Sodium hyaluronate-fullerene 5D complex, and preparation method and application thereof

Abstract

The application discloses a sodium hyaluronate-fullerene 5D compound and a preparation method and application thereof, and belongs to the technical field of compounds, and solves the problem of poor water solubility of the existing fullerene. The preparation method comprises the following steps: ball-milling fullerene, sodium hyaluronate and sodium hydroxide; adding H2O2 solution and hydrolyzing sodium hyaluronate in the first mixture, and continuously ball-milling; adding water in the second mixture, and ultrasonic treatment to obtain a suspension; continuously adding acetylated sodium hyaluronate and water in the suspension, and stirring and uniformly mixing; removing insoluble substances from the third mixture to obtain a mixed solution, and spray drying to obtain sodium hyaluronate-fullerene dry powder; and mixing the sodium hyaluronate-fullerene dry powder, sodium hyaluronate, a sodium hyaluronate crosslinking polymer and water, stirring and uniformly mixing, continuously adding 1,2-pentanediol and ethylhexylglycerin, and stirring to obtain the sodium hyaluronate-fullerene 5D compound. The sodium hyaluronate-fullerene 5D compound has good water solubility.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of composite, and particularly relates to a sodium hyaluronate-fullerene 5D composite as well as a preparation method and application thereof. BACKGROUND

[0002] Fullerene is a hollow spherical, ellipsoidal and cylindrical molecule composed of carbon. The surface of fullerene has a large number of covalent double bonds which are easy to react with free radicals, so it can be used as an antioxidant in vivo. However, fullerene is not soluble in water, which limits its application. SUMMARY

[0003] In view of the above analysis, the present application aims to provide a sodium hyaluronate-fullerene 5D composite as well as a preparation method and application thereof, which can at least solve one of the following technical problems: (1) poor water solubility of fullerene, which limits its application; (2) the existing method improves the water solubility of fullerene by adding a large amount of PVP or butanediol surfactant, and the PVP or butanediol solubilizer can have certain toxicity to the human body, and even has the risk of causing cancer; (3) direct solubilization of butanediol and PVP increases the distance between fullerene and free radicals, greatly reducing the free radical scavenging efficiency of fullerene.

[0004] The purpose of the present application is mainly achieved by the following technical solutions:

[0005] The present application provides a preparation method of a sodium hyaluronate-fullerene 5D composite, comprising the following steps:

[0006] Step 1, ball-milling fullerene, sodium hyaluronate and sodium hydroxide to obtain a first mixture;

[0007] Step 2, adding H2O2 solution and hydrolyzing sodium hyaluronate in the first mixture, and continuing to ball-mill to obtain a second mixture;

[0008] Step 3, adding water in the second mixture, and ultrasonic treatment to obtain a suspension;

[0009] Step 4, continuously adding acetylated sodium hyaluronate and water in the suspension, and stirring to mix uniformly to obtain a third mixture;

[0010] Step 5, filtering or centrifuging the third mixture to remove insoluble substances to obtain a mixed solution, and spray drying the mixed solution to obtain a sodium hyaluronate-fullerene dry powder;

[0011] Step 6, mixing the sodium hyaluronate-fullerene dry powder, sodium hyaluronate, sodium hyaluronate cross-linked polymer and water, stirring to mix uniformly, then continuously adding 1,2-pentanediol and ethylhexyl glycerin, stirring to mix uniformly to obtain a sodium hyaluronate-fullerene 5D composite.

[0012] Further, in step 1, the molecular weight of the sodium hyaluronate is 1M-1.8M Da.

[0013] Further, in step 1, the mass ratio of fullerene, sodium hyaluronate and sodium hydroxide is 4-6:0.5-2:0.6-1.2.

[0014] Further, in step 2, the molecular weight of the hydrolyzed sodium hyaluronate is 5000-8000 Da.

[0015] Further, in step 2, the mass-volume ratio of the hydrolyzed sodium hyaluronate and the H2O2 solution is 0.3-1g:5-17ml.

[0016] Further, in steps 1 and 2, the mass ratio of the hydrolyzed sodium hyaluronate and the fullerene is 0.1-6:0.5-6.

[0017] Further, the mass ratio of the hydrolyzed sodium hyaluronate and the acetylated sodium hyaluronate is 0.3-1g:1.5-2g.

[0018] Further, in step 6, the mass-volume ratio of the sodium hyaluronate fullerene dry powder, sodium hyaluronate, sodium hyaluronate cross-linked polymer, 1,2-pentanediol, ethylhexyl glycerol and water is 50-125g:8-15g:8-14g:100-500g:1-5g:10L.

[0019] The application also provides a sodium hyaluronate-fullerene 5D complex prepared by the above preparation method.

[0020] The application also provides an application of a sodium hyaluronate-fullerene 5D complex, wherein the sodium hyaluronate-fullerene 5D complex is used as a raw material for cosmetics, medicines or health products.

[0021] Compared with the prior art, the application can at least achieve one of the following beneficial effects:

[0022] a) The method of the application can make the reactants fully contact and mix by mechanical energy by first ball-milling fullerene, sodium hyaluronate and sodium hydroxide, so that the high molecular weight sodium hyaluronate is degraded, the addition of fullerene can accelerate the degradation while being mixed with sodium hyaluronate, and the antioxidant property of fullerene can protect sodium hyaluronate from being oxidized during the hydrolysis process; the small molecular weight sodium hyaluronate after hydrolysis can wrap the fullerene molecules, thereby physically solubilizing the fullerene and solving the water-solubility problem of fullerene.

[0023] b) In the method of the present application, four different molecular weight and different function sodium hyaluronate are synthesized with fullerene to form water-soluble sodium hyaluronate-fullerene 5D complex, the macromolecular sodium hyaluronate in the sodium hyaluronate-fullerene 5D complex has the effects of skin outer layer moisturizing and improving product skin feeling; the small and medium molecular sodium hyaluronate can enhance the permeability of fullerene and is not sticky; the sodium hyaluronate cross-linked polymer can disperse fullerene and prevent dust and haze; the acetylated sodium hyaluronate can double moisturize and improve the skin residence of fullerene; the fullerene can resist oxidation, anti-wrinkle, remove spots and whiten, prevent hair loss and remove acne; therefore, the sodium hyaluronate-fullerene 5D complex prepared by the method of the present application has the effects of good water solubility, multiple moisturizing, antioxidant and anti-aging, and can be directly used in cosmetics, medicines and health products.

[0024] c) The sodium hyaluronate-fullerene 5D complex prepared by the method of the present application does not contain PVP, butylene glycol and other substances which can have certain toxicity to human body and even have the risk of causing cancer, is safe and effective, and has good effect.

[0025] d) The sodium hyaluronate-fullerene 5D complex prepared by the method of the present application is in the form that macromolecular sodium hyaluronate wraps fullerene and degrades and shortens the long chain of macromolecular sodium hyaluronate, compared with the fullerene directly wrapped by PVP, the wrapping is smaller and more uniform, and the contact probability of fullerene and free radicals is greatly increased, so that the clearance rate of free radicals is improved.

[0026] e) The synthesis method of the present application is simple, has few impurities, and has short whole chain time, and can be suitable for large-scale industrial production.

[0027] Other features and advantages of the present application will be set forth in the following description of the application, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the application. The objects and other advantages of the present application can be realized and attained by the written description. BRIEF DESCRIPTION OF DRAWINGS

[0028] The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the principles of the application.

[0029] Figure 1 is a process diagram of the preparation method of the sodium hyaluronate-fullerene 5D complex provided by the present application;

[0030] Figure 2 is a macroscopic photo of the sodium hyaluronate-fullerene 5D complex provided by the present application;

[0031] Figure 3 is a macroscopic photo of the mixture of Comparative Example 1;

[0032] Figure 4is a macroscopic photo of the mixture of Comparative Example 2;

[0033] Figure 5 is a graph of the results of the radical ABTS scavenging experiment;

[0034] Figure 6 is a schematic diagram of the results of the cytotoxicity experiment of the sodium hyaluronate-fullerene 5D complex of the present application;

[0035] Figure 7 is a schematic diagram of the results of the radical scavenging experiment at the cellular level of the sodium hyaluronate-fullerene 5D complex of the present application. DETAILED DESCRIPTION

[0036] The sodium hyaluronate-fullerene 5D complex, the preparation method and the application thereof are described in further detail below in conjunction with specific examples, which are only for the purpose of comparison and explanation, and the present application is not limited to these examples.

[0037] Fullerene can be used as one of the raw materials for skin care products, however, fullerene is not soluble in water, and generally needs to add a solubilizer. The existing water-solubilization technology of fullerene is mostly to improve the water solubility of fullerene by adding a large amount of PVP and butylene glycol surfactants. This method has three shortcomings: 1. Long-term contact of the skin with PVP, butylene glycol and other solubilizers will produce certain toxicity to the human body, and even have the risk of causing cancer; 2. Poor bioavailability, direct solubilization of butylene glycol and PVP will increase the distance between fullerene and free radicals, greatly reducing the efficiency of fullerene radical scavenging; 3. Poor water solubility.

[0038] The present application provides a sodium hyaluronate-fullerene 5D complex, and the components of the sodium hyaluronate-fullerene 5D complex include macromolecular sodium hyaluronate, small and medium molecular weight sodium hyaluronate, sodium hyaluronate cross-linked polymer, acetylated sodium hyaluronate and fullerene.

[0039] The present application provides a preparation method of a sodium hyaluronate-fullerene 5D complex, which comprises the following steps:

[0040] Step 1, ball-milling fullerene, sodium hyaluronate and sodium hydroxide to obtain a first mixture;

[0041] Step 2, adding H2O2 solution and hydrolyzing sodium hyaluronate in the first mixture, and continuing to ball-mill to obtain a second mixture;

[0042] Step 3, adding water in the second mixture, and ultrasonic treatment to obtain a suspension;

[0043] Step 4, continuously adding acetylated sodium hyaluronate and water in the suspension, and stirring to mix uniformly to obtain a third mixture;

[0044] Step 5, filtering or centrifuging the third mixture to remove insoluble substances to obtain a mixed solution, and spray drying the mixed solution to obtain a sodium hyaluronate-fullerene dry powder;

[0045] Step 6, mixing the sodium hyaluronate-fullerene dry powder, sodium hyaluronate, sodium hyaluronate cross-linked polymer and water, stirring and mixing uniformly, then continuously adding 1,2-pentanediol and ethylhexyl glycerin, stirring and mixing uniformly to obtain a sodium hyaluronate-fullerene 5D complex, and the sodium hyaluronate-fullerene 5D complex can be canned to obtain the product.

[0046] Specifically, in the above step 1, the fullerene can be C 60 , C 70 , C 78 , C 82 , C 84 , C 90 , C 96 , C 28 or C 32 , etc.

[0047] Specifically, in the above step 1, the molecular weight of sodium hyaluronate is 1M-1.8M Da (Da is Dalton, 1M Da is 1 million Da).

[0048] Specifically, in the above step 1, the addition of sodium hydroxide can effectively degrade the high molecular weight sodium hyaluronate, and the main principle is that in an alkaline environment, the carboxylic acid structure of the glucuronic acid in the hyaluronic acid structure is easy to react with the base, and the glucuronic acid is opened; ball milling is a physical degradation auxiliary sodium hyaluronate degraded by sodium hydroxide, and ball milling can make sodium hyaluronate and sodium hydroxide fully contact and mix while generating a large amount of heat to further promote the degradation of high molecular weight sodium hyaluronate; the addition of fullerene can accelerate the degradation while mixing with sodium hyaluronate, and the antioxidant property of fullerene can also protect sodium hyaluronate from being oxidized during the hydrolysis process; in step 1, the chemical method and the physical method cooperate to promote the degradation of high molecular weight sodium hyaluronate.

[0049] Specifically, the reaction involved in step 1 is as follows: the mechanical force of ball milling causes the covalent bond in the middle of the sodium hyaluronate macromolecular chain to break, and the high temperature generated by ball milling causes the molecular chain to break, in addition, sodium hydroxide can promote the molecular chain to break more easily.

[0050] Specifically, in step 1, in order to reduce the introduction of impurities, the higher the purity of sodium hydroxide, the better, therefore, the electronic grade sodium hydroxide is used.

[0051] Specifically, in step 1, too much sodium hydroxide causes unnecessary waste and makes the final solution alkaline instead of neutral, and neutralizes the H2O2 added later, which cannot play the role of hydrogen peroxide degradation; if the amount of sodium hydroxide is too small, it cannot provide an alkaline environment and cannot effectively break the molecular chain in the structure of hyaluronic acid, therefore, the mass ratio of fullerene, sodium hyaluronate and sodium hydroxide is controlled to be 4-6:0.5-2:0.6-1.2.

[0052] Specifically, in step 1, ball milling can be carried out in a agate ball mill jar, the ball milling time is too short, the ball milling speed is too small to play the role of physical stirring and mixing, and the degradation effect is not good, however, the ball milling time is too long, and the ball milling speed is too high to destroy the carbon cage structure of fullerene, thereby affecting the effect of removing free radicals; therefore, the ball milling speed is controlled to be 500-700 r / min, and the ball milling time is controlled to be 2-5 h. For example, the ball milling speed is 500 r / min, 530 r / min, 550 r / min, 600 r / min, 630 r / min, 650 r / min; the ball milling time is 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h.

[0053] Specifically, in step 1, the small and medium molecular weight hyaluronic acid sodium after hydrolysis can wrap up the fullerene molecules, and play a physical solubilization role, solving the problem of water solubility of fullerene.

[0054] Specifically, in step 1, the first mixture mainly includes small and medium molecular weight hyaluronic acid sodium (the molecular weight of small and medium molecular weight hyaluronic acid sodium powder is detected according to QB / T4416-2012 “Cosmetic Raw Material Hyaluronic Acid Sodium”) with a molecular weight of 20000-40000 Da and fullerene, and the small and medium molecular weight hyaluronic acid sodium wraps the fullerene; because in the reaction process of step 1, the large molecular weight hyaluronic acid sodium is degraded and shortened while wrapping the fullerene, compared with the fullerene wrapped by PVP directly, the wrapping is smaller and more uniform, and the dispersibility of fullerene is improved.

[0055] Specifically, in step 1, the first mixture is a uniform yellow-brown powder.

[0056] Specifically, in step 2, the molecular weight of the hydrolyzed hyaluronic acid sodium is 5000-8000 Da.

[0057] Specifically, in step 2, the mass concentration of the H2O2 solution is 25%-35%, for example, 25%, 28%, 30%, 33%, 35%.

[0058] Specifically, in step 2, the mass concentration of the H2O2 solution is 30%.

[0059] Specifically, in step 2, if the amount of H2O2 solution is too small, it cannot effectively degrade the sodium hyaluronate after neutralization with excess sodium hydroxide, and if the amount is too large, it will cause waste. Therefore, the mass-volume ratio of hydrolyzed sodium hyaluronate to H2O2 solution is controlled to be 0.3-1 g:5-17 ml.

[0060] Specifically, in steps 1 and 2, the mass ratio of hydrolyzed sodium hyaluronate to fullerene is controlled to be 0.1-6:0.5-6; for example, 0.3-1:4-6.

[0061] Specifically, in step 2, if the ball milling time is too short or the ball milling speed is too low, the physical stirring and mixing of ball milling cannot be achieved, the reaction is not sufficient, and the degradation effect cannot be achieved. However, if the ball milling time is too long or the ball milling speed is too high, the carbon cage structure of fullerene will be damaged, thereby affecting the effect of scavenging free radicals; therefore, the ball milling speed is controlled to be 200-600 r / min, and the ball milling time is controlled to be 0.5-5 h. For example, the ball milling speed is 300 r / min, 350 r / min, 400 r / min, 450 r / min, 500 r / min, 550 r / min; the ball milling time is 0.7 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h.

[0062] Specifically, in step 2, H2O2 is added, which can neutralize the excess sodium hydroxide in step 1, and H2O2 can also generate H·, OH·, O· and other free radicals. The action of free radicals on the molecular chain of sodium hyaluronate activates the reaction site, thereby further degrading the sodium hyaluronate. In addition, the degraded sodium hyaluronate uniformly wraps the fullerene, and the excess H2O2 is finally decomposed into water. Hydrolyzed sodium hyaluronate is added to provide small molecule sodium hyaluronate to enhance the permeability of fullerene.

[0063] Specifically, in step 2, the second mixture is a brown / brownish liquid.

[0064] Specifically, in steps 3 and 4, in order to reduce impurity pollution, water can be ultrapure water.

[0065] Specifically, in step 3, the solution on the agate pot and agate beads is washed off during ultrasonic treatment. The mechanical bond breaking of ultrasonic waves is the main physical effect of ultrasonic waves, which can make the material particles have extremely high motion acceleration in the ultrasonic field, thereby producing intense and rapid mechanical motion. The molecular chains of high polymers are broken under the combined action of high-speed vibration and shear force, thereby achieving the effect of degradation. The mechanical bond breaking plays a leading role when the viscosity of the high polymer solution is low, but when degrading high-concentration high-viscosity solutions, the mechanical bond breaking will be limited due to the weakening of the ultrasonic vibration effect.

[0066] Specifically, in step 3, the amount of ultrapure water is 300-500 ml.

[0067] Specifically, in step 3, the frequency of the ultrasonic wave is 40-100 kHz, and the processing time is 10-30 min.

[0068] Specifically, in step 3, the suspension mainly includes water, small molecule sodium hyaluronate, sodium hyaluronate-coated fullerene, and a small amount of unsuccessfully coated fullerene.

[0069] Specifically, the mass ratio of the hydrolyzed sodium hyaluronate and acetylated sodium hyaluronate to the total volume of water in steps 3 and 4 is 0.3-1 g:1.5-2 g:1 L.

[0070] Specifically, in step 4, the molecular weight of the acetylated sodium hyaluronate is 50-100 thousand Da.

[0071] Specifically, in the above step 5, a filter membrane with a pore size of 0.45-5 um is used to filter the third mixture to remove insoluble substances to obtain a mixed solution; or a centrifugal method is used, with a centrifugal speed of 4000-15000 rpm / min, and a duration of 5-30 min, to remove insoluble substances.

[0072] Specifically, in step 5, the insoluble substances are mainly a small amount of unsuccessfully coated fullerene.

[0073] Specifically, in step 5, the water content of the sodium hyaluronate fullerene dry powder needs to be controlled below 30%.

[0074] Specifically, in step 5, the spray drying controls the air supply temperature to be 120-180℃.

[0075] Specifically, in step 6, the molecular weight of the sodium hyaluronate is 1M-1.8M Da (Da is Dalton).

[0076] Specifically, in step 6, the mass-volume ratio of the sodium hyaluronate fullerene dry powder, sodium hyaluronate, sodium hyaluronate cross-linked polymer, 1,2-pentanediol, ethylhexyl glycerol, and water is controlled to be 50-125 g:8-15 g:8-14 g:100-500 g:1-5 g:10 L.

[0077] Specifically, the sodium hyaluronate-fullerene 5D complex obtained in step 6 has good water solubility, good antioxidant properties, and good moisturizing properties, and is safe and effective.

[0078] The sodium hyaluronate-fullerene 5D complex of the present application has good water solubility, good antioxidant properties, and good biological safety, and can be used as an antioxidant and moisturizing raw material, directly used in cosmetics, pharmaceuticals, and health products.

[0079] Compared with the prior art, the method of the present application can make the reactants fully contact and mix by mechanical energy by first ball-milling the fullerene, sodium hyaluronate and sodium hydroxide, so that the high molecular sodium hyaluronate is degraded, the addition of fullerene can accelerate the degradation while being uniformly mixed with sodium hyaluronate, and the antioxidant property of fullerene can protect sodium hyaluronate from being oxidized during the hydrolysis process; the small molecular sodium hyaluronate after hydrolysis can wrap up the fullerene molecules to play a physical solubilization role, thus solving the water solubility problem of fullerene.

[0080] In the method of the present application, four different molecular weight and different function sodium hyaluronate and fullerene are synthesized into water-soluble sodium hyaluronate-fullerene 5D complex, the large molecular sodium hyaluronate in the sodium hyaluronate-fullerene 5D complex has the effect of moisturizing the outer layer of the skin and improving the skin feel of the product; the medium and small molecular sodium hyaluronate can enhance the permeability of fullerene and not be sticky; the sodium hyaluronate cross-linked polymer can disperse fullerene and prevent dust and haze; the acetylated sodium hyaluronate can double moisturize and improve the skin retention of fullerene; fullerene can be antioxidant, anti-wrinkle, spot-removing and whitening, hair loss prevention and acne removal; therefore, the sodium hyaluronate-fullerene 5D complex prepared by the method of the present application has the effects of multiple moisturizing, antioxidant and anti-aging, and can be directly used in cosmetics, drugs and health products.

[0081] The sodium hyaluronate-fullerene 5D complex prepared by the method of the present application does not contain PVP, butylene glycol and other substances that can have certain toxicity to the human body and even have the risk of causing cancer, and is safe and effective.

[0082] The sodium hyaluronate-fullerene 5D complex prepared by the method of the present application is in the form of large molecular sodium hyaluronate wrapping fullerene and degrading and shortening the long chain of large molecular sodium hyaluronate, compared with the fullerene directly wrapped by PVP, the wrapping is smaller and more uniform, greatly increasing the contact probability of fullerene and free radicals and thus improving the clearance rate of free radicals, the sodium hyaluronate-fullerene 5D complex of the present application has excellent antioxidant property; the improvement of the antioxidant property of the sodium hyaluronate-fullerene 5D complex of the present application benefits from the fact that the improvement of the water solubility of fullerene by sodium hyaluronate greatly improves the contact probability of fullerene and free radicals, and the antioxidant effect of sodium hyaluronate itself is superimposed, so as to achieve the synergistic antioxidant effect of fullerene and sodium hyaluronate.

[0083] The synthesis method of the present application is simple, has few impurities and short total chain time, and can be suitable for large-scale industrial production.

[0084] Example 1

[0085] The present embodiment provides a preparation method of sodium hyaluronate-fullerene 5D complex, as shown in Figure 1 the figure, comprising:

[0086] Step 1, take 5g of fullerene, 1g of sodium hyaluronate with a molecular weight of 1M-1.8M Da, and 0.9g of electronic grade sodium hydroxide into a 500ml agate ball mill jar, ball mill for 3h at a speed of 600r / min;

[0087] Step 2, add 16.8ml of 30% H2O2 solution and 0.45g of hydrolyzed sodium hyaluronate, continue to ball mill for 1h at a speed of 600r / min;

[0088] Step 3, wash out the sample in the agate ball mill jar with 400ml of ultrapure water, ultrasonic treatment for 15min at a frequency of 80kHz;

[0089] Step 4, add 1.8g of acetylated sodium hyaluronate, add ultrapure water to 1L, and homogenize under normal pressure to obtain a mixture;

[0090] Step 5, filter out the insoluble precipitate, and then spray dry to obtain sodium hyaluronate fullerene dry powder;

[0091] Step 6, homogenize 6g of sodium hyaluronate fullerene dry powder, 1.44g of sodium hyaluronate, 1.2g of sodium hyaluronate cross-linked polymer and 1L of ultrapure water under normal pressure, and mix 12g of 1,2-pentanediol and 0.2g of ethylhexyl glycerol, and finally canning to form the product.

[0092] Specifically, the fullerene is C 60 .

[0093] As Figure 2 shown is a macroscopic photo of the sodium hyaluronate-fullerene 5D complex prepared in this embodiment. The sodium hyaluronate-fullerene 5D complex prepared in this embodiment has good water solubility, antioxidant properties, good moisturizing properties, and is safe and effective.

[0094] Example 2

[0095] The present embodiment provides a preparation method of a sodium hyaluronate-fullerene 5D complex, comprising:

[0096] Step 1, take 5g of fullerene, 1.5g of sodium hyaluronate with a molecular weight of 1M-1.8M Da, and 1.2g of electronic grade sodium hydroxide into a 500ml agate ball mill jar, ball mill for 3h at a speed of 600r / min;

[0097] Step 2, add 15ml of 30% H2O2 solution and 0.5g of hydrolyzed sodium hyaluronate, continue to ball mill for 1h at a speed of 600r / min;

[0098] Step 3, wash out the sample in the agate ball mill tank with 400 ml ultrapure water, ultrasonic treatment for 15 min, frequency 80 kHz;

[0099] Step 4, add 1.8 g of acetylated sodium hyaluronate, add ultrapure water to 1 L, and obtain a mixture by stirring and homogenizing under normal pressure;

[0100] Step 5, filter or centrifuge the mixture to remove insoluble precipitates (purification, purification), and then spray dry to obtain a dry sodium hyaluronate fullerene powder;

[0101] Step 6, stir and homogenize 5 g of sodium hyaluronate fullerene dry powder, 1.26 g of sodium hyaluronate, 1.05 g of sodium hyaluronate cross-linked polymer and 1 L of ultrapure water under normal pressure, and mix 12 g of 1,2-pentanediol and 0.18 g of ethylhexyl glycerol, and finally canning to form a product.

[0102] Specifically, the fullerene is C 60 .

[0103] The sodium hyaluronate-fullerene 5D complex prepared in this embodiment has good water solubility, antioxidant properties, good moisturizing properties, and is safe and effective.

[0104] Example 3

[0105] The present embodiment provides a preparation method of a sodium hyaluronate-fullerene 5D complex, comprising:

[0106] Step 1, weigh 5 g of fullerene, 0.5 g of sodium hyaluronate with a molecular weight of 1M-1.8M Da, and 1 g of electronic grade sodium hydroxide into a 500 ml agate ball mill tank, and mill for 3 h at a speed of 600 r / min;

[0107] Step 2, add 17 ml of 30% H2O2 solution and 0.6 g of hydrolyzed sodium hyaluronate, and continue to mill for 1 h at a speed of 600 r / min;

[0108] Step 3, wash out the sample in the agate ball mill tank with 400 ml ultrapure water, ultrasonic treatment for 15 min, frequency 80 kHz;

[0109] Step 4, add 1.8 g of acetylated sodium hyaluronate, add ultrapure water to 1 L, and obtain a mixture by stirring and homogenizing under normal pressure;

[0110] Step 5, filter or centrifuge the mixture to remove insoluble precipitates (purification, purification), and then spray dry to obtain a dry sodium hyaluronate fullerene powder;

[0111] Step 6, the sodium hyaluronate fullerene dry powder 5.5 g, 1.3 g sodium hyaluronate, 1.05 g sodium hyaluronate cross-linked polymer and 1 L ultrapure water are stirred and homogenized under normal pressure, and 12 g 1,2-pentanediol, 0.18 g ethylhexyl glycerol are mixed, and finally canned to form a product.

[0112] Specifically, the fullerene is C 60 .

[0113] The sodium hyaluronate-fullerene 5D complex prepared in the embodiment has good water solubility, antioxidant property and moisturizing property, and is safe and effective.

[0114] The inventors have conducted a large number of studies during the research process, and some of the schemes will be compared with the comparative examples as follows.

[0115] Comparative Example 1

[0116] In this comparative example, 50 mg of fullerene and 10 mg of hydrolyzed sodium hyaluronate (the average molecular weight of the hydrolyzed sodium hyaluronate is 5000-8000 Da) are directly weighed, 100 ml of distilled water is added after mixing, and then homogenized for 5 minutes with a homogenizer; observation and photography are performed as shown in Figure 3 ; it can be seen that the fullerene in this comparative example is directly precipitated and cannot be water-soluble. The obtained sample solution is marked as sample No. 1. The sample solution obtained in Example 1 is marked as sample No. 3.

[0117] Comparative Example 2

[0118] In this comparative example, 50 mg of fullerene and 10 mg of pvp (the molecular weight of pvp is 58000 Da) are directly weighed, 100 ml of distilled water is added after mixing, and then homogenized for 5 minutes with a homogenizer; observation and photography are performed as shown in Figure 4 ; it can be seen that the fullerene in this comparative example is directly precipitated and cannot be water-soluble. The obtained sample solution is marked as sample No. 2.

[0119] Comparative Example 3

[0120] In this comparative example, 0.5 g of fullerene and 0.1 g of hydrolyzed sodium hyaluronate (the average molecular weight of the hydrolyzed sodium hyaluronate is 5000-8000 Da) are directly weighed, ball-milled for 3 hours at a speed of 600 rpm, and then 1.68 ml of water is added and ball-milled for 1 hour at a speed of 600 rpm. The obtained sample solution is marked as sample No. 4.

[0121] Comparative Example 4

[0122] In this comparative example, 0.5 g of fullerene and 0.1 g of pvp (the molecular weight of pvp is 58000 Da) are directly weighed, ball-milled for 3 hours at a speed of 600 rpm, and then 1.68 ml of water is added and ball-milled for 1 hour at a speed of 600 rpm. The obtained sample solution is marked as sample No. 5.

[0123] Comparative Example 5

[0124] In this comparative example, 0.5 g of fullerene and 0.1 g of sodium hyaluronate with an average molecular weight of 1.32 M Da were directly weighed, 1.68 ml of water was added, and ball milling was performed at 600 rpm for 4 hours. The obtained sample solution was labeled as Sample No. 6.

[0125] Comparative Example 6

[0126] In this comparative example, 0.5 g of fullerene and 0.1 g of pvp with a molecular weight of 58000 Da were directly weighed, 1.68 ml of water was added, and ball milling was performed at 600 rpm for 4 hours. The obtained sample solution was labeled as Sample No. 7.

[0127] Comparative Example 7

[0128] In this comparative example, 0.5 g of fullerene and 0.1 g of hydrolyzed sodium hyaluronate with an average molecular weight of 5000-8000 Da were directly weighed, 1.68 ml of water was added, and ball milling was performed at 600 rpm for 4 hours. The obtained sample solution was labeled as Sample No. 8.

[0129] Comparative Example 8

[0130] In this comparative example, 0.5 g of fullerene and 0.1 g of sodium hyaluronate with an average molecular weight of 1.32 M Da were directly weighed, ball milling was performed at 600 rpm for 3 hours, 1.68 ml of water was added, and ball milling was performed at 600 rpm for 1 hour. The obtained sample solution was labeled as Sample No. 9.

[0131] The above Sample Nos. 1, 2, 3, 4, 5, 6, 7, 8, and 9 were diluted to three concentration gradients of 50 ppm, 100 ppm, and 200 ppm according to the concentration of fullerene, and free radical ABTS scavenging experiments were performed. The results are shown in Table 1. Figure 5 It can be seen that the free radical ABTS scavenging rate of Sample No. 3 of Example 1 is as high as 99.75% at a concentration of 200 ppm, 78.89% at a concentration of 100 ppm, and 49.1% at a concentration of 50 ppm, all of which are much higher than the free radical ABTS scavenging rates of other samples. The sodium hyaluronate-fullerene 5D complex prepared in Example 2 and Example 3 can also achieve a comparable free radical ABTS scavenging rate, which will not be described here. It can be seen that the sodium hyaluronate-fullerene 5D complex has good antioxidant performance, and the improvement of antioxidant performance is due to the fact that the water solubility of fullerene is improved after sodium hyaluronate is added, which greatly improves the probability of contact between fullerene and free radicals, and the effect of sodium hyaluronate itself on scavenging free radicals is superimposed, thereby achieving the effect of synergistic antioxidant of fullerene and sodium hyaluronate.

[0132] Table 1 Radical ABTS scavenging experiment

[0133]

[0134] Cytotoxicity experiment:

[0135] The cells were routinely cultured to prepare a cell suspension with a density of 3.0-3.5*10 4 The cell suspension was inoculated in a 96-well cell culture plate at 100 μL per well, and cultured for 18-24 h. The original culture solution in the well was discarded, 100 μL of the sample (TA) of Example 1 with different concentrations was added to each well, and the plate was returned to the incubator for incubation for 72 h. The plate was taken out, 20 μL of MTT solution was added to each well, and the plate was incubated in the incubator for 3-4 h. The liquid in the well was removed, 100 μL of DMSO was added to each well, and the plate was placed on a shaker for 10-15 min. The absorbance was measured at a wavelength of 570 nm on an enzyme-labeled instrument.

[0136] Data analysis: the cell viability of the negative control group (NC) was 100%, and the relative cell viability of each group was calculated.

[0137] Viability (%) = (OD TA - OD Blank ) / (OD NC - OD Blank )*100%

[0138] The experimental results are shown in Table 2 and Figure 6 When the concentration of the sodium hyaluronate-fullerene 5D complex of Example 1 was 6.329 mg / mL, the relative cell viability was 90.86±5.76%; when the concentration of the sodium hyaluronate-fullerene 5D complex of Example 1 was 20 mg / mL, the relative cell viability was 73.95±5.25%, which fully embodied the good biological safety of the sodium hyaluronate-fullerene 5D complex of the application.

[0139] Table 2 Cytotoxicity experiment results

[0140] Concentration (mg / mL) OD 570 ]]> Relative cell viability (%) NC 1.1491±0.0783 100.00±7.08 20.000 0.8498±0.0603 73.95±5.25 6.329 1.0441±0.0662 90.86±5.76 2.003 1.0569±0.0928 91.97±8.08 0.634 1.0765±0.0843 93.67±7.34 0.201 1.1058±0.0859 96.23±7.48 0.063 1.0517±0.1017 91.52±8.85 0.020 1.0751±0.1162 93.56±10.12 0.006 1.1049±0.1245 96.15±10.83

[0141] Microbial detection:

[0142] According to the relevant provisions of the Cosmetics Safety Technical Specifications 2015 Chapter 5 2 Colony Count Test Method, Cosmetics Safety Technical Specifications 2015 Chapter 5 6 Mold and Yeast Test Method, Cosmetics Safety Technical Specifications 2015 Chapter 5 3 Heat-resistant Coliform Test Method, Cosmetics Safety Technical Specifications 2015 Chapter 5 5 Staphylococcus aureus Test Method, Cosmetics Safety Technical Specifications 2015 Chapter 5 4 Pseudomonas aeruginosa Test Method, etc.; the colony count, mold and yeast, heat-resistant coliform, Staphylococcus aureus, Pseudomonas aeruginosa and other indicators of the sodium hyaluronate-fullerene 5D complex of Example 1 were determined.

[0143] The results are shown in Table 3: the colony count, mold and yeast, heat-resistant coliform, Staphylococcus aureus, Pseudomonas aeruginosa and other indicators are all qualified.

[0144] The colony count, mold and yeast, heat-resistant coliform, Staphylococcus aureus, Pseudomonas aeruginosa and other indicators of the sodium hyaluronate-fullerene 5D complex of Example 2-3 were also determined, and the indicators were all qualified.

[0145] Table 3 Test Results

[0146]

[0147]

[0148] Heavy metal detection:

[0149] According to the Cosmetics Safety Technical Specifications 2015 Chapter 4 1.6 Inductively Coupled Plasma Mass Spectrometry, the mercury, lead, arsenic, cadmium and other heavy metal element contents of the sodium hyaluronate-fullerene 5D complex of Example 1 were determined.

[0150] The results are shown in Table 4: the mercury, lead, arsenic, cadmium and other heavy metal element contents are all less than the corresponding detection limit, indicating that the sodium hyaluronate-fullerene 5D complex of Example 1 does not contain mercury, lead, arsenic, cadmium and other heavy metal elements.

[0151] The sodium hyaluronate-fullerene 5D complex of Example 2-3 was also detected, and did not contain mercury, lead, arsenic, cadmium and other heavy metal elements.

[0152] Table 4 Test Results

[0153]

[0154]

[0155] Skin occlusive patch test:

[0156] Method according to: “Cosmetic Safety Technical Specifications 2015 Edition”. Test purpose: to detect the potential possibility of causing skin adverse reactions by the test substance. Testee information: a total of 30 people, 1 male and 29 females, aged 21 to 42 years old, with an average age of 32 years old. Test site: back; frequency of use: once.

[0157] Patch test method: select qualified patch test equipment with an area not exceeding 50 mm 2 and a depth of about 1 mm, apply about 0.020-0.025 mg of the test substance in the patch test equipment chamber, and apply a special adhesive tape to the back of the testee. When the test sample is a cosmetic product as it is, the control is a blank control (without any substance). When the test substance is a diluted cosmetic product, the control is the diluent used for the cosmetic product.

[0158] Experimental procedure: remove the patch test equipment after 24 hours, gently wipe off any remaining product with a paper towel, and observe the skin reaction at 0.5 hours, 24 hours, and 48 hours after removing the patch test equipment, respectively. Record the results according to the skin reaction grading standard in “Cosmetic Safety Technical Specifications” (2015).

[0159] Table 5 Skin reaction grading standard for skin closed patch test:

[0160]

[0161]

[0162] Result determination standard

[0163] According to the cumulative calculation of the test scores, the results should meet the following criteria: the number of people with grade 1 skin adverse reactions is less than or equal to 5, the number of people with grade 2 skin adverse reactions is less than or equal to 2, and the number of people with grade 3 or above skin adverse reactions is 0 (the total number of skin adverse reactions is less than or equal to 5).

[0164] The skin reaction summary of the sodium hyaluronate-fullerene 5D complex of Example 1 in the human skin patch test is shown in Table 6; the experimental results show that 2 out of 30 people had grade 1 skin adverse reactions. This indicates that the test substance, sodium hyaluronate-fullerene 5D complex, is basically not likely to cause an allergic reaction.

[0165] The sodium hyaluronate-fullerene 5D complex of Examples 2-3 was also tested, indicating that the test substance, sodium hyaluronate-fullerene 5D complex, is basically not likely to cause an allergic reaction.

[0166] Table 6 Results of skin closed patch test

[0167]

[0168] Dioxane, phenol, diethylene glycol detection:

[0169] According to the Cosmetic Safety Technical Specification 2015 Chapter 4 2.20 gas chromatography, Cosmetic Safety Technical Specification 2015 Chapter 4 2.26 first method high performance liquid chromatography-diode array detector method, Cosmetic Safety Technical Specification 2015 Chapter 4 2.19 second method gas chromatography-mass spectrometry, the content of dioxane, phenol, diethylene glycol and the like of the sodium hyaluronate-fullerene 5D complex of Example 1 is tested.

[0170] The test results are shown in Table 7; the contents of dioxane, phenol, diethylene glycol and the like are all less than the corresponding detection limit, and the sodium hyaluronate-fullerene 5D complex of Examples 2-3 is also detected, and the contents of dioxane, phenol, diethylene glycol and the like are all less than the corresponding detection limit, indicating that the sodium hyaluronate-fullerene 5D complex of the application does not contain dioxane, phenol, diethylene glycol.

[0171] Table 7 test results

[0172]

[0173] Radical scavenging experiment at the cell level under ultraviolet irradiation:

[0174] HaCaT cells were placed in confocal culture dishes, each containing 1.5 x 10 5 cells, and cultured in complete culture medium (37℃, 5% CO2) for 24 hours. Then 20 μg mL -1 of the sodium hyaluronate-fullerene 5D complex of Example 1 (referred to as 5D fullerene) was added, and incubated for another 24 hours. After dilution with DCFH-DA at 1:1000, staining was performed, and after incubation at 37℃ in the dark for 20 minutes, ultraviolet lamp 20W was irradiated for 10 minutes. Finally, a laser scanning confocal microscope (Nikon, LU-M4) was used to observe the fluorescence image.

[0175] The experimental results are shown in Figure 7 The blue color in the picture represents the cell nucleus, and the green color represents the free radicals; in the control group and the 5D fullerene group, no green color was observed because there was no ultraviolet lamp irradiation, indicating that no free radicals were generated. In the group irradiated only with the ultraviolet lamp, a large number of green free radicals were observed in the cells. In the ultraviolet lamp irradiation + 5D fullerene group, almost no green free radicals were observed, indicating that the 5D fullerene has good free radical scavenging ability at the cell level.

[0176] The above description is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any changes or replacements within the technical range disclosed by the present application can be easily thought of by those skilled in the art, which should be covered within the protection scope of the present application.

Claims

1. A method for preparing a sodium hyaluronate-fullerene 5D complex, characterized by, It comprises the following steps: Step 1, ball-milling fullerenes, sodium hyaluronate and sodium hydroxide to obtain a first mixture; Step 2, adding H2O2 solution and hydrolyzing sodium hyaluronate in the first mixture, and continuing ball-milling to obtain a second mixture; Step 3, adding water in the second mixture, and ultrasonic treatment to obtain a suspension; Step 4, continuously adding acetylated sodium hyaluronate and water in the suspension, and stirring to obtain a third mixture; Step 5, filtering or centrifuging the third mixture to remove insoluble substances to obtain a mixed solution, and spray-drying the mixed solution to obtain a sodium hyaluronate-fullerene dry powder; Step 6, mixing the sodium hyaluronate-fullerene dry powder, sodium hyaluronate, sodium hyaluronate cross-linked polymer and water, stirring to mix, then continuously adding 1,2-pentanediol and ethylhexylglycerin, and stirring to mix to obtain a sodium hyaluronate-fullerene 5D complex; In the step 1, the molecular weight of the sodium hyaluronate is 1M-1.8M Da; In the step 1, the mass ratio of the fullerenes, sodium hyaluronate and sodium hydroxide is 4-6:0.5-2:0.6-1.2; In the step 2, the mass-volume ratio of the hydrolyzed sodium hyaluronate to the H2O2 solution is 0.3-1g:5-17ml.

2. The method for preparing the sodium hyaluronate-fullerene 5D complex according to claim 1, characterized in that, In the step 2, the molecular weight of the hydrolyzed sodium hyaluronate is 5000-8000 Da.

3. The method for preparing the sodium hyaluronate-fullerene 5D complex according to claim 1, characterized in that, In the step 1 and step 2, the mass ratio of the hydrolyzed sodium hyaluronate to the fullerenes is 0.1-6:0.5-6.

4. The method for preparing the sodium hyaluronate-fullerene 5D complex according to claim 1, characterized in that, The mass ratio of the hydrolyzed sodium hyaluronate to the acetylated sodium hyaluronate is 0.3-1g:1.5-2g.

5. The method for preparing the sodium hyaluronate-fullerene 5D complex according to claim 1, characterized in that, In the step 6, the mass-volume ratio of the sodium hyaluronate-fullerene dry powder, sodium hyaluronate, sodium hyaluronate cross-linked polymer, 1,2-pentanediol, ethylhexylglycerin and water is 50-125g:8-15g:8-14g:100-500g:1-5g:10L.

6. A sodium hyaluronate-fullerene 5D complex, characterized in that, The sodium hyaluronate-fullerene 5D complex is prepared by the preparation method of any one of claims 1-5.

7. Use of a sodium hyaluronate-fullerene 5D complex, characterized in that, The sodium hyaluronate-fullerene 5D complex of claim 6 is used as a raw material for cosmetics.

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