A ginsenoside composition and its preparation method and application

Abstract

The present invention relates to a ginsenoside composition and a preparation method and application thereof, belonging to the technical field of natural products. The ginsenoside composition of the present invention comprises the following components in mass percentage: ginsenoside R-Rg3 24.07-25.57%, ginsenoside F4 23.09-27.69%, ginsenoside S-Rg3 12.35-14.08%, ginsenoside Rk1 8.45-9.71%, ginsenoside Rg6 6.25-7.85%, ginsenoside Rg5 5.13-7.88%, and ginsenoside Rd 2.32-4.68%. The present invention selects ginsenosides R-Rg3, F4, S-Rg3, Rk1, Rg6, Rg5, Rd, Rg2, Rk3, Rh4, Rg1 and Re to form a ginsenoside composition, which has the advantages of whitening, anti-wrinkle, anti-ultraviolet and anti-inflammatory.

Description

Technical Field

[0001] The present invention relates to the technical field of natural products, and in particular to a ginsenoside composition and a preparation method and application thereof. Background Art

[0002] Ginseng fruit is the mature fruit of ginseng, and its main active component is ginsenosides, of which ginsenoside Re content is as high as 30%, which has a wide range of biological activities. The total ginsenosides in ginseng fruit are about 4 times that of ginseng root, and ginsenoside Re is the monomer saponin with the highest content in ginseng fruit, about 30 times that of ginseng root. Therefore, using an effective treatment method to convert prototype saponins into rare saponin compositions has important development significance and economic value in the fields of medicine, food and cosmetics.

[0003] Ginseng fruit extract contains a variety of rare saponins that are not found in ginseng. They have multiple functions such as beauty and skin care, and are one of the important natural raw materials for the development of whitening and skin care cosmetics. However, there is no report on a ginseng fruit glycoside composition rich in rare saponins with beauty and skin care functions and a preparation method. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a ginsenoside composition containing a variety of rare ginsenosides of ginseng and its preparation method and application. The ginsenoside composition has the advantages of whitening, anti-wrinkle, anti-ultraviolet and anti-inflammatory.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In the first aspect, the present invention provides a ginsenoside composition, which includes the following components in mass percentage: ginsenoside R-Rg3 24.07-25.57%, ginsenoside F4 23.09-27.69%, ginsenoside S-Rg312.35-14.08%, ginsenoside Rk1 8.45-9.71%, ginsenoside Rg6 6.25-7.85%, ginsenoside Rg5 5.13-7.88%, and ginsenoside Rd 2.32-4.68%.

[0007] The present invention selects ginsenoside R-Rg3, ginsenoside F4, ginsenoside S-Rg3, ginsenoside Rk1, ginsenoside Rg6, ginsenoside Rg5, and ginsenoside Rd to form a ginsenoside composition, which has the advantages of whitening, anti-wrinkle, anti-ultraviolet and anti-inflammatory. Rg6 and F4 have stronger antioxidant activity than prototype saponins Re, Rg1, etc., and Rg3 can inhibit the expression of MITF and TYR proteins by regulating the ERK signaling pathway to inhibit the melanin content in B16-F10 cells. It can be seen that the ginsenoside composition of the present invention is a combination of multiple rare ginsenosides to synergistically improve the effects of whitening, anti-wrinkle, anti-ultraviolet and anti-inflammatory.

[0008] As a preferred embodiment of the ginsenoside composition of the present invention, it also includes the following components in mass percentage: ginsenoside Rg2 1.66-2.45%, ginsenoside Rk3 1.25-1.46%, ginsenoside Rh4 0.72-0.88%, ginsenoside Rg1 0.51-0.69%, ginsenoside Re 0.22-0.37%. The inventors found in the experiment that the trace amount of ginsenoside monomers contained in the ginsenoside composition can also further improve the whitening, anti-wrinkle, anti-ultraviolet and anti-inflammatory effects of the ginsenoside composition.

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned ginsenoside composition, which comprises the following steps: mixing a ginseng fruit extract with fruit acid, stirring at 60-120°C for 60-180min, cooling and post-processing by a one-step precipitation method to obtain a ginsenoside composition. The present invention can effectively improve the hydrolysis efficiency of the prototype ginsenoside in the ginseng fruit extract and significantly increase the proportion of rare ginsenosides in the ginsenoside composition by mixing the ginseng fruit extract with the fruit acid and then heating the mixture. The present invention uses a one-step precipitation method for post-processing, which has the advantages of simple operation, rapid and efficient, energy saving, and no loss caused by the participation of organic solvents.

[0010] As a preferred embodiment of the method for preparing the ginsenoside composition of the present invention, the ratio of ginseng fruit extract to fruit acid is ginseng fruit extract: fruit acid = (1-5) kg: (1-2.5) L. Under the preferred ratio range, the content of rare ginsenosides and total ginsenosides in the ginsenoside composition of the present invention is significantly improved.

[0011] As a preferred embodiment of the preparation method of the ginsenoside composition of the present invention, the ratio of ginsenoside extract to fruit acid is ginsenoside extract: fruit acid = 1 kg: (1.5-2.5) L. Under the preferred ratio range, the content of rare ginsenosides and total ginsenosides in the ginsenoside composition of the present invention is significantly improved.

[0012] As a preferred embodiment of the method for preparing the ginsenoside composition of the present invention, the fruit acid includes at least one of citric acid, malic acid and gluconic acid.

[0013] As a preferred embodiment of the preparation method of the ginsenoside composition of the present invention, the total content of ginsenosides in the ginseng fruit extract is 5-95%. The method of the present invention is applicable to ginseng fruit extracts with different ginsenoside contents, and can maximize the use of ginsenoside resources in the ginseng fruit extract.

[0014] As a preferred embodiment of the method for preparing the ginsenoside composition of the present invention, the one-step precipitation method is to retain the precipitate after centrifuging the product, and wash the precipitate with water for 2-4 times.

[0015] In a third aspect, the present invention provides the use of the ginsenoside composition in the preparation of cosmetics.

[0016] In a fourth aspect, the present invention provides the use of the above-mentioned ginsenoside composition in the preparation of whitening, anti-ultraviolet, anti-wrinkle and / or anti-inflammatory products. The inventors have verified through cell experiments and animal experiments that the ginsenoside composition of the present invention has the ability to inhibit melanin synthesis, improve skin tissue keratin accumulation, epidermal hyperplasia and traumatic wounds caused by ultraviolet rays, inhibit collagen degradation and improve the phenomenon of cell active oxygen burst, and at the same time has the effects of whitening, anti-ultraviolet, anti-wrinkle and anti-inflammatory, and has a wide range of uses.

[0017] As a preferred embodiment of the application of the ginsenoside composition of the present invention, the product is a medicine.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention selects ginsenoside R-Rg3, ginsenoside F4, ginsenoside S-Rg3, ginsenoside Rk1, ginsenoside Rg6, ginsenoside Rg5, ginsenoside Rd, ginsenoside Rg2, ginsenoside Rk3, ginsenoside Rh4, ginsenoside Rg1 and ginsenoside Re to form a ginsenoside composition, which has the advantages of whitening, anti-wrinkle, anti-ultraviolet and anti-inflammatory.

[0020] (2) The present invention can effectively improve the hydrolysis efficiency of the prototype ginsenosides in the ginseng fruit extract by mixing the ginseng fruit extract with the fruit acid and then heating the mixture, and significantly increase the proportion of the rare ginsenosides in the ginseng fruit glycoside composition. The prepared ginsenoside composition has clear effective components and controllable quality, and is suitable for large-scale production. In addition, the present invention uses a one-step precipitation method for post-treatment, which has the advantages of simple operation, rapid and efficient, energy saving, and no loss caused by the participation of organic solvents. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of different test groups in effect example 1 on the melanin content in B16F19 cells, where A is not treated with α-MSH, and B is treated with α-MSH;

[0022] Figure 2 The effect of different test groups in Example 1 on tyrosinase activity in B16F10 cells, where A is not treated with α-MSH, and B is treated with α-MSH;

[0023] Figure 3 The effect of different test groups in effect example 1 on the expression level of melanin-related proteins in B16F10 cells, wherein A is MCIR protein, B is Tyrosinase protein, C is p-MITF protein, and D is MITF protein;

[0024] Figure 4 The effect of different test groups on mouse skin photodamage in effect example 2, where A is the thickness of the epidermis, B is the thickness of the dermis, C is the collagen area, and D is the thickness of the dermis and epidermis;

[0025] Figure 5 The ultraviolet absorption spectra of the ginsenoside composition and the ginseng fruit extract in Effect Example 3;

[0026] Figure 6 The effects of different test groups in effect example 3 on the expression level of matrix metalloproteinases in L929 cells, where A is MMP-1 and B is MMP-9;

[0027] Figure 7 The effect of different test groups in effect example 4 on the release of NO from RAW 264.7 cells;

[0028] Figure 8 The effect of different ginsenoside monomers on L929 cell viability in effect example 5;

[0029] Fig. 9 The effect of different ginsenoside monomers on the ROS level of L929 cells in effect example 5;

[0030] In the above figure, "*" indicates a significant difference compared with the control group (P < 0.05), "**" indicates a significant difference compared with the control group (P < 0.01), "***" indicates a significant difference compared with the control group (P < 0.001), "##" indicates a significant difference compared with the model group (P < 0.01), and "###" indicates a significant difference compared with the model group (P < 0.001). DETAILED DESCRIPTION

[0031] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0032] Unless otherwise specified, other materials, reagents, etc. used in the following embodiments and effect examples can be obtained from commercial sources.

[0033] In the following examples, the methods for detecting the types and contents of ginsenosides in ginsenoside extracts are all HPLC technology, and the specific operations are as follows:

[0034] Accurately weigh 20 mg of the ginsenoside composition sample, place it in a 10 mL volumetric flask, add 5 mL of chromatographic methanol, ultrasonicate for 10 min, and then dilute to 10 mL with chromatographic methanol. Filter the ginsenoside composition solution with a 0.45 μm organic microporous filter membrane for later use;

[0035] Accurately weigh 1 mg each of ginsenoside Rg1, Re, Rg2, Rd, Rg6, F4, Rk3, Rh4, S-Rg3, R-Rg3, Rk1, and Rg5 reference substances, place them in a 1 mL volumetric flask, add methanol to dissolve and make up to volume to prepare a standard solution, prepare a solution of the above 12 reference substances mixed in a 1:1 ratio, filter through a 0.45 μm organic microporous filter membrane, and set aside.

[0036] The chromatographic conditions were as follows: the chromatographic column was Agilent Zorbax Eclipse XDB C18 (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile (A)-water (B), the volume flow rate was 1.0 mL / min; the injection volume was 20 μL; the column temperature was 25°C; the detection wavelength was 203 nm; the elution conditions were 0-20 min, 20% A; 20-45 min, 20-46% A; 45-55 min, 46-55% A; 55-60 min, 55% A;

[0037] The detection was carried out according to the chromatographic conditions to obtain the peak area of ​​the reference substance, and then the peak area of ​​the sample corresponding to the retention time was measured, and the content of the component to be tested in the sample was calculated according to the external standard method. Ginsenoside content (%) = Ax*Cr / Ar*Cx; Ax: sample peak area; Cr: reference substance concentration; Ar: reference substance peak area; Cx: sample concentration.

[0038] C57 BL / 6 mice were purchased from Liaoning Changsheng Animal Co., Ltd. (Liaoning, China) and maintained in an SPF-level environment with a 12-h light / dark cycle.

[0039] Ginseng fruit extract is provided by Jilin Changqing Ginseng Industry Co., Ltd. with production batch number 20200901. Testing shows that its total ginsenoside content is 5-95%.

[0040] B16F10 cells were provided by Dalian Meilun Biotechnology Co., Ltd. with the catalog number CL0124.

[0041] L929 cells were provided by Dalian Meilun Biotechnology Co., Ltd. with the catalog number CL0136.

[0042] The tyrosinase activity detection kit was provided by Nanjing Jiancheng Biological Co., Ltd., with the catalog number H310-1-2.

[0043] The collagenase activity detection kit was provided by Aladdin Chemical Reagent Co., Ltd., with the catalog number C278962.

[0044] The elastase activity detection kit was provided by Aladdin Chemical Reagent Co., Ltd., with the catalog number E107150.

[0045] Ginsenoside R-Rg3, ginsenoside F4, ginsenoside S-Rg3, ginsenoside Rk1, ginsenoside Rg6, ginsenoside Rg5, ginsenoside Rd, ginsenoside Rg2, ginsenoside Rk3, ginsenoside Rh4, ginsenoside Rg1, and ginsenoside Re were all purchased from Chengdu Purifa Co., Ltd.

[0046] The cell activity detection kit was provided by Dalian Meilun Biotechnology Co., Ltd. with the catalog number MA0198.

[0047] The ROS detection kit was provided by Dalian Meilun Biotechnology Co., Ltd. with the catalog number MA0129.

[0048] Example 1

[0049] In order to better increase the content of rare ginsenosides in the ginsenoside composition, the parameter conditions of the preparation method of the ginsenoside composition are optimized. The specific scheme is as follows:

[0050] 1. Effect of different solid-liquid ratios on the content of ginsenosides in ginsenoside compositions.

[0051] After different weights of ginseng fruit extract were dissolved, the temperature was raised to 90-100°C, and different volumes of citric acid were added respectively. The mixture was heated and stirred for 100 minutes. After cooling, the mixture was centrifuged and the supernatant was removed. The obtained precipitate was washed twice with 20 times purified water, vacuum dried and ground to obtain a ginseng fruit glycoside composition. The type and content of ginsenosides in the obtained ginseng fruit glycoside composition were detected. The ratio of ginseng fruit extract to citric acid, and the type and content of ginsenosides in the ginseng fruit glycoside composition are shown in Table 1.

[0052] Table 1 Effect of different solid-liquid ratios on ginsenoside extracts (by mass percentage)

[0053]

[0054]

[0055] As shown in Table 1, when the ratio of ginseng fruit extract to citric acid is 1kg:(1-2.5)L, the content of rare ginsenosides is greatly increased, and the content of total ginsenosides is also increased accordingly. Among them, when the ratio of ginseng fruit extract to citric acid is 1kg:1.5L, the obtained rare ginsenoside content and total ginsenoside content are the highest, indicating that 1kg:1.5L is the optimal solid-liquid ratio in the preparation method of the ginseng fruit glycoside composition.

[0056] 2. Effect of different heating times on the content of ginsenosides in ginsenoside compositions

[0057] After 1 kg of ginseng fruit extract was dissolved, the temperature was raised to 90-100°C, 1.5 L of citric acid was added, and the mixture was heated and stirred for 40-100 min. After cooling, the mixture was centrifuged and the supernatant was removed. The resulting precipitate was washed twice with 20 times purified water, vacuum dried and ground to obtain a ginseng fruit glycoside composition. The type and content of ginsenosides in the obtained ginsenoside composition were detected. The heating time, the type and content of ginsenosides in the ginseng fruit glycoside extract are shown in Table 2.

[0058] Table 2 Effect of different heating time on the content of ginsenosides in ginsenoside composition (by mass percentage)

[0059]

[0060]

[0061] As shown in Table 2, with the increase of heating time, the content of rare ginsenosides and total ginsenosides in the ginsenoside composition also increases. When the heating time is 60-120 min, the content of rare ginsenosides and total ginsenosides in the ginsenoside composition is higher, and when the heating time is 100 min, the content of rare ginsenosides and total ginsenosides in the ginsenoside composition is the highest, so 100 min is the optimal heating time.

[0062] 3. Optimization of post-processing technology

[0063] (1) One-step precipitation method: 1 kg of ginseng fruit extract was dissolved and heated to 90-100°C, 1.5 L of citric acid was added, and the mixture was heated and stirred for 100 min. After cooling, the mixture was centrifuged and the supernatant was removed. The resulting precipitate was washed twice with 20-fold purified water, vacuum dried and ground to obtain a ginseng fruit glycoside composition. The types and contents of ginsenosides in the obtained ginsenoside composition were detected. The types and contents of ginsenosides in the ginsenoside extract are shown in Table 3.

[0064] (2) Organic solvent extraction and concentration: 1 kg of ginseng fruit extract was dissolved and heated to 90-100°C, 1.5 L of citric acid was added, heated and stirred for 100 min, and allowed to stand in a flask at 90°C for precipitation, and cooled to room temperature using normal temperature tap water;

[0065] Add purified water to the flask to rinse the precipitate, pour out the purified water and add 150-200 mL of 95% ethanol to dissolve the brown solid at the bottom of the flask, evaporate at 80°C until the liquid is viscous, transfer the liquid to a stainless steel plate, rinse the flask with a small amount of ethanol, and transfer the rinsed liquid to a stainless steel plate;

[0066] The stainless steel plate with the liquid was evaporated in a water bath at 100°C until no bubbles were generated in the liquid, and then transferred to a 100°C forced air drying oven for drying for 3 hours. The material in the stainless steel plate was scraped off and powdered. The resulting powder was the ginsenoside composition. The types and contents of ginsenosides in the obtained ginsenoside composition were detected. The types and contents of ginsenosides in the ginsenoside composition are shown in Table 3.

[0067] Table 3 Effects of different post-treatment processes on ginsenosides in ginsenoside compositions

[0068]

[0069]

[0070] As shown in Table 3, the types and contents of ginsenosides contained in the ginsenoside composition obtained by post-treatment with the one-step precipitation method are close to those of the ginsenoside composition obtained by post-treatment with an organic solvent. However, the one-step precipitation method has the advantages of simple operation, no need for concentration and no organic solvent participation, which can greatly improve the efficiency of the post-treatment process without causing the loss of ginsenoside types and components. In addition, the ginsenoside composition post-treated by the one-step precipitation method also contains the rare ginsenoside F4 that is not obtained by post-treatment with an organic solvent. Therefore, the one-step precipitation method is preferably used for post-treatment of the ginseng fruit extract after acid treatment.

[0071] Example 2

[0072] The present embodiment provides a ginsenoside composition and a preparation method thereof. The preparation method of the ginsenoside composition comprises the following steps: mixing 1 kg of ginseng fruit extract with 10-20 L of hot water, stirring at 300 r / min for 3-5 min until the ginseng fruit extract is dissolved, heating to 90-100° C., adding 1.5 L of citric acid, heating to 90-100° C., stirring at 300 r / min for 100 min, cooling and centrifuging, removing the supernatant, washing the obtained precipitate twice with 20 times purified water, vacuum drying and grinding to obtain a ginsenoside composition, and detecting the type and content of ginsenosides in the obtained ginsenoside composition. The results are shown in Table 4.

[0073] The composition of ginsenosides in the ginsenoside composition was identified by LC-MS technology, and the specific operation was as follows:

[0074] The chromatographic conditions were as follows: the chromatographic column was Cica MERCK RP18 (2.0 mm × 100 mm, 3 μm); the mobile phase was acetonitrile (A)-0.1% formic acid water (B), the volume flow rate was 0.25 mL / min; the injection volume was 5 μL; the column temperature was 35°C; the detection wavelength was 203 nm;

[0075] Gradient elution was used, and the elution conditions were: 0-3min, 19-81% A; 3-6min, 22% A; 6-9min, 30% A; 9-16min, 35% A; 16-18min, 55% A; 18-22min, 55% A; 22-26min, 70% A; 26-19min, 90% A; 29-29.1min, 19% A; 29.1-32min, 18% A;

[0076] The mass spectrometry results obtained were processed and analyzed by Compound Discovere3.2 software in the full scan range of 100-1500 m / z in the positive ion mode of the sample. The MS / MS fragmentation of the first two ions was combined with the structural identification of the auxiliary components to screen out the secondary spectrum of the molecular ion peak. The detected mass spectrometry fragments and formulas were then compared with the compound library established before the test for component identification. Based on these databases, the fragmentation patterns were summarized, and the structures of components with similar structures were inferred. In addition, for compounds with standard substances, the compounds were determined by comparing the retention time, high-resolution accurate mass and ion fragments with those of the reference substances. The mass spectrometry results are shown in Table 5.

[0077] Table 4 Test results of ginsenoside types and dosages of different ginsenoside compositions (by mass percentage)

[0078] Ginsenoside Types Ginseng Fruit Extract Example 1 Example 2 Example 3 <![CDATA[Rg1]]> 1.73％ 0.69％ 0.61％ 0.51％ Re 30.94％ 0.37％ 0.22％ 0.26％ <![CDATA[Rg2]]> 1.02％ 2.45％ 2.34％ 2.66％ Rd 7.06％ 3.43％ 4.68％ 3.32％ <![CDATA[Rg6]]> 0.56％ 7.77％ 7.85％ 6.25％ F4 1.42％ 27.69％ 27.60％ 23.09％ <![CDATA[Rk3]]> 0.35％ 1.46％ 1.43％ 1.25％ <![CDATA[Rh4]]> 0.01％ 0.88％ 0.72％ 0.79％ <![CDATA[S-Rg3]]> 0.79％ 14.08％ 12.49％ 12.35％ <![CDATA[R-Rg3]]> 0.29％ 24.07％ 14.01％ 25.57％ <![CDATA[Rk1]]> 0.13％ 9.71％ 9.47％ 8.45％ <![CDATA[Rg5]]> 0.10％ 6.66％ 5.13％ 7.88％

[0079] As shown in Table 4, the mass percentages of the effective ingredients in the ginsenoside composition prepared according to the optimal preparation process and different feeding amounts of ginseng fruit extract are as follows: ginsenoside R-Rg3 24.07-25.57%, ginsenoside F4 23.09-27.69%, ginsenoside S-Rg3 12.35-14.08%, ginsenoside Rk1 8.45-9.71%, ginsenoside Rg6 6.25-7.85%, ginsenoside Rg5 5.13-7.88%, ginsenoside Rd 2.32-4.78%, ginsenoside Rg2 1.66-2.45%, ginsenoside Rk3 1.25-1.46%, ginsenoside Rh4 0.72-0.88%, ginsenoside Rg1 0.51-0.69%, ginsenoside Re 0.22-0.37%.

[0080] Table 5 LC-MS detection results of ginsenoside composition

[0081]

[0082]

[0083] As shown in Table 5, 15 ginsenoside components were identified from the ginsenoside composition, namely: ginsenoside PPD, ginsenoside Rh3, Rk3 / Rh4, Rh2, F1, Rh1, Rg6, Rk1, F4, Rg5, Rg3, Rg2, Rg1, Rd and Re. The difference between the theoretical molecular weight and the actual measured value is within 5ppm. Different types of saponins were identified by compound molecular weight, retention time and fragment information, and mass spectrometry fragmentation rules and structure analysis were performed. The parent core structure of ginsenosides is mainly PPD type and PPT type. The former is characterized by the sugar group attached to C-3 and / or C-20. During cleavage, the glycosidic bond at C-20 is first broken, and one molecule of sugar is lost. If it is a disaccharide, two molecules of sugar are lost together. Then the sugar group at C-3 falls off in turn, and the whole process is accompanied by the loss of H2O molecules. The latter is characterized by the sugar group attached to C-6 and / or C-20. During cleavage, the sugar at C-20 is first lost, and then the sugar at C-6 falls off in turn, and the whole process is accompanied by the loss of H2O molecules.

[0084] Example 3

[0085] The present embodiment provides a ginsenoside composition and a preparation method thereof. The preparation method of the ginsenoside composition comprises the following steps: dissolving 3 kg of ginseng fruit extract and heating to 90-100°C, adding 4.5 L of citric acid, heating and stirring for 100 min, cooling and centrifuging, removing the supernatant, washing the obtained precipitate twice with 20 times purified water, vacuum drying and grinding to obtain a ginsenoside composition, and detecting the type and content of ginsenosides in the obtained ginsenoside composition. The results are shown in Table 4.

[0086] Example 4

[0087] The present embodiment provides a ginsenoside composition and a preparation method thereof. The preparation method of the ginsenoside composition comprises the following steps: dissolving 5 kg of ginseng fruit extract and heating to 90-100°C, adding 7.5 L of citric acid, heating and stirring for 100 min, centrifuging after cooling, removing the supernatant, washing the obtained precipitate twice with 20 times purified water, vacuum drying and grinding to obtain a ginsenoside composition, and detecting the type and content of ginsenosides in the obtained ginsenoside composition. The results are shown in Table 4.

[0088] Effect Example 1

[0089] In order to verify the whitening effect of the ginsenoside composition of the present invention, an in vitro experiment was conducted using B16F10 cells to detect the inhibitory effect of the composition sample in Example 1 on the melanin content, tyrosinase, and melanin-related proteins in B16F10 cells. The specific scheme is as follows:

[0090] 1. 2×10 5 / mL cells were inoculated in a 6-well plate and cultured overnight, and then different concentrations (1.5, 3 and 6 μg / mL) of the ginsenoside composition obtained in Example 2 and α-MSH (1 μM, 24h) were added to treat the corresponding groups. After washing the cells with PBS, they were placed on ice, lysed for 30 minutes, centrifuged at 12000r for 15 minutes at 4°C, and the supernatant was retained for the subsequent tyrosinase detection, and the black precipitate was dissolved with 1mol / L NaOH (containing 10% DMSO) at 85°C for 40 minutes, and then the A value was detected at 405nm using an enzyme marker. The obtained A value is the melanin content. The results are shown in Figure 1 .

[0091] like Figure 1 As shown, the ginseng fruit extract has almost no effect on the melanin content, while the ginseng glycoside composition exhibits a better inhibitory effect on melanin. At the same time, the ginseng glycoside composition exhibits a significant inhibitory effect on melanin content even 24 hours after induction by melanocyte-stimulating hormone (α-MSH), indicating that the ginseng glycoside composition of the present invention has a better whitening effect, which is not possessed by the ginseng fruit extract.

[0092] 2. Take the above protein supernatant for tyrosinase detection and perform tyrosinase detection according to the instructions of the kit. Mix the sample with the same protein concentration with 180 μL of L-DOPA, detect the A1 value at 490nm at the 10th second, and then incubate at 37°C for 3 minutes, accurately detect the A2 value at the 3rd minute 10 seconds, calculate the tyrosinase (U / prot) = 90.09*(A2-A1) / Cpr according to the protein concentration, and calculate the tyrosinase activity (%) = (experimental group-control group)*100%, and the tyrosinase activity test results are shown in Figure 2 .

[0093] like Figure 2 As shown, after treating B16F10 cells for 48 hours, the ginsenoside composition showed a better tyrosinase activity inhibitory effect than the ginseng fruit extract. At the same time, under the condition of α-MSH induction (24 hours), the tyrosinase activity increased by more than 1.5 times compared with the control group, and the ginsenoside composition could also significantly reduce the tyrosinase activity in B16F10 cells after α-MSH induction, indicating that the ginsenoside composition of the present invention can significantly inhibit tyrosinase activity.

[0094] 3. 2×10 5 / mL B16F10 cells were inoculated in 6-well plates and cultured overnight. First, α-MSH (1μM) was used to stimulate the cells for 24h, and then 1.5, 3 and 6μg / mL of ginsenoside compositions were used for further treatment for 48h. The cells were collected and washed with cold PBS, RIPA lysis buffer with added protease inhibitors was used, and the cells were fully lysed on ice for 30min, and then centrifuged at 4℃, 12000rpm for 20min and the protein supernatant was collected. Equal amounts of protein lysates were placed in protein buffer and heated at 95℃ for 10min to denature them. The samples were separated by electrophoresis and transferred to nitrocellulose membranes. Then they were blocked with 5% skim milk powder for 1h, and the protein membranes were incubated with the corresponding primary antibodies (melanin-related proteins MC1R, Tyrosinase, MITF, p-MITF, and control β-actin) at 4℃ overnight (18h), where β-actin was used as a control for total protein. In the presence of horseradish peroxidase-conjugated secondary antibody, the ECL reagent was used for reaction and the signal was observed in real time in Tanon 4600. The results of the detection of melanin-related protein expression levels are shown in Figure 3 .

[0095] like Figure 3As shown in the results, the ginsenoside composition had a significant inhibitory effect on the expression of different melanin-producing proteins. After α-MSH stimulation, the expression of MC1R, Tyrosinase and MITF was significantly upregulated, and the ginsenoside composition (6 μg / mL) reversed this effect. In addition, the ginsenoside composition alone also reduced the expression of MC1R, Tyrosinase, p-MITF and MITF, indicating that the ginsenoside composition exhibited a significant anti-melanin effect in B16F10 cells.

[0096] In summary, the ginsenoside composition of the present invention can achieve the effect of reducing the melanin content in cells by inhibiting tyrosinase activity and reducing the expression levels of melanin-related genes (MC1R, Tyrosinase, p-MITF and MITF), and can also inhibit tyrosinase activity and melanin-related gene expression levels under the induction of melanocyte-stimulating hormone, and has excellent whitening effect.

[0097] Effect Example 2

[0098] In order to verify the anti-ultraviolet effect of the ginsenoside composition of the present invention, a mouse model of skin damage induced by ultraviolet irradiation was established to detect the protective effect of the ginsenoside composition obtained in Example 2 on the skin. The specific scheme is as follows:

[0099] After C57 BL / 6 mice were adapted for one week, the back hair of all groups of mice was shaved one day before the experiment. A total of 5 experimental groups were set up in the experiment, and the specific treatments are shown in Table 6. UVB 20mJ / cm 2 The back of mice was irradiated once for 7 days to establish an acute UV skin injury model. 12 hours after the last UVB irradiation, all mice were killed by dislocation of the neck, and the back skin was peeled and collected, fixed in 4% paraformaldehyde, and used for Masson staining (Masson), and the dermal thickness, epidermal thickness, total dermal and epidermal thickness, and collagen area of ​​the mouse skin were analyzed. The statistical results are shown in Figure 4 .

[0100] Table 6 Treatment of mice in each test group in the anti-ultraviolet experiment

[0101]

[0102] like Figure 4As shown in the results, compared with the normal group, the UVB irradiation group showed more inflammatory reactions in the mouse skin, mainly manifested as excessive accumulation of the stratum corneum, epidermal hyperplasia and increased thickness, while pretreatment with the ginsenoside composition improved these tissue morphological changes. At the same time, the Masson staining results observed that UVB irradiation caused traumatic wounds in the dermis, and the reticular fibers in the reticular layer proliferated in large quantities, while the ginsenoside composition significantly restored the distribution of collagen in the dermis and reduced the amount of fibers densely distributed in the reticular layer.

[0103] In summary, the ginsenoside composition of the present invention can effectively improve keratin accumulation, epidermal hyperplasia and traumatic wounds in the skin tissue of ultraviolet-irradiated mice, indicating that the ginsenoside composition of the present invention has anti-ultraviolet efficacy, and the anti-ultraviolet effect is better than that of the positive drug VC.

[0104] Effect Example 3

[0105] In order to verify the anti-wrinkle effect of the ginsenoside composition of the present invention, the ultraviolet absorption of the ginseng fruit extract and the ginsenoside composition obtained in Example 2 was measured by ultraviolet spectrophotometer, and the inhibition of collagenase and elastase was measured. At the same time, the effect of the ginsenoside composition on the expression of MMP1 and MMP9 proteins in L929 cells after ultraviolet irradiation was detected by ultraviolet irradiation. The specific scheme is as follows:

[0106] The ginseng fruit extract and the ginseng fruit glycoside composition were dissolved in chromatographic methanol and diluted to 100 μg / mL, respectively, and their ultraviolet absorption characteristics at 200-400 nm were measured. The ginseng fruit extract and the ginseng fruit glycoside composition were diluted to 1, 2, and 4 μg / mL, respectively, and the inhibition rates of collagenase and elastase were measured according to the instructions of the kit. The ultraviolet absorption spectra are shown in FIG. Figure 5 The inhibition rates of collagenase and elastase are shown in Table 7.

[0107] Using UVB (600mJ / cm 2 ) irradiated L929 cells, and then used the ginsenoside composition and the positive drug vitamin C (VC) for drug intervention for 24 hours, and then determined the protein expression levels of MMP-1 and MMP-9 according to the method of step 3 of effect example 1 (the antibodies were changed to MMP-1 and MMP-9 according to the situation). The expression level detection results of MMP-1 and MMP-9 are shown in Figure 6 .

[0108] Table 7 Effects of different concentrations of ginseng fruit extract and ginsenoside combination on collagen membrane and elastase

[0109]

[0110] like Figure 5As shown in Table 7, compared with the ginseng fruit extract, the ginseng fruit glycoside composition has better ultraviolet absorption properties and better inhibitory effects on collagenase and elastase.

[0111] MMP-9 is matrix metalloproteinase 9, which mainly degrades collagen in the body. After ultraviolet radiation, the expression of MMP9 protein increases, and the collagen secreted by the human body and cells decreases, leading to skin aging, such as Figure 6 As shown, the expression levels of MMP-1 and MMP-9 proteins increased after ultraviolet irradiation, and the expression levels of MMP-1 and MMP-9 proteins decreased significantly after treatment with the ginsenoside composition, indicating that the ginsenoside composition can inhibit the degradation of collagen.

[0112] In summary, the ginsenoside composition of the present invention has a good effect of preventing the degradation of collagen and elastin secreted by cells, thereby achieving an anti-wrinkle effect.

[0113] Effect Example 4

[0114] In order to verify the anti-inflammatory effect of the ginsenoside composition of the present invention, lipopolysaccharide (LPS)-induced RAW 264.7 cells were used to detect the effect of the ginsenoside composition obtained in Example 2 on the NO release of RAW 264.7 cells induced by LPS. The specific scheme is as follows:

[0115] LPS was used as an inducer to treat RAW 264.7 cells to construct a stable inflammatory model. A total of 6 experimental groups were set up, and the treatment of each group is shown in Table 8. RAW 264.7 cells in the logarithmic growth phase were inoculated in a 96-well plate and cultured overnight. After adding LPS (final concentration of 1 μg / mL) for 24 hours, different concentrations of ginsenoside composition were used for treatment for 24 hours, and the NO content in the supernatant was determined by the Griess method. The results are shown in Figure 7 .

[0116] Table 8 Settings of each test group for NO content detection

[0117] Group LPS treatment Treatment with retinoic acid or ginsenoside combination Control group No treatment Equal volume of solvent Model Group 1 μg / mL Equal volume of solvent Retinoic acid group (positive control) 1 μg / mL 20 μg / mL High dose group 1 μg / mL 80 μg / mL Medium dose group 1 μg / mL 60 μg / mL Low dose group 1 μg / mL 40 μg / mL

[0118] like Figure 7 As shown, compared with the control group, the NO level in the model group increased significantly, indicating that the RAW 264.7 cell inflammation model was successfully constructed. Compared with the model group, the NO content in the supernatant of cells treated with the ginsenoside composition and LPS was significantly reduced, and the inhibitory effect of the ginsenoside composition on the release of NO by RAW 264.7 cells induced by LPS was comparable to that of the positive control retinoic acid, indicating that the ginsenoside composition of the present invention has a certain anti-inflammatory effect.

[0119] Effect Example 5

[0120] In order to evaluate the main active ingredients in the ginsenoside composition, the proliferation inhibitory activity of ginsenoside monomers on L929 cells and the H2O2-induced redox imbalance of L929 cells were determined. The specific scheme is as follows:

[0121] 1. Cultivate L929 cells in DMEM medium containing 10% FBS. When the cell density reaches 80%, seed the cells. Inoculate L929 cells in a 96-well plate (cell density 1*10 5 ), when the cell density grows to 80%, a ginsenoside monomer control group is administered. Ginsenoside R-Rg3, ginsenoside F4, ginsenoside S-Rg3, ginsenoside Rk1, ginsenoside Rg6, ginsenoside Rg5, ginsenoside Rd, ginsenoside Rg2, ginsenoside Rk3, ginsenoside Rh4, ginsenoside Rg1, and ginsenoside Re were prepared into 1 μM solutions and added to a 96-well cell plate cultured with L929 cells, and the cells were administered and incubated for 24 hours. After the administration, 10 μL of MTT solution was added to each well, and the culture was continued for 3 hours. The culture medium in the wells was then discarded, and 100 μL of DMSO solution was added to each well. After standing for 20 minutes, the absorbance value was measured at a wavelength of 490 nm, and the activity of ginsenosides on L929 cells was calculated. The results are shown in Figure 8 .

[0122] like Figure 8 As shown in the data, when the concentration of ginsenosides was low (1 μM), ginsenosides promoted the proliferation of L929 cells, among which Rk1, F4, Rg6, and Rd (1 μM) had the strongest promoting effect, suggesting that Rk1, F4, Rg6, and Rd (1 μM) may play a major role in biological activity by promoting the proliferation of fibroblast L929 cells and increasing the secretion of collagen and elastin.

[0123] 2. According to the method in step 1, ginsenoside monomers Rk1, F4, Rg6, and Rd were co-cultured with L929 cells. After incubation for 24 hours, the cellular ROS level was determined according to the instructions of the kit. The results are shown in Fig. 9 .

[0124] like Fig. 9 As shown, Rg6 and F4 significantly inhibited H2O2-induced oxidative stress in L929 cells, and their ability to inhibit ROS production was significantly better than that of Rk1 and Rd. Therefore, Rg6 and F4 may be the main active ingredients in the ginsenoside composition of the present invention.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A ginsenoside composition, characterized in that: The invention comprises the following components in percentage by mass: ginsenoside R-Rg3 24.07-25.57%, ginsenoside F4 23.09-27.69%, ginsenoside S-Rg3 12.35-14.08%, ginsenoside Rk1 8.45-9.71%, ginsenoside Rg6 6.25-7.85%, ginsenoside Rg5 5.13-7.88%, ginsenoside Rd 2.32-4.68%, ginsenoside Rg2 1.66-2.45%, ginsenoside Rk3 1.25-1.46%, ginsenoside Rh4 0.72-0.88%, ginsenoside Rg1 0.51-0.69% and ginsenoside Re 0.22-0.37%.

2. The method for preparing the ginsenoside composition according to claim 1, characterized in that: The following steps are involved: The ginseng fruit extract and the fruit acid are mixed, stirred at 60-100° C. for 60-120 min, and after cooling, post-treated by a one-step precipitation method to obtain a ginseng fruit glycoside composition; the ratio of the ginseng fruit extract to the fruit acid is ginseng fruit extract: fruit acid = 1 kg: (1.5-2.5) L; the operation of the one-step precipitation method is to retain the precipitate after centrifugation of the product, wash the precipitate with water 2-4 times, and grind it into powder after vacuum drying.

3. The preparation method according to claim 2, characterized in that: The fruit acid includes at least one of citric acid, malic acid and gluconic acid.

4. The preparation method according to claim 2, characterized in that: The total content of ginsenoside in the ginseng fruit extract is 5-95%.

5. Use of the ginsenoside composition as claimed in claim 1 in the preparation of cosmetics.

6. Use of the ginsenoside composition as claimed in claim 1 in the preparation of whitening, anti-ultraviolet, anti-wrinkle and / or anti-inflammatory products.

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