Nanoscale ginseng, preparation method and application thereof and cosmetic

Abstract

The invention provides nanoscale ginseng, a preparation method and application thereof and a cosmetic, and relates to the technical field of cosmetics. The preparation method provided by the invention comprises the following steps: carrying out infiltration, microwave radiation pretreatment and dissolution on ginseng to obtain a nano spray drying preparation solution; the nano-scale ginseng is prepared by using a nano spray drying technology. The invention further provides application of the nanoscale ginseng in preparation of cosmetics with anti-inflammatory, anti-oxidation and anti-aging effects. The nanoscale ginseng disclosed by the invention is natural in component, mild and safe, can effectively improve the transdermal absorption of human skin, improve the skin chromaticity and reduce the melanin content of the skin, and can effectively solve the problems of wrinkles and reduced elasticity caused by skin aging. In addition, the preparation method is simple and convenient, and large-scale production and application are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a nano-level ginseng and a preparation method and application thereof, as well as a cosmetic. Background Art

[0002] Ginsenoside, the representative physiologically active ingredient of ginseng, is evenly distributed in the above-ground and underground parts of ginseng. When using ginseng, its root is mainly used. Ginsenoside is also contained in ginseng leaves and ginseng seeds, and has the same effect as ginseng root. So far, ginseng is known to have the following effects: anti-carcinogenic effect on the central nervous system, anti-cancer activity, immune function control, anti-diabetic effect, liver function improvement effect, cardiovascular disorder improvement, anti-atherosclerosis effect, blood pressure control, menopausal improvement, osteoporosis improvement, anti-stress and anti-fatigue effect, antioxidant effect, anti-aging effect, etc.

[0003] The biosynthesis of melanin involves a series of enzymatic reactions, among which tyrosinase is the key rate-limiting enzyme in the biosynthesis pathway of melanin. Tyrosinase is widely distributed in microorganisms, animals, plants and the human body. It is a copper-containing metal enzyme that catalyzes the oxidation of tyrosine into dopa, dopamethine, dopachrome, dihydroxyindole / dihydroxyindolecarboxylic acid, and finally forms melanin. Tyrosinase is one of the key catalytic enzymes in the synthesis of melanin. It plays an important catalytic role in the formation of melanin and then reaches the epidermis. Tyrosinase activity is closely related to the level of melanin synthesis. In view of this, most of the whitening ingredients on the market achieve the effect of skin whitening by inhibiting tyrosinase activity and reducing melanin content. Plant extract ingredients, especially active ingredients of Chinese herbal medicine, have the characteristics of natural sources, mildness and safety, and are receiving more and more attention.

[0004] Chinese patent CN111434325A discloses a method for extracting ginseng antioxidant essence for repair, which includes the following steps: Step 1: Dry and crush the ginseng, put it into a sterilized 800-1000 mesh gauze bag, and place it in a vacuum homogenization emulsification pot; Step 2: Then add 8-12 times the weight of the ginseng, filter and extract at 90-100°C to obtain ginseng extract. This method uses deionized water for extraction, so that the extraction of ginseng antioxidant essence does not require the use of a large amount of ethanol solution, thereby improving the extraction purity of the antioxidant essence. This method adds a preservative phenoxyethanol, so that the essence has antibacterial and antiseptic functions and is not easy to deteriorate. However, its effect is single and the process is complicated.

[0005] Chinese patent CN102512476A relates to a preparation method of a compound ginseng preparation and its antioxidant activity. Ginseng and codonopsis are added to 70% ethanol and extracted at room temperature, then ultrasonicated for 30 minutes at 30°C and 80W, filtered, and the residue is extracted with 6 times and 4 times the amount of 70% ethanol respectively, filtered to obtain filtrate; the filtrates are combined, and ethanol is recovered to obtain ginseng and codonopsis extracts; Porphyridium is centrifuged with a 5000r / min centrifuge for 10 minutes, then suspended in a pH7.4, 50mmol / L phosphate buffer, ultrasonically crushed, centrifuged with a high-speed refrigerated centrifuge, and a supernatant is obtained, freeze-dried, and a dried product is obtained. The dried product is evenly mixed with ginseng and codonopsis extracts to obtain a compound ginseng preparation. The product has an antioxidant effect. However, the preparation process of the above product is too complicated.

[0006] At present, innovative drug delivery systems used in drug preparation have been applied to cosmetic production, including liposomes, solid lipid nanoparticles and nanospheres. These systems can achieve the effect of better delivery of active ingredients to their targets and improve the biocompatibility of different active ingredients with other excipients. However, nanoparticles still have the problem of high cost and unresolved safety. Therefore, the fundamental way to achieve nano-ization of cosmetic raw materials is to directly nano-ize traditional Chinese medicine extracts. Summary of the invention

[0007] In view of the above problems, the present invention provides a method for preparing nano-ginseng, which uses ginseng as raw material, breaks the plant wall by radiation to obtain the physiologically active ingredients and special functional ingredients of the plant, and prepares the nano-ginseng by nano-spray drying to obtain a natural cosmetic product with whitening, anti-oxidation and anti-inflammatory effects.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows: In one aspect, the present invention provides a method for preparing nano-ginseng, comprising the following steps: S1: Infiltration: Ginseng is mixed with solvent, the solid-liquid ratio is 1:2-5, unit is mg:mL; the solvent is an aqueous solution of ethanol; the volume ratio of ethanol to water is 1:0.8-1.2; S2: Microwave radiation pretreatment: The ginseng obtained in step S1 is pretreated by heating in a vacuum microwave device, with a vacuum degree of 50 KPa-90 KPa, a microwave time of 150 s-210 s, and a temperature of 35°C-45°C; S3: membrane filtration: the ginseng pretreated by microwave radiation obtained in step S2 is mixed with a solvent at a solid-liquid ratio of 1:8-12, in units of mg / mL, and filtered through a filter membrane with a pore size of 0.3 μm-0.55 μm to obtain a nano spray drying preparatory solution; S4: Nano spray drying: The nano spray drying preliminary solution obtained in step S3 is subjected to nano spray drying to prepare nano-grade ginseng; the parameters of the nano spray drying are: inlet temperature of 40°C-130°C, spray rate of 90%-100%, gas flow rate of 110L / min-150L / min, and spray cap aperture of 2μm-6μm.

[0009] Preferably, in step S4, the particle size of the nano-sized ginseng is 30nm-150nm.

[0010] Preferably, in step S1, the volume ratio of the ethanol solution to water is 1:1.

[0011] Preferably, in step S1, the material-liquid ratio is 1:2-3, in mg / mL; further preferably, in step S1, the material-liquid ratio is 1:3, in mg / mL.

[0012] Preferably, in step S1, the specific operation of mixing is stirring at 90°C-100°C for 55min-65min; further preferably, in step S1, the specific operation of mixing is stirring at 100°C for 60min.

[0013] Preferably, in step S2, the vacuum degree is 60 KPa-80 KPa, the microwave time is 170 s-190 s, and the temperature is 35° C.-42° C.

[0014] Further preferably, in step S2, the vacuum degree is 60 KPa-70 KPa, the microwave time is 170 s-180 s, and the temperature is 35° C.-40° C.

[0015] More preferably, in step S2, the vacuum degree is 70 KPa, the microwave time is 180 s, and the temperature is 40°C.

[0016] Preferably, in step S3, the material-liquid ratio is 1:9-11, in mg / mL; further preferably, in step S3, the material-liquid ratio is 1:10, in mg / mL.

[0017] Preferably, in step S3, the mixing is performed by stirring at 50°C-70°C for 40min-80min. Further preferably, in step S3, the mixing is performed by stirring at 55°C-65°C for 50min-70min. More preferably, in step S3, the mixing is performed by stirring at 60°C for 60min.

[0018] Preferably, in step S3, the pore size of the filter membrane is 0.4 μm-0.5 μm; further preferably, in step S3, the pore size of the filter membrane is 0.45 μm.

[0019] Preferably, in step S4, the parameters of the nano spray drying are: inlet temperature of 50°C-120°C, spray rate of 95%-100%, gas flow rate of 120L / min-150L / min, and spray cap aperture of 3μm-5μm.

[0020] Further preferably, in step S4, the parameters of the nano spray drying are: inlet temperature of 50°C-110°C, spray rate of 98%-100%, gas flow rate of 120L / min-140L / min, and spray cap aperture of 3μm-4μm.

[0021] More preferably, in step S4, the parameters of the nano spray drying are: inlet temperature of 100° C., spray rate of 100%, gas flow rate of 130 L / min, and spray cap aperture of 4 μm.

[0022] In another aspect, the present invention provides a nano-sized ginseng prepared by the above-mentioned preparation method.

[0023] In another aspect, the present invention provides the use of the nano-sized ginseng prepared by the above preparation method in the preparation of cosmetics with anti-inflammatory, antioxidant and anti-aging effects.

[0024] In another aspect, the present invention provides a cosmetic, wherein the effective ingredient of the cosmetic comprises the nano-sized ginseng prepared by the above preparation method.

[0025] Preferably, the cosmetic is selected from at least one of a facial mask, an essence, a facial cream, a shower gel, and an essence water.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention uses an infiltration operation to select a specific solvent and its ratio to fully infiltrate ginseng, which can greatly improve the effect of microwave radiation pretreatment and increase the yield and activity of nano-level ginseng; 2. The present invention destroys the cell wall from the two aspects of thermal effect and non-thermal effect through microwave radiation pretreatment under specific parameters, which greatly improves the extraction of ginseng functional components and improves the yield and activity of nano-ginseng; 3. The present invention optimizes the filtration pore size and specific operation through membrane filtration operation, thereby significantly improving the activity of nano-level ginseng; 4. The present invention uses nano spray drying technology under specific parameters to significantly improve the activity of nano ginseng while ensuring the yield of nano ginseng; 5. The nano-ginseng prepared by the overall technical solution of the present invention has high yield, high activity, better transdermal absorption, anti-inflammatory, antioxidant and whitening effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are transmission electron microscope images of the nano-sized ginseng prepared in Example 1 and the non-nano-sized ginseng prepared in Comparative Example 1; the left image is the non-nano-sized ginseng, and the right image is the nano-sized ginseng.

[0028] Figure 2 This is a comparison chart of the transdermal absorption test of the nano-sized ginseng prepared in Example 1 and the non-nano-sized ginseng prepared in Comparative Example 1.

[0029] Figure 3 These are experimental results of the cytotoxic effects of the nano-sized ginseng prepared in Example 1 and the non-nano-sized ginseng prepared in Comparative Example 1 on RAW264.7 cells; the left picture is the nano-sized ginseng, and the right picture is the non-nano-sized ginseng.

[0030] Figure 4 This is an anti-inflammatory evaluation graph of the nano-sized ginseng prepared in Example 1 and the non-nano-sized ginseng prepared in Comparative Example 1; wherein **** indicates P < 0.0001.

[0031] Figure 5 This is a graph showing the antioxidant efficacy of the nano-sized ginseng prepared in Example 1 and the non-nano-sized ginseng prepared in Comparative Example 1; the left graph is nano-sized ginseng, and the right graph is non-nano-sized ginseng; wherein **** indicates P < 0.0001.

[0032] Figure 6 This is a graph showing the effect of the nano-grade ginseng prepared in Example 1 and the non-nano-grade ginseng prepared in Comparative Example 1 on the melanin content; the left graph is nano-grade ginseng, and the right graph is non-nano-grade ginseng; wherein, **** indicates P < 0.0001; wherein, **** indicates P < 0.0001.

[0033] Figure 7 This is a graph showing the effect of the nano-sized ginseng prepared in Example 1 and the non-nano-sized ginseng prepared in Comparative Example 1 on the tyrosinase activity; the left graph is nano-sized ginseng, and the right graph is non-nano-sized ginseng; wherein, **** indicates P < 0.0001; wherein, **** indicates P < 0.0001.

[0034] Figure 8 This is a graph showing the toxic effect of the nano-ginseng prepared in Example 1 on zebrafish.

[0035] Fig. 9 The experimental results of the effect of the nano-ginseng prepared in Example 1 on the melanin content of zebrafish are shown in Figure a; Figure b is a diagram of the relative melanin content of zebrafish under the action of nano-ginseng, where **** indicates P < 0.0001.

[0036] Fig.10The experimental results of the effect of the nano-ginseng prepared in Example 1 on the activity of zebrafish tyrosinase are shown in FIG. 1 , wherein **** indicates P < 0.0001.

[0037] Fig.11 The graphs are the experimental results of evaluating the antioxidant effect of nano-ginseng prepared in Example 1 on zebrafish; wherein, FIG. a is a fluorescence image of zebrafish embryos; and FIG. b is a relative level image of zebrafish ROS, wherein *** indicates P < 0.001.

[0038] Fig.12 The graph is an experimental result diagram of the anti-inflammatory effect evaluation of the nano-ginseng prepared in Example 1 on zebrafish; wherein, Figure a shows the distribution and number of neutrophils in the tail of zebrafish; and Figure b shows the number of zebrafish macrophages, wherein **** indicates P < 0.0001. DETAILED DESCRIPTION

[0039] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further explained below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without making creative work all belong to the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their sources are not specifically limited. The technology and scientific terms used in the embodiments have the meanings commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0040] Example 1 Nano-level ginseng, the preparation process is as follows: S1: Infiltration: Weigh 10 mg of common ginseng raw material, put it into a conical flask, and add 10 mL of 50% ethanol aqueous solution at a solid-liquid ratio of 1:3 (mg / mL). Add the solution at 100°C while stirring for 60 minutes. Let it stand for a while to allow the solution to fully penetrate the cell tissue and obtain fully infiltrated common ginseng.

[0041] S2: Microwave radiation pretreatment: Put the fully soaked common ginseng into a vacuum microwave device for rapid heating pretreatment, with a vacuum degree of 70KPa, a microwave time of 180s, and a temperature of 40°C. The solvent that has penetrated into the sample cell tissue is vaporized, and the plant cell tissue is fully destroyed, which is convenient for subsequent extraction.

[0042] S3: membrane filtration: the ginseng pretreated by microwave radiation was dissolved in 100 mL of an aqueous solution of 50% ethanol by volume, stirred at 60°C for 60 min, cooled to room temperature, and filtered with a filter membrane with a pore size of 0.45 μm to obtain a nanospray drying preparatory solution; S4: Nano spray drying: The obtained nano spray drying preliminary solution was subjected to nano spray drying. The process parameters of nano spray drying were set as follows: the inlet temperature of the spray drying cylinder was 50°C, 70°C, 100°C, and 120°C, the spray rate was 100%, the gas flow rate was 130 L / min, and the spray cap aperture was 4 μm. Nano-ginseng was obtained, and the yield of the collected nano-ginseng particles was calculated by weighing the mass before and after, as shown in Table 1 below.

[0043] Yield (%) = mass of collected nano-ginseng powder ÷ mass of ordinary ginseng raw material × 100%.

[0044] Table 1. Effect of inlet temperature on yield of nano spray drying technology

[0045] From the data in Table 1 above, it can be seen that the yield of nano-ginseng is affected by the inlet temperature of the nano-spray drying, and the best yield can be obtained when the inlet temperature is 100°C.

[0046] Comparative Example 1 Compared with Example 1, the only difference is that the preparation process does not undergo S4 nano spray drying technology.

[0047] Specifically: S1: Infiltration: Weigh 10 mg of common ginseng raw material, put it into a conical flask, add 30 mL of 50% ethanol aqueous solution at a solid-liquid ratio of 1:3 (mg / mL), stir at 100°C for 60 minutes, spread and let stand for a while to allow the solution to fully penetrate into the cell tissue to obtain fully infiltrated common ginseng.

[0048] S2: Microwave radiation pretreatment: Put the fully soaked common ginseng into a vacuum microwave device for rapid heating pretreatment, with a vacuum degree of 70KPa, a microwave time of 180s, and a temperature of 40°C. The solvent that has penetrated into the sample cell tissue is vaporized, and the plant cell tissue is fully destroyed, which is convenient for subsequent extraction.

[0049] S3: Dissolution: The ginseng pretreated by microwave radiation was dissolved in 100 mL of 50% ethanol aqueous solution, stirred at 60°C for 60 min, cooled to room temperature, filtered with a 0.45 μm pore size filter membrane, and dried at 100°C to obtain non-nanoscale ginseng with a yield of 87%.

[0050] Comparative Example 2 Compared with Example 1, the only difference is that the solvent in the S1 infiltration is replaced by deionized water from 50% by volume ethanol aqueous solution, and the remaining operations are the same as those in Example 1, and nano-ginseng is obtained with a yield of 48%.

[0051] Comparative Example 3 Compared with Example 1, the only difference is that the solvent in the infiltration of S1 is replaced by 50% by volume ethanol solution from 50% by volume ethanol aqueous solution.

[0052] The remaining operations are the same as those in Example 1 to obtain nano-sized ginseng; The yield was 64%.

[0053] Comparative Example 4 Compared with Example 1, the only difference is that the solvent in the S1 infiltration is replaced by an ethanol aqueous solution with a volume fraction of 33% instead of an ethanol aqueous solution with a volume fraction of 50%.

[0054] The remaining operations were the same as those in Example 1 to obtain nano-ginseng with a yield of 71%.

[0055] Comparative Example 5 Compared with Example 1, the only difference is that the specific parameters in S2 are changed: S2: Microwave radiation pretreatment: The soaked common ginseng is placed in a vacuum microwave device for rapid heating pretreatment, with a vacuum degree of 100KPa, a microwave time of 180s, and a temperature of 40°C. The solvent that has penetrated into the sample cell tissue is vaporized, and the plant cell tissue is fully destroyed, which is convenient for subsequent extraction.

[0056] The remaining operations were the same as those in Example 1 to obtain nano-ginseng with a yield of 63%.

[0057] Comparative Example 6 Compared with Example 1, the only difference is S3, which does not have a membrane filtration operation: S3: dissolving the ginseng pretreated by microwave radiation in 100 mL of 50% ethanol aqueous solution, stirring at 60° C. for 60 min, and cooling to room temperature to obtain a nano spray drying preparatory solution; The remaining operations were the same as those in Example 1 to obtain nano-ginseng with a yield of 90%.

[0058] Comparative Example 7 Compared with Example 1, the only difference is that the S3 membrane filtration is replaced by activated carbon filtration. The remaining operations were the same as those in Example 1 to obtain nano-ginseng with a yield of 85%.

[0059] Comparative Example 8 Compared with Example 1, the only difference is that the pore size of the S3 membrane filtration is replaced from 0.45 μm to 0.6 μm. The remaining operations were the same as those in Example 1 to obtain nano-ginseng with a yield of 88%.

[0060] Comparative Example 9 Compared with Example 1, the only difference is that the specific parameters of S4 are changed: S4: Nano spray drying: The obtained nano spray drying preliminary solution was subjected to nano spray drying, and the process parameters of nano spray drying were set as follows: the inlet temperature of the spray drying cylinder was 150°C, the spray rate was 100%, the gas flow rate was 130L / min, the spray cap aperture was 4μm, and nano-grade ginseng was obtained with a yield of 41%.

[0061] Table 2. Yield and particle size of Example 1 and Comparative Examples 1 to 9

[0062] Test Example 1: Transmission Electron Microscopy The nano-sized ginseng prepared in Example 1 and the non-nano-sized ginseng prepared in Comparative Example 1 were observed using a transmission electron microscope (TEM). Figure 1 shown.

[0063] pass Figure 1 It can be seen that the pore size of nano-sized ginseng is smaller than that of non-nano-sized ginseng, which is beneficial to increase its penetration and improve the solubility of the substance, facilitating further development and utilization.

[0064] As shown in Table 2, the nano-ginseng prepared under the preparation conditions of Example 1 has a high yield and a small particle size, and its effect can be further verified by experiments.

[0065] Test Example 2: Transdermal absorption test The transdermal absorption test used a modified Franz diffusion apparatus, with pig skin fixed between the drug administration pool and the receiving pool. The effective permeation area of ​​the diffusion pool was 2.54 cm 2 , the stratum corneum faces the supply chamber, and the dermis faces the receiving chamber. Weigh 2 mg of the sample and dissolve it in 1 mL of ethanol, place it in the dosing pool, and apply it evenly on the ex vivo pig skin of the same size. Fix the upper and lower chambers, keep the temperature at 37℃±0.5℃, set the diffusion cell speed to 200r / min, stir continuously, and take 3.0 mL of receiving solution after 1.0h, 6.0h, 20.0h, and 39.0h, respectively, and replenish the same temperature and equal volume of receiving solution at the same time. Centrifuge the receiving solution, take the supernatant and filter it with a microporous filter membrane, measure the absorbance of the filtrate according to the given chromatographic conditions, and calculate the cumulative permeation per unit area Q of the drug at each time point.

[0066] ； Where Cn is the drug concentration measured at the nth sampling point; V is the volume of the receiving pool; and S is the effective diffusion area.

[0067] Cumulative permeability K = [Q × S ÷ m] × 100%; where m is the mass of the sample in 1 mL of solution.

[0068] Experimental process: pig skin was used as in vitro transdermal absorption skin, and the diffusion cell method was used to perform transdermal test on the in vitro transdermal absorption skin. When used, 10 portions of in vitro transdermal absorption skin were fixed on Franz diffusion cells, physiological saline was used as the receiving solution, and the temperature was 36.5°C-37.5°C. The products prepared in Example 1 and Comparative Examples 1-9 were added to the corresponding supply chambers for measurement, and the cumulative permeation per unit area of ​​the drug at each time point was calculated. The results are shown in Table 3: Table 3. Transdermal absorption test

[0069] Figure 2 This is a comparison chart of the transdermal absorption test of the nano-sized ginseng prepared in Example 1 and the non-nano-sized ginseng prepared in Comparative Example 1; According to the above Table 3 and Figure 2 It can be learned from the content that: the 5h permeability of the nano-grade ginseng prepared in Example 1 calculated by the above-mentioned cumulative permeability formula per unit area is 33.80%; the 5h permeability of the non-nano-grade ginseng prepared in Comparative Example 1 is 12.37%, that is, the 5h permeability of the nano-grade ginseng is about 3 times that of the non-nano-grade ginseng. The permeability of the nano-ginseng prepared in Comparative Example 2 was 33.67% in 5 hours, the permeability of the nano-ginseng prepared in Comparative Example 3 was 31.97% in 5 hours, the permeability of the non-nano-ginseng prepared in Comparative Example 4 was 31.81% in 5 hours, the permeability of the nano-ginseng prepared in Comparative Example 5 was 32.47% in 5 hours, the permeability of the nano-ginseng prepared in Comparative Example 6 was 21.09% in 5 hours, the permeability of the nano-ginseng prepared in Comparative Example 7 was 21.09% in 5 hours, the permeability of the nano-ginseng prepared in Comparative Example 8 was 27.57% in 5 hours, and the permeability of the nano-ginseng prepared in Comparative Example 9 was 32.12% in 5 hours.

[0070] Experimental Example 3: Using lipopolysaccharide to induce RAW246.7 cells to establish an inflammation model to evaluate the anti-inflammatory effect of nano-ginseng Cell culture: RAW246.7 cells were cultured in DMEM medium (containing 10% FBS) at 37°C and 5% CO 2 Culture in an incubator, replace the culture medium every 24h-48h, gently blow off the cells with DEME culture medium containing 10% FBS, collect the cell suspension, centrifuge (1000r / min, 5min), discard the supernatant, add 1mL of DEME cell culture medium containing 10% FBS to resuspend the cells, and inoculate them into a new culture dish at a volume ratio of 1:3 for cell culture.

[0071] Experimental operation: Toxicity of Nano-Ginseng to RAW264.7 Cells: The MTT method was used to detect cell viability to evaluate the toxic effects of the products prepared in Example 1 and Comparative Examples 1 to 9 on RAW264.7 cells. 4 The cells were inoculated into a 96-well plate at a density of 100 cells, cultured overnight, and the next day, the culture solution containing different concentrations of the products prepared in Example 1 and Comparative Examples 1-9 (final concentrations of 0 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1.00 mg / mL) was added and cultured for 48 hours. After full action, the culture medium in the well plate was carefully aspirated, 100 μL PBS and 10 μL MTT solution were added to each well, and after 4 hours of culture, 100 μL SDS solution was added to each well and incubated overnight. The absorbance value (OD value) of the well plate at 570 nm was tested with an ELISA instrument. The cell survival rate (%) was calculated at a dosing concentration of 0.5 mg / mL.

[0072] Cell survival rate (%) = average OD value of the drug-treated group / average OD value of the control group × 100%.

[0073] Table 4. Cell survival rates of Example 1 and Comparative Examples 1 to 9

[0074] Figure 3 The experimental results of the cytotoxicity of the nano-sized ginseng prepared in Example 1 and the non-nano-sized ginseng prepared in Comparative Example 1 on RAW264.7 cells are shown; Figure 3 As shown, with the increase of the concentration of non-nano ginseng and nano ginseng, the growth of RAW264.7 can be inhibited. When the concentration is 0.25 and 0.5 mg / mL, the survival rate of RAW264.7 cells is above 95%, so 0.25 mg / mL and 0.5 mg / mL are selected as the subsequent experimental concentrations.

[0075] The data in Table 4 show that when the concentration is 0.5 mg / mL, the survival rate of RAW264.7 cells is above 95% and can be used for subsequent experiments.

[0076] Evaluation of anti-inflammatory effect: Cells in logarithmic growth phase were collected at a rate of 1×10 5 The number of cells / well was inoculated into a 24-well plate and placed in a 37°C, 5% CO 2After culturing in the incubator for 24 hours, a blank group, a model group, and a drug administration group were set up. The drug administration group was added with 500 μL / well of 0.25 μg / mL and 0.5 μg / mL of the products obtained in Example 1, Comparative Examples 1 to Comparative Examples 9, and 0.5 μg / mL dexamethasone (positive control), followed by the addition of LPS solution. The model group was added with 500 μL / well of culture medium followed by the addition of LPS solution. The blank group was added with the same volume of LPS solution. The supernatant was collected after continuing to be cultured for 24 hours under the same conditions. The OD value was determined according to the instructions of the nitric oxide synthase kit. The results are shown in Table 5 and Figure 4 As shown. Compared with the model group ( Figure 4 There was a statistically significant difference compared with the LPS group in the experiment, P<0.0001, indicated by **** in the figure.

[0077] The results show that the release of NO decreases with the increase of the concentration of the products obtained in Example 1 and Comparative Examples 1 to Comparative Examples 9, and the release of NO is the lowest at 0.5 mg / mL. Compared with Example 1, at a concentration of 0.5 mg / mL, each comparative example can reduce the release of NO by 49% after converting ginseng into nano-ginseng. Changing the solvent for extraction will affect the anti-inflammatory effect of the nano-ginseng extract; without membrane filtration or activated carbon filtration, the release of NO will be slightly reduced; Comparative Example 4 only changes the volume of the infiltration solvent, which mainly affects the yield of nano-ginseng and has little effect on the anti-inflammatory effect; changing the parameters in the preparation process, such as Comparative Example 9, does not have a good effect. Since the nitric oxide content is negatively correlated with the anti-inflammatory activity, the nano-ginseng obtained by this experimental operation has a significant anti-inflammatory effect.

[0078] Table 5. Anti-inflammatory effect

[0079] Test Example 4, H 2 O 2 Construction of an induced RAW264.7 cell oxidative damage model to evaluate the antioxidant effect of nano-ginseng RAW264.7 cells were plated in 96-well plates. RAW264.7 cells were divided into a blank group, a damaged group, and a sample group, with 3 parallel wells in each group. The blank group was given normal culture medium, and the damaged group was given H 2 O 2 (800 µmol / L); the sample group was first pretreated with the products prepared in Example 1 and Comparative Examples 1 to Comparative Examples 9 at different concentrations (0.5 mg / mL and 0.25 mg / mL) for 24 h, and then H 2 O 2 , (800µmol / L) for oxidative damage for 2h. Then the culture medium was replaced and incubated for 24h. Finally, CCK-8 was used to detect cell survival rate and calculate the H2 O 2 The effect of concentration on cell survival rate. The calculation formula is as follows: Cell survival rate (%) = (A 1 -A 2 ) / (A 3 -A 2 )×100%; Where: A 1 :Contains cells, CCK-8, samples and H 2 O 2 The absorbance value of A 2 : Contains CCK-8, but does not contain cells, samples, or H 2 O 2 The absorbance value of A 3 : Contains cells, CCK-8, but does not contain samples and H 2 O 2 The absorbance value.

[0080] Table 6. Cell viability of examples and comparative examples

[0081] As shown in Table 6, the nano-sized ginseng obtained by Example 1 has the best oxidative damage protection ability, and changing other conditions will reduce its effect. Changing the microwave radiation pretreatment method and the extraction solvent will greatly reduce its oxidative damage protection ability.

[0082] Depend on Figure 5 As shown in the figure, compared with the blank group, H 2 O 2 The cell survival rate in the model group was significantly reduced, and there was a statistical difference compared with the blank group (P < 0.0001), indicating that the model was successfully established. 2 O 2 Compared with the model group, the cell survival rates treated with 0.25 mg / mL nano-ginseng and 0.5 mg / mL nano-ginseng were significantly increased, which were (76.62±1.18)% and (82.07±2.11)%, respectively. Compared with the model group, they were increased by 26.32% (P<0.0001) and 34.84% (P<0.0001), respectively, which were statistically significant, indicating that nano-ginseng has good oxidative damage protection ability.

[0083] Experimental Example 5: Determination of the effect of nano-ginseng on melanin inhibition by measuring the effect of B16F10 mouse melanoma cells Effect of Nano-Ginseng on Melanin Content: B16F10 cells were used to measure the melanin content, and the melanin content was intervened by the products prepared in Example 1 and Comparative Examples 1 to Comparative Examples 9. The experiment was divided into 4 groups: a blank group (an experimental group in which an equal volume of normal culture medium was added but no other substances were added), an arbutin group (0.5 mg / mL), products prepared in Example 1 and Comparative Examples 1 to Comparative Examples 9 (0.25 mg / mL), and products prepared in Example 1 and Comparative Examples 1 to Comparative Examples 9 (0.5 mg / mL).

[0084] The specific experimental methods are as follows: The content of melanin in B16F10 cells was determined by NaOH lysis method. 4 The cells were inoculated into 6-well plates at a density of 100 cells / mL, and 2 mL of culture medium was added to each well. 37°C, 5% CO 2 , incubator, after culturing for 24 h, 200 μL of each of DMEM medium, DMEM medium containing 0.25 mg / mL arbutin, DMEM medium containing 0.5 mg / mL arbutin, DMEM medium containing 0.25 mg / mL of the products prepared in Example 1 and Comparative Examples 1 to Comparative Examples 9, and DMEM medium containing 0.5 mg / mL of the products prepared in Example 1 and Comparative Examples 1 to Comparative Examples 9 were added according to the grouping, with 3 parallels in each group, 37°C, 5% CO 2 After 24 hours of incubation in the incubator, the supernatant was discarded, and the cells were collected by trypsin digestion. 300 μL of 1 mol / L NaOH (containing 10% DMSO) was added and reacted in an 80°C water bath for 40 minutes to completely dissolve the melanin particles. The cells were collected in a 96-well plate with 200 μL in each well, and the absorbance of each well was measured at a wavelength of 405 nm on a microplate reader.

[0085] like Figure 6 As shown in Table 7, the nano-ginseng has a significant inhibitory effect on the production of melanin, which is significantly different from the arbutin group and the non-nano-ginseng group. The tyrosinase activity of B16F10 cells was measured by L-DOPA oxidation method, and the tyrosinase activity was intervened by the products prepared in Example 1 and Comparative Examples 1 to 9. The experiment was divided into 7 groups: a blank group (an experimental group in which an equal volume of normal culture medium was added but no other substances were added), a positive control group (arbutin 0.25 mg / mL), a positive control group (arbutin 0.5 mg / mL), products prepared in Example 1 and Comparative Examples 1 to 9 (0.25 mg / mL), and products prepared in Example 1 and Comparative Examples 1 to 9 (0.5 mg / mL).

[0086] B16F10 cells were plated in a 96-well plate according to the above method. After culturing for 24 hours, 200 μL of DMEM medium, DMEM medium containing 0.25 mg / mL arbutin, DMEM medium containing 0.5 mg / mL arbutin, DMEM medium containing 0.25 mg / mL of the product prepared in Example 1 and Comparative Examples 1 to Comparative Examples 9, and DMEM medium containing 0.5 mg / mL of the product prepared in Example 1 and Comparative Examples 1 to Comparative Examples 9 were added according to the grouping, 3 in parallel in each group, 37°C, 5% CO 2 After 24 hours of incubation in the incubator, discard the supernatant, add 100 μL of cell lysis solution, and place in a -80℃ freezer for 2 hours. After removal, thaw in 37℃ hot water, add 10 μL of 0.5% L-DOPA solution to each well, incubate at 37℃ for 30 minutes, and measure the absorbance of each well at 492nm using an enzyme reader. Calculate the intracellular tyrosinase activity of B16F10 cells according to the following formula: Tyrosinase activity = (OD sample ÷ OD blank) × 100%.

[0087] Table 7. Melanin content and tyrosinase activity of Examples and Comparative Examples

[0088] The results are shown in Table 7 and Figure 7 As shown, in the Example 1 group, the tyrosinase activity at a concentration of 0.25 mg / mL of the product prepared in Example 1 and the tyrosinase activity at a concentration of 0.5 mg / mL of the product prepared in Example 1 were 50.42% and 36.53%, respectively, with statistical differences (P<0.0001). As the concentration of nano-ginseng increases, its inhibitory effect on the activity of tyrosinase in B16F10 melanoma cells is enhanced and dose-dependent. The trend of melanin content is consistent with the trend of tyrosinase activity. Compared with the blank group, it was reduced by 26.54% and 44.61%, respectively, with statistical differences (P<0.0001). However, in Comparative Examples 1 to Comparative Example 9, the tyrosinase activity and melanin inhibition effect at a concentration of 0.25 mg / mL of the product and the tyrosinase activity and melanin inhibition effect at a concentration of 0.5 mg / mL of the product are not as good as in Example 1. As the concentration of nano-ginseng increases, its inhibitory effect on the synthesis of melanin in B16F10 melanoma cells is enhanced and dose-dependent. Therefore, the whitening mechanism of nano-ginseng is to inhibit tyrosinase activity, thereby reducing melanin production and reducing skin pigmentation.

[0089] Experimental Example 6: Animal experiment based on zebrafish model to study the efficacy of the nano-ginseng prepared in Example 1 (1) Zebrafish culture Wild-type zebrafish were raised in an environment with a water temperature of 28.5℃ and a pH of 7.5-8.5. Ten groups of zebrafish were placed in a breeding tank with a partition at a breeding ratio of 1:2 between male and female. The males and females were separated and treated in the dark for 12 hours before being restored to light treatment. The partition was removed to allow the zebrafish to mate and obtain eggs. Healthy zebrafish with a fertilization rate of ≥80% were selected for the experiment.

[0090] (2) Effect of Nano-ginseng on the Survival Rate of Zebrafish Zebrafish eggs are highly sensitive, so toxicity verification was performed before the activity experiment. Healthy zebrafish eggs were collected in a 96-well plate, and plated at a density of 1 per well. 180 μL of zebrafish culture water containing different concentrations (25 ug / mL, 50 ug / mL, 75 ug / mL, 100 ug / mL) of nano-ginseng was added to each well. Embryonic death and teratogenicity were observed every 24 hours for 5 consecutive days to observe the effects of different concentrations of nano-ginseng on zebrafish development. The results are as follows: Figure 8 As shown. Figure 8 It can be seen that within the concentration range of 25mg / mL-100mg / mL, all zebrafish treated with nano-ginseng survived and were in normal physiological state. Based on this, the whitening effect of nano-ginseng will be studied.

[0091] (3) Effects of Nano-Ginseng on Melanin Content and Tyrosinase Activity in Zebrafish The zebrafish eggs produced are transparent. As the fertilized eggs develop, melanin is gradually produced on the surface and inside the zebrafish eggs. Eventually, the eggs develop into fish. The black production on the surface of the zebrafish can be directly observed, reflecting the effect of the sample on the synthesis of melanin in zebrafish, and then the whitening effect can be evaluated. Glutathione (GSH) is a short peptide with antioxidant and whitening effects. It is often used in functional foods or cosmetics and can be used as a positive control for whitening active peptides.

[0092] Healthy zebrafish eggs were collected 24 hours after fertilization and plated in a 12-well plate at a density of 20 per well. Nano-ginseng groups were set up, and 2 mL of zebrafish culture water (100 ug / mL) containing nano-ginseng active peptides was added; blank group (2 mL culture water), positive control group (culture water containing 2 mL GSH, 100 ug / mL). After constant temperature treatment at 28.5°C for 48 hours, the zebrafish were observed for morphology and melanin distribution under a stereoscope (Nikon SMZ460, Nikon corporation, Japan) and dorsal and side views were photographed. The results are shown in Figure 2. Fig. 9 shown.

[0093] The melanin accumulation sites were selected and the melanin area was counted by ImageJ software, and the data were analyzed. The calculation formula is as follows. The result obtained was that the melanin content of the nano-ginseng group was 28.4% lower than that of the normal group.

[0094] Melanin inhibition rate (%) = .

[0095] The remaining zebrafish were collected, washed with distilled water, and then frozen (-80°C) to kill. 200 μL of Western and IP cell lysis buffer (containing 1% (volume fraction) 1 mM PMSF) after ice bath was added and the fish bodies were broken by repeated freezing and thawing for 3 times. The fish bodies were centrifuged at 4000 r / min for 10 min to obtain the supernatant and precipitate.

[0096] The precipitate was used to determine the melanin content. 250 μL NaOH solution (1M, 10% DMSO) was added to the precipitate, and the precipitate was incubated at 85°C for 2 h. 100 μL was added to a 96-well plate and the absorbance was measured at 405 nm. Each sample was normalized using a BCA kit (Biyuntian). The relative melanin content was calculated according to the following formula.

[0097] Relative melanin content (%) = (A t / A 0 )×100%; Where: A t is the absorbance value of the nano-ginseng treatment group or the positive control (GSH) group with whitening effect; A 0 A blank group.

[0098] from Fig. 9 It can be seen that compared with the blank group, at a concentration of 100 μg / mL, the melanin in the eye area of ​​the zebrafish treated with nano-ginseng was slightly lighter in the side view, and the fish body was more transparent than the blank group.

[0099] The test results of relative melanin content are as follows Fig. 9 As shown, from Fig. 9 It can be seen that the melanin content of zebrafish treated with nano-ginseng, which has whitening effect, is significantly lower than that of the blank group (p<0.0001). Under the treatment of nano-ginseng at a concentration of 100μg / mL, the melanin content in zebrafish decreased by 34.63% compared with the blank group. This shows that nano-ginseng has a good ability to inhibit melanin.

[0100] The supernatant was used to determine the tyrosinase activity. 20 μL of the supernatant and 180 μL of L-DOPA (1 mg / mL) were mixed in a 96-well plate and incubated at 37°C for 2 h in the dark. The absorbance was measured at 75 nm, and the relative tyrosinase activity was calculated according to the following formula.

[0101] Relative tyrosinase activity (%) = (A t ’ / A 0 ’ )×100%; Where At ’ is the absorbance value of the nano-ginseng treatment group or the positive control (GSH) group with whitening effect. 0 ’ is the absorbance value of the blank group.

[0102] The relative tyrosinase activity test results are as follows Fig.10 As shown in the figure, compared with the blank group, nano-ginseng can significantly reduce the tyrosinase activity in zebrafish (p<0.0001). Under the treatment of nano-ginseng at a concentration of 100μg / mL, the tyrosinase activity of zebrafish was reduced by 25.35% compared with the blank group. This shows that nano-ginseng exhibits good tyrosinase inhibition ability. The above results show that nano-ginseng has good tyrosinase inhibition activity in the zebrafish model, and can reduce the amount of melanin production by inhibiting enzyme activity, thereby achieving a whitening effect.

[0103] (4) Evaluation of the antioxidant effect of nano-ginseng on zebrafish Under a stereomicroscope, 9hpf wild-type zebrafish embryos with normal development were randomly selected and placed in a 6-well cell culture plate, 20 per well, and the tested zebrafish were divided into four groups, namely the normal group, model group, positive control group and nano-ginseng group. The normal group did not ingest menadione, while the model group, positive control group and nano-ginseng group ingested the same amount of menadione (menadione was ingested into the zebrafish by dissolving it in the fish farming water). The nano-ginseng group ingested reduced glutathione oxidant after ingesting menadione. The positive control group ingested arbutin after ingesting menadione. According to the safety evaluation results, the concentration of the test sample group was set to 0.15%.

[0104] The culture water in the 6-well plate was aspirated, 5.0 mL of buffer water was added to each well of the blank control group, 4500.0 μL of buffer water was added to each well of the model control group, and 4500.0 μL of sample solution was added to each well of the nano-ginseng group. After pretreatment for 1 h, 500.0 μL of menadione was added to each well of the model control group and the nano-ginseng group to construct an oxidative stress model, and the plates were incubated in a constant temperature incubator at 28.5 °C for 24 h.

[0105] After incubation for 24 h, the embryos in each well were transferred to a new six-well plate, each well was washed three times with culture water, and finally 5.0 mL of culture water was added and cultured in a 37°C constant temperature incubator until 3 dpf.

[0106] After 3 dpf, the egg membranes in each well were aspirated, and 5.0 mL (20 ug / mL) of fluorescent probe (DCHF-DA) was added to each well to detect ROS content. The embryos were cultured in the dark for 50 min. The embryos were then washed three times with culture water, anesthetized with tricaine, observed and recorded under a fluorescence microscope, and the fluorescence intensity was analyzed with ImageJ. The results are shown in the figure. Fig.11 shown.

[0107] like Fig.11 As shown, the white areas are active oxygen. It can be seen that the fluorescence intensity of the nano-ginseng group is stronger than that of the normal group, but significantly weaker than that of the model group.

[0108] (5) Evaluation of the anti-inflammatory effect of nano-ginseng on zebrafish Zebrafish tail transection inflammation model test 3dpf zebrafish larvae in good condition were selected, anesthetized with 0.03% tricaine solution, placed on a glass slide, and the tail fin of the larvae was cut off with a scalpel. After resuscitation in culture water at 28.5°C, they were randomly assigned to the model group and the nano-ginseng group, with 15 fish in each group, and 2mL of nano-ginseng (100ug / mL) was given respectively. The zebrafish in the model group did not undergo tail fin transection. 4h after administration, the larvae were placed on an agar plate, and the distribution and number of neutrophils in the zebrafish tail were observed using a stereo fluorescence microscope. The neutrophils in the zebrafish tail were photographed and counted, and statistical analysis was performed. The results are shown in Fig.12 shown.

[0109] from Fig.12 It can be seen that compared with the model group, the number of neutrophils in the nano-ginseng tail was less, and Fig.12 It can be seen that nano-ginseng can effectively reduce neutrophils, that is, nano-ginseng has potential anti-inflammatory effects.

[0110] Test Example 7: 5α-reductase inhibition test The 5α-reductase inhibition test was carried out using non-nano ginseng and nano ginseng as the test groups.

[0111] Experimental method: Prepare control group solution and test group solution respectively and incubate them; Control group solution: The control group solution is an enzyme reaction system, and the enzyme reaction system is incubated at 37°C for 30 minutes.

[0112] Detection group solution: Dissolve the sample to be tested in 50% ethanol to prepare a 1% detection group solution. Take 0.5 mL of the detection group solution and place it in the enzyme reaction system and incubate it at 37°C for 30 minutes.

[0113] The formula of the enzyme reaction system is as follows: 5 mL of deionized water, 2 μL of 1 mmol / L testosterone, 600 μL of 10 mg / mL rat steroid 5α-reductase solution, 200 μL of 1 mmol / L NADPH solution Detection method: UV-visible spectrophotometer was used to detect the decrease in absorbance of the control group solution and the test group solution before and after incubation at a wavelength of 340 nm. The decrease in absorbance of the control group solution was recorded as ∆A1 The decrease in absorbance of the test group solution is recorded as ∆A n (∆A 纳米级人参 , ∆A 非纳米级人参 ), and the enzyme activity inhibition rate was calculated.

[0114] Enzyme activity inhibitor (%) = (∆A 1 -∆A n ) / ∆A 1 ×100%; Where: A 1 is the decrease in absorbance of the control solution, ∆A n =∆A 纳米级人参 , ∆A 非纳米级人参 , which are the absorbance decrease values ​​of the test group solutions prepared with nano-grade ginseng and non-nano-grade ginseng, respectively.

[0115] Note: 5α-reductase can convert testosterone into dihydrotestosterone under the action of NADPH. After NADPH participates in the reaction, it will become oxidized NADP+. DADP has characteristic absorption at 340nm. + There is no characteristic absorption at 340nm. Therefore, by comparing the absorbance change values ​​of the control group solution and the test group solution, the activity inhibition of 5α-reductase can be determined. Test results: The test results are shown in Table 8.

[0116] Table 8. 5α-reductase inhibition test results of nano-sized and non-nano-sized ginseng

[0117] According to the test results in Table 8 above, both nano-ginseng and non-nano-ginseng have significant inhibitory effects on 5α-reductase; the inhibitory effect of nano-ginseng on 5α-reductase is better than that of non-nano-ginseng, that is, nano-ginseng can be used in shampoo to improve the problem of seborrheic alopecia.

[0118] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing nano-ginseng, characterized in that: The following steps are involved: S1: Infiltration: Ginseng is mixed with solvent, the solid-liquid ratio is 1:2-5, unit is mg:mL; the solvent is an aqueous solution of ethanol; the volume ratio of ethanol to water is 1:0.8-1.2; S2: Microwave radiation pretreatment: The soaked ginseng obtained in step S1 is pretreated by heating in a vacuum microwave device, with a vacuum degree of 50 KPa-90 KPa, a microwave time of 150 s-210 s, and a temperature of 35°C-45°C; S3: membrane filtration: the ginseng pretreated by microwave radiation obtained in step S2 is mixed with a solvent at a solid-liquid ratio of 1:8-12, in units of mg / mL, and filtered through a filter membrane with a pore size of 0.3 μm-0.55 μm to obtain a nano spray drying preparatory solution; S4: Nano spray drying: The nano spray drying preliminary solution obtained in step S3 is subjected to nano spray drying to prepare nano-grade ginseng; the parameters of the nano spray drying are: inlet temperature of 40°C-130°C, spray rate of 90%-100%, gas flow rate of 110L / min-150L / min, and spray cap aperture of 2μm-6μm.

2. The preparation method according to claim 1, characterized in that: In step S1, the volume ratio of ethanol to water is 1:

1.

3. The preparation method according to claim 1, characterized in that: In step S1, the specific operation of mixing is stirring at 90°C-100°C for 55min-65min.

4. The preparation method according to claim 1, characterized in that: In step S2, the vacuum degree is 60 KPa-80 KPa, the microwave time is 170 s-190 s, and the temperature is 35° C.-42° C.

5. The preparation method according to claim 1, characterized in that: In step S3, the mixing is carried out by stirring at 50° C.-70° C. for 40 min-80 min.

6. The preparation method according to claim 1, characterized in that: In step S4, the parameters of the nano spray drying are: inlet temperature of 50°C-120°C, spray rate of 95%-100%, gas flow rate of 120L / min-150L / min, and spray cap aperture of 3μm-5μm.

7. A nano-level ginseng, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the nano-ginseng prepared by the preparation method according to any one of claims 1 to 6 in the preparation of cosmetics with anti-inflammatory, antioxidant and anti-aging effects.

9. A cosmetic, characterized in that: The effective ingredient of the cosmetic comprises nano-sized ginseng prepared by the preparation method according to any one of claims 1 to 6.

10. The cosmetic according to claim 9, characterized in that: The cosmetic is selected from at least one of a facial mask, an essence, a facial cream, a shower gel, and an essence water.

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