Application of ginsenoside Rg5-containing composition in preparation of whitening, freckle-removing and antioxidant products

Abstract

The invention discloses application of a composition containing ginsenoside Rg5 in preparation of whitening, freckle removing and oxidation resisting products, and belongs to the technical field of biology. By combining rare ginsenoside Rg5 with alpha-arbutin, it is found that the composition can effectively resist oxidation, prevent, relieve or treat melanin deposition and inhibit melanin generation, and the effects of whitening and removing freckles are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a composition containing ginsenoside Rg5 in the preparation of whitening, freckle-removing and anti-oxidation products. Background Art

[0002] With the progress of society and the improvement of living standards, people have higher and higher requirements for the beautification of their appearance. However, the changes in eating habits and the increase in mental stress of modern people can easily lead to functional disorders of the body, causing excessive production of oxidative free radicals and the generation of a large amount of melanin in the basal layer of the skin, leading to skin problems such as redness, dullness, and accumulation of spots, which have a negative impact on people's quality of life. Among them, excessive melanin deposition is a common and difficult-to-cure skin disease. Its formation is related to genetic factors, ultraviolet radiation, estrogen, progesterone, thyroid and other factors. The most common ones are chloasma, freckles, age spots and sun spots.

[0003] The Yellow Emperor's Classic of Internal Medicine - Suwen points out that "the five colors of the mind are the brilliance of qi", and the dullness and spots on the face are caused by qi and blood deficiency, liver and spleen disorders, obstruction of qi and blood circulation, invasion of external evils, etc., and it is necessary to use drugs that can replenish qi and blood, promote metabolism and regeneration for conditioning. However, the whitening ingredients of common whitening agents on the market, such as hydroquinones and kojic acid, have obvious short-term effects, but they all have the disadvantages of high recurrence rate, strong irritation, easy to cause skin inflammation and allergies, and potential safety hazards. Therefore, using natural plants as the main raw materials, researching and developing a whitening and freckle-removing effect composition with mild effects, few side effects, high safety and significant effects has important positive significance for the physical and mental health of contemporary people, especially women.

[0004] Ginseng Panax ginseng CAMey. is a plant of the genus Panax in the Araliaceae family. It has been known as the "king of herbs" since ancient times. "Shennong's Herbal Classic" records: "Ginseng is sweet and slightly cold. It mainly nourishes the five internal organs, calms the spirit, calms the soul, stops panic, removes evil spirits, improves eyesight, makes people happy and intelligent, and long-term use can make the body light and prolong life." "Qianjin Fang" and "Shengji Zonglu" both record that ginseng and its prescriptions have beauty effects. Modern research has shown that ginseng has the effects of moisturizing the skin, tightening and removing wrinkles, and can effectively inhibit the reduction of melanin, making the skin white and smooth. Rare ginsenoside Rg5, as one of the main active ingredients of ginseng, is a secondary saponin obtained by regioselective hydrolysis of ginsenosides Rb1, Rb2, Rb3, Rc, Rd (PPD saponins for short) in ginseng, and then stereoselective dehydration. It is currently mainly used in the field of anti-cancer research.

[0005] α-Arbutin is a glycoside substance derived from green plants. Due to its medicinal activities such as whitening, freckle removal and anti-oxidation, it has been widely used in the cosmetics and pharmaceutical industries. Chinese patent CN 115154351A discloses a skin whitening composition containing arbutin or its derivatives, specifically a composition containing α-arbutin or β-arbutin and mesenchymal stem cell secretions, wherein the mesenchymal stem cell secretions are obtained by resuscitating, culturing and digesting mesenchymal stem cells. The operation process is cumbersome and must be carried out in a clean bench with harsh experimental conditions. Patent CN112245342 A discloses a ginseng series skin care extract and its preparation method and application, which achieves a whitening effect through ginseng extracts, but does not introduce the role of the rare ginsenoside Rg5.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide an application of a composition containing ginsenoside Rg5 in the preparation of whitening and anti-freckle and antioxidant products. By combining the rare ginsenoside Rg5 with α-arbutin, it can effectively resist oxidation, prevent, alleviate or treat melanin deposition, inhibit the production of melanin, and achieve the effect of whitening and anti-freckle.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides use of a composition containing ginsenosides in the preparation of a whitening and anti-freckle product, wherein the composition comprises rare ginsenoside Rg5 and arbutin.

[0010] In a second aspect, the present invention provides use of a composition containing ginsenosides in preparing a product for inhibiting tyrosinase activity, wherein the composition comprises rare ginsenoside Rg5 and arbutin.

[0011] In a third aspect, the present invention provides use of a composition containing ginsenosides in the preparation of a product for inhibiting melanin production, wherein the composition comprises rare ginsenoside Rg5 and arbutin.

[0012] In a fourth aspect, the present invention provides use of a composition containing ginsenosides in the preparation of an antioxidant product, wherein the composition comprises rare ginsenoside Rg5 and arbutin.

[0013] In some embodiments, the above applications include scavenging DPPH free radicals and ABTS free radicals.

[0014] In some embodiments, the arbutin is α-arbutin.

[0015] In some embodiments, the composition comprises 0.2-2.0 parts of rare ginsenoside Rg5 and 0.5-5.0 parts of α-arbutin, by weight.

[0016] In some embodiments, the composition comprises 0.5-1.5 parts of rare ginsenoside Rg5 and 1.0-3.0 parts of α-arbutin, by weight.

[0017] In some embodiments, the composition comprises 1.0 parts of rare ginsenoside Rg5 and 2.0 parts of α-arbutin, by weight.

[0018] In some embodiments, the above-mentioned products include food, medicine and cosmetics.

[0019] The present invention has the following beneficial effects:

[0020] (1) The present invention combines rare ginsenoside Rg5 and α-arbutin for the first time to obtain a new composition, which can significantly inhibit tyrosinase activity and reduce the production of melanin. Its synergistic effect is stronger than that of using rare ginsenoside Rg5 or α-arbutin alone.

[0021] (2) The rare ginsenoside Rg5 and α-arbutin used in the whitening and anti-freckle composition of the present invention are both derived from natural plants, and the rare ginsenoside Rg5 is derived from plants of the genus Panax, such as ginseng, notoginseng and American ginseng, etc., which is green, safe, non-toxic or low-toxic.

[0022] (3) The preparation process of the whitening and anti-freckle composition of the present invention is simple and easy to operate, does not require the investment of professional technicians and large-scale instruments and equipment, has a low production cost, and is easy to achieve industrial development. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 This is the molecular structure diagram of the rare ginsenoside Rg5. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] Ginseng extract is commonly used in existing whitening and anti-freckle products, and ginsenoside Rg5 (structural formula: Figure 1 In the present invention, the inventors combined α-arbutin with rare ginsenoside Rg5 for the first time, and found that the combination of the two in a certain proportion can play a synergistic role in whitening and anti-freckle and anti-oxidation effects.

[0027] Based on this, the present invention obtains a new composition, which includes rare ginsenoside Rg5 and α-arbutin.

[0028] During the experiment, the inventors found that the whitening and anti-freckle effects of rare ginsenoside Rg5 and α-arbutin at different proportions were significantly different. Preferably, when the weight proportions of the two components were 0.2-2.0 parts of rare ginsenoside Rg5 and 0.5-5.0 parts of α-arbutin, the combination of rare ginsenoside Rg5 and α-arbutin showed a synergistic effect, and beyond this range, the combination of the two showed an antagonistic effect.

[0029] In order to obtain better effects, the inventors optimized the above-mentioned weight proportions and found that when the weight proportions of the two components are: 0.5-1.5 parts of rare ginsenoside Rg5 and 1.0-3.0 parts of α-arbutin, the synergistic effect of the combination of rare ginsenoside Rg5 and α-arbutin is better.

[0030] Furthermore, when the weight proportions of the two components are: 1.0 part of rare ginsenoside Rg5 and 2.0 parts of α-arbutin, the synergistic effect of the combination of rare ginsenoside Rg5 and α-arbutin is the best, which can more effectively whiten, remove spots and resist oxidation.

[0031] Among them, the rare ginsenoside Rg5 and α-arbutin can be obtained by conventional preparation methods in the art, or can be purchased, and the preparation methods or sources thereof are not limited in the present invention.

[0032] The whitening and freckle removal of the present invention is specifically manifested in that the composition can significantly inhibit the activity of tyrosinase and reduce the generation of melanin; the anti-oxidation is specifically manifested in that the composition can significantly remove ABTS free radicals and DPPH free radicals.

[0033] The composition of the invention can be used to prepare related whitening and freckle removal and anti-oxidation products, including foods, medicines and cosmetics.

[0034] In some embodiments, the compositions of the present invention can be formulated into medicines suitable for topical administration using techniques known to those skilled in the art.

[0035] In some embodiments, the drug prepared by the composition of the present invention may also include pharmaceutically acceptable excipients, such as: buffers, emulsifiers, suspending agents, decomposers, disintegrants, dispersants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, etc.

[0036] In some embodiments, the medicament prepared from the composition of the present invention can be in the following dosage forms: emulsion, gel, ointment, cream, patch, wipe, powder, aerosol, spray, lotion, serum, paste, foam, drop, suspension, ointment, bandage and oil.

[0037] In some embodiments, the composition of the present invention can be made into cosmetics using techniques known to those skilled in the art.

[0038] In some embodiments, the cosmetics prepared from the composition of the present invention may also include cosmetically acceptable adjuvants, such as solvents, gelling agents, active agents, preservatives, antioxidants, masking agents, chelating agents, surfactants, coloring agents, thickeners, fillers, fragrances, and odor absorbers.

[0039] In some embodiments, the cosmetics prepared from the composition of the present invention can be of the following types: emulsions, gels, ointments, creams, masks, powders, aerosols, sprays, lotions, serums, pastes, foams, dispersions, drops, sunscreens, lotions, foundations, eye shadows, makeup removers, soaps, and the like.

[0040] In some embodiments, the composition of the present invention can be made into food using techniques known to those skilled in the art.

[0041] In some embodiments, the food prepared from the composition of the present invention may further include food-acceptable auxiliary materials, such as solvents, adhesives, fillers, sugars, sweeteners, and the like.

[0042] In some embodiments, the food prepared from the composition of the present invention can be in the following types: tablets, capsules, pills, granules, decoctions, pastes, lotions, oral solutions, drop pills or syrups.

[0043] The present invention will be further described with reference to the following examples. However, it should be understood that the examples are only for illustration and should not be construed as limitations on the implementation of the present invention.

[0044] Example 1

[0045] According to parts by weight, 0.2 parts of the rare ginsenoside Rg5 and 0.5 parts of α-arbutin are accurately weighed and mixed, and then passed through a 200-mesh sieve to obtain a composition.

[0046] Example 2

[0047] According to parts by weight, 1.0 part of the rare ginsenoside Rg5 and 2.0 parts of α-arbutin are accurately weighed and mixed, and then passed through a 200-mesh sieve to obtain a composition.

[0048] Example 3

[0049] According to parts by weight, 0.5 parts of the rare ginsenoside Rg5 and 1.0 parts of α-arbutin are accurately weighed and mixed, and then passed through a 200-mesh sieve to obtain a composition.

[0050] Example 4

[0051] According to parts by weight, 2.0 parts of the rare ginsenoside Rg5 and 5.0 parts of α-arbutin are accurately weighed and mixed, and then passed through a 200-mesh sieve to obtain a composition.

[0052] Example 5

[0053] According to parts by weight, 0.1 parts of the rare ginsenoside Rg5 and 6.0 parts of α-arbutin are accurately weighed and mixed, and then passed through a 200-mesh sieve to obtain a composition.

[0054] Example 6

[0055] According to parts by weight, 1.0 part of the rare ginsenoside Rg5 and 6.0 parts of α-arbutin are accurately weighed and mixed, and then passed through a 200-mesh sieve to obtain a composition.

[0056] Example 7

[0057] According to parts by weight, 2.0 parts of the rare ginsenoside Rg5 and 3.0 parts of α-arbutin are accurately weighed and mixed, and then passed through a 200-mesh sieve to obtain a composition.

[0058] Example 8

[0059] According to parts by weight, 1.5 parts of the rare ginsenoside Rg5 and 5.0 parts of α-arbutin are accurately weighed and mixed, and then passed through a 200-mesh sieve to obtain a composition.

[0060] The weight parts of Examples 1-8 are summarized, and the results are shown in Table 1:

[0061] Table 1 Summary of weight parts of Examples 1-8

[0062] Example Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Composition Composition 1 Composition 2 Composition 3 Composition 4 Composition 5 Composition 6 Composition 7 Composition 8 Rare Ginsenoside Rg5 0.2 1.0 0.5 2.0 0.1 1.0 2.0 1.5 α-Arbutin 0.5 2.0 1.0 5.0 6.0 6.0 3.0 5.0

[0063] Experimental example

[0064] The compositions prepared in Examples 1-6, rare ginsenoside Rg5 and α-arbutin were respectively subjected to effect verification experiments to further illustrate the whitening, freckle removal and antioxidant effects of the composition containing rare ginsenoside Rg5 provided by the present invention.

[0065] 1. Antioxidant experiments of rare ginsenoside Rg5 and α-arbutin alone and in combination

[0066] 1. ABTS free radical scavenging rate test

[0067] 7.4mM ABTS free radical solution and 2.6mM persulfate solution were reacted at room temperature in the dark for 12-16h to prepare ABTS working solution. 2mL ABTS working solution was added to 1mL of the above sample solutions of different concentrations and mixed evenly. The samples were placed in a water bath in the dark at 30℃ for 6min. The absorbance of each sample solution at a wavelength of 734nm was measured with an ELISA reader to calculate the ABTS free radical scavenging rate. Ascorbic acid was used as a positive control.

[0068] 2. DPPH free radical scavenging rate detection

[0069] Add 0.2 mM methanol solution of DPPH free radical to 1 mL of the above sample solutions of different concentrations, mix evenly, and place at room temperature in the dark for 30 minutes. Measure the absorbance of each sample solution at a wavelength of 517 nm with an enzyme marker to calculate the DPPH free radical scavenging rate. Ascorbic acid was used as a positive control.

[0070] 3. Calculation of the joint index

[0071] Combination index (CI) is used to measure the interaction between different drugs. CI values ​​can be calculated based on the IC50 values ​​of different drugs acting alone or in combination. CI>1 indicates that the drug combination has an antagonistic effect; when CI<1, it indicates that the drug combination has a synergistic effect; when CI=1, it indicates an additive effect; the smaller the CI value, the stronger the synergistic effect. Chou-talalay software was used for CI value analysis.

[0072] 4. Experimental results and analysis

[0073] The rare ginsenoside Rg5 of Example 1-6 was prepared to have a mass concentration of 0.2 mg / mL to 2.0 mg / mL, and a mass concentration of α-arbutin was prepared to have a mass concentration of 0.5 mg / mL to 5.0 mg / mL.

[0074] As can be seen from Table 2, compared with rare ginsenoside Rg5 and α-arbutin, when composition 1, composition 2, composition 3 and composition 4 are used in combination, CI is less than 1, and any adjustment of the mass ratio of rare ginsenoside Rg5 to α-arbutin in the composition shows a good synergistic effect on the scavenging ability of ABTS free radicals and DPPH free radicals; among them, composition 2 has the lowest combination index, CI <0.7, indicating the strongest synergistic effect. When any one monomer or two monomers in the composition are used in combination beyond their respective mass concentration ranges, CI>1, indicating that the combination has an antagonistic effect (composition 5, composition 6). In summary, the combination of composition 1, composition 2, composition 3 and composition 4 has a synergistic effect on the scavenging of ABTS free radicals and DPPH free radicals, respectively, and the combination of composition 2 has the strongest effect, which is better than rare ginsenoside Rg5 and α-arbutin, while the combination of composition 5 and composition 6 does not have a synergistic effect.

[0075] Table 2 Analysis of ABTS free radical and DPPH free radical scavenging rates

[0076]

[0077]

[0078] 2. In vitro cell experiments on the effects of rare ginsenoside Rg5 and α-arbutin alone and in combination

[0079] 1. Materials and Instruments

[0080] α-Arbutin (purity 98%) was purchased from Xi'an Tianguangyuan Biotechnology Co., Ltd.; rare ginsenoside Rg5 (purity 95%) was from Shaanxi Juzi Biotechnology Co., Ltd.; mouse melanoma cells (batch number: B16F10) were obtained through primary and subculture of cells by Subcon (Shanghai) Biotechnology Co., Ltd. CO2 incubator (Thermo, 150i), clean bench (Sujing Antai, SW-CJ-1F), inverted microscope (Olympus, CKX41), and microplate reader (BioTek, Epoch).

[0081] 2. Experimental Methods

[0082] 2.1B16 cell culture

[0083] B16 cells were cultured in a constant temperature incubator at 37°C and 5% CO2 using DMEM medium containing 10% FBS, and the medium was changed every 24 hours. When the cells covered 70% of the bottom of the culture flask, they were digested with 0.25% trypsin (containing 0.05% EDTA), subcultured at a volume ratio of 1:3, and logarithmic phase cells were taken for subsequent experiments.

[0084] 2.2 Detection of relative melanin content in B16 cells by using rare ginsenoside Rg5 and α-arbutin alone or in combination

[0085] The relative melanin content in cells was determined by NaOH lysis method. B16 cells in the logarithmic growth phase of 2.1 were taken and 2×10 5 The cells were inoculated in a 96-well culture plate at a density of 10 μg / mL and placed in a 37°C, 5% CO2 constant temperature incubator for 12 h (6 replicate wells were set vertically for each group). After inoculation, each plate was set up with a blank group, a rare ginsenoside Rg5 (0.1, 0.2, 0.5, 1.0, 2.0 mg / mL) group, an α-arbutin (0.5, 1.0, 2.0, 5.0, 6.0 mg / mL) group, and a combination (0.2+0.5 mg / mL, 1.0+2.0 mg / mL, 2.0+5.0 mg / mL, 0.5+1.0 mg / mL, 0.1+6.0 mg / mL, 1.0+6.0 mg / mL) group. 100 μL of drug was administered to each well, and the culture medium was discarded after culturing for 48 hours. The cells were washed twice with PBS, and 1 mL of NaOH (1 mol / L) solution was added to each well to lyse the cells. The cell lysate was collected in a centrifuge tube and placed in an 80°C water bath for 30 minutes. The tube was centrifuged at 4°C and 3000 r / min for 5 minutes, and 100 μL of the supernatant was aspirated into a 96-well plate. The cells without drug administration were used as the blank control group, and the absorbance was measured at 405 nm. The relative content of melanin was calculated as follows (1):

[0086]

[0087] Where: OD 空白组 With OD 样品组 They are the absorbance values ​​of the blank group and the sample group at a wavelength of 405 nm.

[0088] 2.3 Detection of the inhibition of tyrosinase activity in B16 cells by rare ginsenoside Rg5 and α-arbutin alone or in combination

[0089] The DOPA oxidation method was used to determine the inhibition of intracellular tyrosinase activity. The operation was the same as in 2.2. B16 cells in the logarithmic growth phase were taken, and after drug administration in groups, the cells were cultured for 48 hours and the culture medium was discarded. The cells were washed twice with PBS, and 100 μL of lysis solution was added to each well. The cells were scraped and collected in a centrifuge tube. The cells were centrifuged at 4°C and 4000 r / min for 10 minutes. 50 μL of cell supernatant was taken to a 96-well plate, and 100 μL of 1% L-DOPA was added to each well. The cells were incubated at 37°C for 1 hour, and the absorbance was measured at 475 nm. The calculation formula for tyrosinase activity inhibition is as follows (2):

[0090]

[0091] Where: OD 空白组With OD 样品组 They are the absorbance values ​​of the blank group and the sample group at a wavelength of 475 nm.

[0092] 2.4 Experimental Results and Analysis

[0093] 2.4.1 Effects of rare ginsenoside Rg5 and α-arbutin alone and in combination on the relative content of melanin in B16 cells

[0094] The rare ginsenoside Rg5 of Example 1-6 was prepared to have a mass concentration of 0.2 mg / mL to 2.0 mg / mL, and a mass concentration of α-arbutin was 0.5 mg / mL to 5.0 mg / mL.

[0095] As shown in Table 3, compared with the blank group, rare ginsenoside Rg5, α-arbutin and the combination can reduce the relative content of melanin in B16 cells to varying degrees; among them, when composition 1, composition 2, composition 3 and composition 4 are used in combination, CI <1, and any adjustment of the mass concentration ratio of rare ginsenoside Rg5 and α-arbutin in the composition can show a good synergistic effect. The combination index of composition 2 is the lowest, CI = 0.39, CI <1, indicating that it has the strongest synergistic effect. When the rare ginsenoside Rg5 and / or α-arbutin in the combination group exceed the corresponding mass concentration range and are used in combination, CI>1 when the combination is used in combination, indicating that the combination does not have a synergistic effect. In summary, the combination of composition 1, composition 2, composition 3 and composition 4 has a synergistic effect on reducing the relative content of melanin in B16 cells, and the combination of composition 2 has the strongest effect, which is better than rare ginsenoside Rg5 and α-arbutin, and the combination of composition 5 and composition 6 does not have a synergistic effect.

[0096] Table 3 Effects of rare ginsenoside Rg5 and α-arbutin alone and in combination on the relative content of melanin and inhibition of tyrosinase activity in B16 cells

[0097]

[0098] 2.4.2 Effects of rare ginsenoside Rg5 and α-arbutin alone and in combination on the inhibition of tyrosinase activity in B16 cells

[0099] The melanin production process is regulated by multiple enzymes, and tyrosinase is a copper ion-dependent metalloenzyme and the rate-limiting enzyme involved in the melanin production process. Therefore, reducing the production and accumulation of melanin by inhibiting tyrosinase activity is an important research direction for achieving skin whitening and freckle removal effects. As shown in Table 3, compared with the blank group, when the mass concentration of rare ginsenoside Rg5 was in the range of 0.2 mg / mL to 2.0 mg / mL and the mass concentration of α-arbutin was in the range of 0.5 mg / mL to 5.0 mg / mL, rare ginsenoside Rg5, α-arbutin and the combination could inhibit the tyrosinase activity in B16 cells to varying degrees; among them, the combination of composition 1, composition 2, composition 3 and composition 4 had a synergistic effect (CI < 1), and any adjustment of the mass concentration ratio of rare ginsenoside Rg5 to α-arbutin in the composition also had the same effect, and the combination index of composition 2 was the lowest, CI = 0.40, indicating that it had the strongest synergistic effect. When composition 5 and composition 6 were used in combination, CI> 1, indicating that the combination did not have a synergistic effect. In summary, the combination of composition 1, composition 2, composition 3 and composition 4 has a synergistic effect on improving the inhibitory effect of tyrosinase activity in B16 cells, the synergistic effect of composition 2 is the strongest, which is better than rare ginsenoside Rg5 and α-arbutin, and the combination of composition 5 and composition 6 has no synergistic effect.

[0100] 3. Study on the whitening and freckle removal effects of rare ginsenoside Rg5 and α-arbutin alone and in combination on experimental animals

[0101] 3.1 Animal husbandry of zebrafish

[0102] AB strain zebrafish aged 3-4 months were kept in 28℃ fish farming water (water quality requirements: 200mg instant sea salt per 1L reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-7.5; hardness 50-100mg / LCaCO3), light and dark cycles of 14h and 10h. They were provided and bred by Shaanxi Juzi Biotechnology Co., Ltd., and the feeding and management met the requirements of the International Laboratory Animal Care Assessment and Accreditation Committee.

[0103] 3.2 Experimental methods

[0104] (1) Zebrafish grouping and dosing: The experiment set up a blank control group and a sample group, including the rare ginsenoside Rg5 (0.1, 0.2, 0.5, 1.0, 2.0 mg / mL) group, α-arbutin (0.5, 1.0, 2.0, 5.0, 6.0 mg / mL) group and the combination (0.2+0.5 mg / mL, 1.0+2.0 mg / mL, 2.0+5.0 mg / mL, 0.5+1.0 mg / mL, 0.1+6.0 mg / mL, 1.0+6.0 mg / mL) group. Fifteen zebrafish embryos were randomly selected and placed in a 6-well plate, and 5.0 mL of different concentrations of rare ginsenoside Rg5, α-arbutin solution and combination solution were added. The blank control group was added with culture water and incubated at 28°C for 72 h (the solution was changed every 24 h). The fish embryos were washed twice with aquaculture water, 150 μL of 5.0 mg / mL sodium deoxycholate solution was added, and ultrasonically disrupted at 4°C. After centrifugation for 5 min (4°C, 10000 rpm), 100 μL of the supernatant and 5.0 mmol / L of levodopa solution were respectively taken and placed in a 96-well plate for mixing. The plate was incubated in a 37°C water bath for 1 h, and the absorbance was measured at 405 nm. The relative content of melanin was calculated as follows (3):

[0105]

[0106] Where: OD 样品组 With OD 空白组 They are the absorbance values ​​of the sample group and the blank group at a wavelength of 405 nm.

[0107] Continuing the above experiment, the remaining supernatant after centrifugation of the zebrafish embryo disruption solution was discarded, and 150 μL of 1 mol / L sodium hydroxide was added to the remaining precipitate to fully dissolve it. 100 μL was then drawn and placed in a 96-well plate, and sodium hydroxide was used as the blank zero adjustment group. The absorbance value was measured at 475 nm. The calculation formula for tyrosinase activity inhibition is as follows (4):

[0108]

[0109] Where: OD 样品组 With OD 空白组 They are the absorbance values ​​of the sample group and the blank group at a wavelength of 475 nm.

[0110] 3.3 Experimental Results and Analysis

[0111] 3.3.1 Effects of rare ginsenoside Rg5 and α-arbutin alone and in combination on the relative content of melanin in zebrafish embryos

[0112] The rare ginsenoside Rg5 of Example 1-4 and Comparative Example 1-2 was prepared to have a mass concentration of 0.2 mg / mL to 2.0 mg / mL, and a mass concentration of α-arbutin was 0.5 mg / mL to 5.0 mg / mL.

[0113] As shown in Table 4, compared with the blank group, the rare ginsenoside Rg5, α-arbutin and the combination can reduce the relative content of melanin in B16 cells to varying degrees; among them, when composition 1, composition 2, composition 3 and composition 4 are used in combination, CI < 1, and any adjustment of the mass concentration ratio of rare ginsenoside Rg5 and α-arbutin in the composition also has a synergistic effect. Composition 2 has the lowest combination index, CI = 0.40, CI < 1, indicating that it has the strongest synergistic effect; when composition 5 and composition 6 are used in combination, CI > 1, indicating that the combination does not have a synergistic effect. In summary, the combination of composition 1, composition 2, composition 3 and composition 4 has a synergistic effect on reducing the relative content of melanin in zebrafish embryos, and the synergistic effect of composition 2 is the strongest, which is better than rare ginsenoside Rg5 and α-arbutin, and the combination of composition 5 and composition 6 has no synergistic effect.

[0114] Table 4 Effects of rare ginsenoside Rg5 and α-arbutin alone and in combination on the relative content of melanin and inhibition of tyrosinase activity in zebrafish embryos

[0115]

[0116] 3.3.2 Effects of rare ginsenoside Rg5 and α-arbutin alone and in combination on the inhibition of tyrosinase activity in zebrafish embryos

[0117] As shown in Table 4, compared with the blank group, when the mass concentration of rare ginsenoside Rg5 was in the range of 0.2mg / mL to 2.0mg / mL and the mass concentration of α-arbutin was in the range of 0.5mg / mL to 5.0mg / mL, rare ginsenoside Rg5, α-arbutin and the combination could inhibit tyrosinase activity to varying degrees; among them, when composition 1, composition 2, composition 3 and composition 4 were used in combination, CI < 1, and any adjustment of the mass concentration ratio of rare ginsenoside Rg5 to α-arbutin in the composition also had the effect of synergistically improving the inhibition of tyrosinase activity. Composition 2 had the lowest combination index, CI < 0.40, indicating that it had the strongest synergistic effect; when composition 5 and composition 6 were used in combination, CI > 1, indicating that the combination had no synergistic effect. In summary, the combination of composition 1, composition 2, composition 3 and composition 4 has a synergistic effect on improving the tyrosine inhibition rate in zebrafish embryos, and the synergistic effect of composition 2 is the strongest, which is better than rare ginsenoside Rg5 and α-arbutin. The combination of composition 5 and composition 6 has no synergistic effect.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The use of a composition containing ginsenosides in the preparation of a whitening and freckle removing product, characterized in that: The composition comprises rare ginsenoside Rg5 and arbutin.

2. Use of a composition containing ginsenosides in the preparation of a product that inhibits tyrosinase activity, characterized in that: The composition comprises rare ginsenoside Rg5 and arbutin.

3. Use of a composition containing ginsenosides in the preparation of a product for inhibiting melanin production, characterized in that: The composition comprises rare ginsenoside Rg5 and arbutin.

4. Use of a composition containing ginsenosides in the preparation of an antioxidant product, characterized in that: The composition comprises rare ginsenoside Rg5 and arbutin.

5. The use according to claim 4, characterized in that: The applications include scavenging DPPH free radicals and ABTS free radicals.

6. The use according to any one of claims 1 to 5, characterized in that: The arbutin is α-arbutin.

7. The use according to claim 6, characterized in that: The composition comprises 0.2 to 2.0 parts of rare ginsenoside Rg5 and 0.5 to 5.0 parts of α-arbutin by weight.

8. The use according to claim 7, characterized in that: The composition comprises 0.5 to 1.5 parts of rare ginsenoside Rg5 and 1.0 to 3.0 parts of α-arbutin by weight.

9. The use according to claim 8, characterized in that: By weight, the composition comprises 1.0 part of rare ginsenoside Rg5 and 2.0 parts of α-arbutin.

10. The use according to claim 9, characterized in that: The products include food, medicine and cosmetics.

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