138] Application Example 1

[0139] The paeonol-hyaluronic acid-fullerene complex prepared in Example 1 was prepared into a lotion according to the formula in Table 10. The whitening effect of the lotion was evaluated using the Lab colorimetric system. 30 young women with no history of photoallergy or cosmetic allergy were randomly selected and signed an informed consent form. Paeonol-hyaluronic acid-fullerene complex whitening lotion was used continuously for 90 days, and lotion was applied to the cheek skin after washing the face every morning and evening, and the amount of application was based on the slight appearance of the smear layer. The changes in the color values ​​of the cheek skin were observed before use and at 30, 60, and 90 days after use, respectively, and the results of the changes are shown in Table 11.

[0140] Table 10 Formula of Paeonol-Hyaluronic Acid-Fullerene Complex Lotion

[0141] Component name Mass fraction % glycerin 2.5 Sorbitol 2.0 Zinc sulfate 0.2 Citric Acid 0.1 Dodecylphenol polyoxyethylene ether 0.8 Anhydrous ethanol 15.0 Lemon essential oil 0.4 Propylparaben 0.1 Paeonol-hyaluronic acid-fullerene complex 0.8 Sodium hydroxide 0.3 Deionized water Add to 100%

[0142] Table 11 Comparison of cheek skin color values ​​at different times of lotion

[0143] index 0d 30d 60d 90d L 59.18±1.73 61.94±2.20 64.47±1.53** 65.26±1.98** a 10.97±1.67 9.78±1.98** 8.47±1.74** 8.65±1.31** b 19.82±1.46 21.36±2.25 23.32±1.75* 25.07±2.35**

[0144] Among them, compared with before use, *P<0.05; **P<0.01.

[0145] As can be seen from Table 11, when the lotion is used for 30 days, the L value of the cheek skin increases, which is significantly different from before use. When the lotion is used for 60 days, the L value of the cheek skin increases, the a value decreases, and the b value increases, which is significantly different from before use. The L value represents brightness, and the larger the value, the more the color tends to be white. It shows that when the lotion is used for 60 days, the whitening effect on the cheek skin is significantly improved. When the lotion is used for 90 days, the L value of the cheek skin increases, the a value decreases, and the b value increases, which shows extremely significant differences compared with before use, indicating that continuous use of lotion can have a very obvious whitening effect on the cheek skin.

[0146] It can be seen from the above examples that the paeonol-hyaluronic acid-fullerene complex provided by the present invention has good water solubility, stability and biosafety; has no cytotoxicity and can promote cell growth; has a good scavenging effect on DPPH and ABTS free radicals; has an inhibitory effect on tyrosinase, hyaluronidase and elastase; can increase the water content of the skin, and the effect is better than that of commercially available lotions; the paeonol-hyaluronic acid-fullerene complex is used to prepare lotions, which has an obvious whitening effect.

[0147] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A paeonol-hyaluronic acid-fullerene complex, comprising fullerene and an ester coated on the surface of the fullerene, wherein the ester is an ester of paeonol and hyaluronic acid.

2. The paeonol-hyaluronic acid-fullerene complex according to claim 1, characterized in that: The mass ratio of the fullerene to the ester is 1:(1-50).

3. The method for preparing the paeonol-hyaluronic acid-fullerene complex according to claim 1 or 2, characterized in that: The following steps are involved: 1) mixing a paeonol aqueous solution and a sodium hyaluronate aqueous solution to perform an esterification reaction to obtain an esterified aqueous solution; the temperature of the paeonol aqueous solution is 60 to 80° C.; the pH value of the sodium hyaluronate aqueous solution is 4 to 6; 2) The esterified aqueous solution obtained in step 1) is mixed with fullerene and then intermittently ball-milled to obtain a paeonol-hyaluronic acid-fullerene complex; the temperature of the intermittent ball-milling is 15-20°C.

4. The preparation method according to claim 3, characterized in that: The intermittent ball milling in step 2) is performed by stopping the ball mill for 20 to 40 minutes after running the ball mill for 1 to 20 minutes.

5. The preparation method according to claim 3 or 4, characterized in that: The rotation speed of the intermittent ball milling in the step 2) is 500 to 800 rpm, and the time of the intermittent ball milling is 6 to 12 hours.

6. The preparation method according to claim 3, characterized in that: In the step 1), the mass ratio of paeonol in the paeonol aqueous solution to sodium hyaluronate in the sodium hyaluronate aqueous solution is (0.5-5): (20-60).

7. The preparation method according to claim 3, characterized in that: The ball milling medium in the intermittent ball milling in step 2) is 1,3-butanediol, and the volume ratio of the ball milling medium to the ball milling material in the intermittent ball milling is (2-4):

1.

8. The preparation method according to claim 3, characterized in that: The temperature of the esterification reaction in step 1) is 60-80°C.

9. The preparation method according to claim 3, characterized in that: The molecular weight of sodium hyaluronate in step 1) is 42.5-145 kD.

10. Use of the paeonol-hyaluronic acid-fullerene complex according to claim 1 in the preparation of cosmetics.

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