Rare saponin-rich ginseng extract as well as preparation method and application thereof

Abstract

The invention discloses a ginseng extract rich in rare saponins and preparation and anti-aging application thereof, and belongs to the technical field of oral beauty and skin care products, the ginseng extract is obtained by fermentation treatment of ginseng powder through phytobacterium plantarum, and the preservation number of the phytobacterium plantarum is GDMCC No.64374. The ginseng extract rich in rare saponin is fermented by specific plant lactobacillus, ginsenoside is efficiently converted into CK and F1, various natural plant components such as ginseng polysaccharide are reserved, and a specific synergistic effect is generated. Through verification, the prepared ginseng extract is high in bioavailability, can more effectively inhibit cell inflammation, and can more effectively recover the mitochondrial function of senescent cells and inhibit cell senescence. The preparation method provided by the invention is high in operability and easy to standardize and intellectualize. Compared with a traditional extraction and purification method, the adopted fermentation method is more environmentally friendly and efficient, and large-scale production can be achieved in industrial production.

Description

Technical Field

[0001] The present invention relates to a ginseng extract rich in rare saponins, its preparation method and application in anti-aging, belonging to the technical field of oral beauty and skin care products. Background Art

[0002] The skin is the largest organ of the body exposed to the external environment and is easily affected by internal and external factors, leading to aging. The main features of skin aging are skin relaxation and wrinkle formation, which not only affect appearance but also may impact an individual's mental health and social interaction. In recent years, anti-skin aging health products and skin care products have emerged one after another, but they all have defects such as unsatisfactory anti-skin aging effects and difficulty in exerting the effects of anti-skin aging active ingredients. Therefore, there is an urgent need to develop a raw material with high safety, no irritation, and obvious anti-skin aging effects.

[0003] Existing studies have found that ginsenosides can promote the synthesis of collagen, improve skin elasticity and luster, and have broad application potential in the fields of healthcare products, beauty and skin care, food products, and daily necessities. However, the prototype components of ginsenosides extracted by traditional methods, such as Rb1, Rb2, Rc, Rd, Re, and Rg1, account for more than 80% of the total ginsenosides in ginseng, while the proportion of secondary saponins F1 and CK in the total ginsenosides of ginseng is extremely low. The bioavailability of the prototype components of saponins is relatively low, and their biological activities are poor. In contrast, secondary saponins can be better absorbed and utilized. Therefore, exploring a suitable treatment process to convert the prototype ginsenosides in ginseng into secondary saponins and aglycones with relatively high bioavailability and retaining other natural active ingredients in ginseng to maximize the bioavailability and biological activities of the active ingredients in ginseng has become the key to the in-depth processing research of ginseng. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides a ginseng extract rich in rare saponins, its preparation method and application in anti-aging, which can convert most of the prototype components of saponins into secondary saponins, and the conversion products have higher bioavailability and lower cytotoxicity.

[0005] The technical solution adopted by the present invention to solve its technical problems is as follows: In a first aspect, the present application provides a ginseng extract rich in rare saponins, which is obtained by fermenting ginseng powder with Lactiplantibacillus plantarum, and the preservation number of the Lactiplantibacillus plantarum is GDMCC No.64374. The Lactiplantibacillus plantarum is preserved in the Guangdong Provincial Culture Collection of Microorganisms, located on the 5th floor of Building 59, No. 100, Middle Xianlie Road, Guangzhou, and the preservation date is February 5, 2024.

[0006] The ginseng extract provided by this application not only contains higher concentrations of rare ginsenosides F1 and CK, but also the various plant components contained in the ginseng extract can produce a stronger synergistic effect. Compared with single-component ginseng polysaccharide, F1, CK, or a mixture of these three, the ginseng extract obtained by fermentation in this application has higher bioavailability and lower cytotoxicity, can more effectively inhibit cell inflammation, more effectively restore the mitochondrial function of senescent cells and inhibit cell senescence.

[0007] Further, by mass, the content of rare ginsenosides is 0.2% - 2%, and the content of ginseng polysaccharide is 10% - 30%.

[0008] Further, the rare ginsenosides include CK and F1, the total amount of CK and F1 accounts for more than 99wt% of the rare ginsenosides, and the mass ratio of CK to F1 is 1:1 - 5.

[0009] In a second aspect, this application provides a method for preparing a ginseng extract rich in rare ginsenosides, which ferments ginseng powder with Lactiplantibacillus plantarum, and the preservation number of the Lactiplantibacillus plantarum is GDMCC No. 64374.

[0010] Fermentation with a specific Lactiplantibacillus plantarum can efficiently convert protopanaxadiol ginsenosides into rare ginsenosides while retaining other active components of ginseng, ensuring the high bioavailability and low cytotoxicity of the extract, and is suitable for the development of anti-aging products.

[0011] The steps include: Freeze-dry fresh ginseng and powder it to obtain the ginseng powder; Disperse the ginseng powder in water, sterilize it, and cool it to 37°C to obtain a fermentation substrate; Inoculate the seed liquid of the Lactiplantibacillus plantarum into the fermentation substrate, ferment and culture it, sterilize it after monitoring the reaction is complete, and collect the supernatant to obtain the ginseng extract rich in rare ginsenosides.

[0012] Further, in the step of dispersing the ginseng powder in water, the mass ratio of the ginseng powder to the water is 1 - 5:100.

[0013] Controlling the ratio of ginseng powder to water ensures an appropriate concentration of the fermentation substrate, which not only guarantees the sufficient supply of nutrients during the fermentation process but also avoids affecting the fermentation efficiency and product quality due to too high or too low concentration.

[0014] Further, the concentration of the seed liquid is OD 600 = 0.8 - 2.0, and the volume ratio of the seed liquid to the fermentation substrate is 1:20 - 100.

[0015] Controlling the concentration of the seed liquid and the inoculation ratio ensures the activity of the strains and the fermentation efficiency during the fermentation process, and avoids the problems of incomplete fermentation or unstable product quality caused by too many or too few strains.

[0016] Furthermore, the temperature of the fermentation culture is 30°C to 50°C, and the time is 8h to 16h.

[0017] Controlling the fermentation temperature and the culture time ensures that Lactobacillus plantarum ferments under the optimal conditions, maximally converting protopanaxadiol saponins in ginseng into rare saponins, and at the same time avoiding the degradation of active ingredients caused by too long time.

[0018] Furthermore, the sterilization temperature for sterilizing after monitoring the completion of the reaction is 85°C to 110°C, and the time is 15min to 30min.

[0019] Controlling the sterilization temperature and time ensures the aseptic state of the fermentation product, and at the same time avoids the loss of active ingredients caused by high temperature or long-term sterilization, ensuring the stability and safety of the extract.

[0020] In the third aspect, the present application provides the use of the ginseng extract rich in rare saponins described in the first aspect for making foods or skin care products with anti-aging functions.

[0021] The beneficial effects of the present invention are as follows: The ginseng extract rich in rare saponins of the present invention is fermented by specific Lactobacillus plantarum, efficiently converting ginsenosides into CK and F1, and also retaining various natural plant components such as ginseng polysaccharides, resulting in a unique synergistic effect. It has been verified that the prepared ginseng extract has high bioavailability, can more effectively inhibit cell inflammation, more effectively restore the mitochondrial function of senescent cells and inhibit cell senescence. The preparation method provided by the present invention has high operability and is easy to standardize and intelligentize. The fermentation method adopted is more environmentally friendly and efficient than the traditional extraction and purification methods, and can achieve large-scale production in industrial production.

[0022] Other features and advantages of the present application will be described in the subsequent specification, and part of them will become obvious from the specification or be understood by implementing the present application. The objectives and other advantages of the present application can be realized and obtained by the written specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a comparison chart of the saponin composition before and after the fermentation of the ginseng extract.

[0024] Figure 2 It is a comparison chart of the bioavailability of each example and comparative example.

[0025] Figure 3It is a comparison chart of the toxicity test results of each example and comparative example on RAW264.7 cells.

[0026] Figure 4 It is a comparison chart of the anti-inflammatory activity test results of each example and comparative example on RAW264.7 cells.

[0027] Figure 5 It is a comparison chart of the toxicity test results of each example and comparative example on Hacat cells.

[0028] Figure 6 It is a comparison chart of the statistical results of the mitochondrial membrane potential of each example and comparative example on Hacat cells.

[0029] Figure 7 It is a comparison chart of the fluorescence staining results of the mitochondrial membrane potential of each example and comparative example on Hacat cells.

[0030] Figure 8 It is a comparison chart of the toxicity test results of each example and comparative example on HSF cells.

[0031] Figure 9 It is a comparison chart of the statistical results of the cell blue staining rate of each example and comparative example on HSF cells.

[0032] Figure 10 It is a comparison chart of the β-galactosidase staining results of each example and comparative example on HSF cells. Detailed implementation manners

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present disclosure clearer, the technical solutions of the embodiments of the present disclosure will be described clearly and completely below. Obviously, the described embodiments are some, but not all, of the embodiments of the present disclosure. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present disclosure without creative efforts fall within the scope of protection of the present invention.

[0034] It should be understood that, on the premise of no conflict, any and all implementation manners of the present invention can be combined with the technical features in any other implementation manner or multiple other implementation manners to obtain additional implementation manners. The present invention includes such implementation manners obtained by combination.

[0035] Unless otherwise specified, all technical terms and scientific terms used herein have the standard meanings in the field to which the claimed subject matter belongs. If there are multiple definitions for a certain term, the definition in this article shall prevail.

[0036] The embodiments of the present application disclose a ginseng extract rich in rare saponins, which is prepared by the following steps: S1: Freeze-dry fresh ginseng and powder it to obtain ginseng powder.

[0037] S2: Disperse ginseng powder in water, sterilize it, and cool it down to 37 °C to obtain a fermentation substrate.

[0038] S3: Inoculate the fermentation substrate with the seed liquid of Lactiplantibacillus plantarum, conduct fermentation culture, sterilize it after monitoring the completion of the reaction, and collect the supernatant to obtain a ginseng extract rich in rare saponins.

[0039] Specifically, in step S1, after powdering, it is sieved through a 20-mesh sieve. In step S2, the mass ratio of ginseng powder to water is 1 - 5:100; the sterilization requirement can be sterilization at 85 °C for 30 min. In step S3, the Lactiplantibacillus plantarum used is preserved in the Guangdong Provincial Microbial Culture Collection Center, with the GDMCC number being No. 64374; the concentration of the seed liquid is OD 600 = 0.8 - 2.0, and the volume ratio of the seed liquid to the fermentation substrate is 1:20 - 100; the temperature for fermentation culture is 30 °C - 50 °C, and the time is 8 h - 16 h; the way to monitor the reaction can be to monitor the reaction with HPLC (High Performance Liquid Chromatography); the sterilization temperature after monitoring the completion of the reaction is 85 °C - 110 °C, and the time is 15 min - 30 min. Preferably, the supernatant collected in step S3 can be further embedded, filtered, and dried.

[0040] The obtained ginseng extract rich in rare saponins has a rare saponin content of 0.2% - 2% and a ginseng polysaccharide content of 10% - 30%. The rare saponins include CK and F1, and the total amount of CK and F1 accounts for more than 99 wt% of the rare saponins, and the mass ratio of CK to F1 is 1:1 - 5.

[0041] This ginseng extract rich in rare saponins can be used to produce foods or skin care products with anti-aging functions. The foods include but are not limited to biscuits, bread, gummy candies, compressed tablets, and can also be health products such as capsules and oral liquids. The types of skin care products include but are not limited to facial cleansers, eye creams, facial masks, lotions, and creams. The addition ratio of the ginseng extract in the skin care products is 0.2% - 2%.

[0042] In the following examples, the raw materials, equipment, etc. used can be obtained by purchase except for special limitations.

[0043] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0044] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0045] The ginseng in the following examples is from the market.

[0046] The fermenting bacterium in the following examples is Lactobacillus plantarum subsp. plantarum, which is self-screened and preserved in the Guangdong Provincial Culture Collection of Microorganisms, with the GDMCC number being No. 64374 and the preservation date being February 5, 2024.

[0047] Example 1 Fresh ginseng (Panax ginseng) was freeze-dried, powdered, and sieved to obtain ginseng powder (20 mesh). 10 g of ginseng powder was taken and added to 500 g of water. The mass ratio of ginseng powder to water was 2:100. After mixing evenly, it was sterilized to obtain a fermentation substrate. 10.2 mL of Lactobacillus plantarum seed liquid was inoculated into 510 g of the fermentation substrate. The volume ratio of Lactobacillus plantarum seed liquid to the fermentation substrate was 1:50 to obtain a fermentation system. The fermentation system was placed in a shaker at 45 °C for 12 hours to obtain a fermentation product. The fermentation product was sterilized at 85 °C for 30 min to inactivate the bacteria, filtered, the precipitate was discarded, and the supernatant was collected to obtain ginseng extract.

[0048] Example 2 The difference between Example 2 and Example 1 is that the mass ratio of ginseng powder to water is 1:100, and the volume ratio of Lactobacillus plantarum seed liquid to the fermentation substrate is 1:100, and the other conditions are the same as those in Example 1.

[0049] Example 3 The difference between Example 3 and Example 1 is that the mass ratio of ginseng powder to water is 5:100, and the volume ratio of Lactobacillus plantarum seed liquid to the fermentation substrate is 1:20, and the other conditions are the same as those in Example 1.

[0050] Comparative Example 1 Fresh ginseng was freeze-dried, powdered, and sieved to obtain ginseng powder (20 mesh). 10 g of ginseng powder was taken and added to 500 g of water. The mass ratio of ginseng powder to water was 2:100. After mixing evenly, it was sterilized at 85 °C for 30 min, then placed in a shaker at 45 °C for 12 hours, filtered, the precipitate was discarded, and the supernatant was collected to obtain unfermented ginseng extract.

[0051] Liquid phase detection was carried out on the ginseng extracts prepared in Example 1, Example 2, Example 3, and Comparative Example 1 to determine the substance content in the ginseng extracts. The liquid phase conditions were as follows: Agilent 1100 Series; the chromatographic column was Diamonsil® C18 (250 mm × 4.6 mm × 5 μm); the detector was a DAD detector, the detection wavelength was 370 nm; the flow rate was 1 mL / min; the column temperature was 30 °C; the injection volume was 10 μL; the mobile phase was 0.5% acetic acid aqueous solution: acetonitrile = 73:27.

[0052] The detection results are asFigure 1 as shown in Table 1 Figure 1 At point A in it is Comparative Example 1, representing the saponin composition before the fermentation of ginseng extract, and at point B is Example 1, representing the saponin composition after the fermentation of ginseng extract. The results show that after the fermentation by Lactiplantibacillus plantarum, ginsenosides Rb1, Rb2, Rc, Rd, Re and Rg1 ( Figure 1 in which Re / Rg1 represents Re and Rg1, and the liquid phase detection under the above conditions cannot separate Re and Rg1 individually) are basically converted into ginsenosides CK and F1. This shows that under the fermentation cooperation of specific Lactiplantibacillus plantarum, the saponin components in ginseng can be directionally converted into rare saponins with higher biological activities. And the rare saponins obtained in the present invention only contain CK and F1.

[0053] Table 1: Determination results of rare saponins and ginseng polysaccharides in Examples 1 - 3 Sample CK content F1 content Polysaccharide content Example 1 0.25% 0.75% 15% Example 2 0.03% 0.17% 10% Example 3 1% 1% 30% As shown in Table 1, the content of rare saponins in the ginseng extract of Example 1 is 1%, the mass ratio of CK to F1 is 1:3 (the content of CK is 0.25% and the content of F1 is 0.75%), and the content of ginseng polysaccharides is 15%; the content of rare ginsenosides in Example 2 is 0.2%, the mass ratio of CK and F1 is about 1:5 (the content of CK is 0.03% and the content of F1 is 0.17%), and the content of ginseng polysaccharides is 10%; the content of rare saponins in the ginseng extract of Example 3 is 2%, the mass ratio of CK to F1 is 1:1 (the content of CK is 1% and the content of F1 is 1%), and the content of ginseng polysaccharides is 30%.

[0054] Next, a bioavailability test was carried out, and the specific implementation method is as follows.

[0055] Comparative Example 2 Prepare a 1% CK monomer solution (Aladdin, G170096).

[0056] Comparative Example 3 Prepare a 1% F1 monomer solution (Aladdin, G170877).

[0057] Comparative Example 4 Prepare a ginseng polysaccharide solution containing 15% (Guchen Biotech, GC51908).

[0058] Comparative Example 5 Prepare a compound solution, which contains a compound solution of 0.25% CK monomer (Aladdin, G170096), 0.75% F1 monomer (Aladdin, G170877), and 15% ginseng polysaccharide (Guchen Biotech, GC51908).

[0059] When the Caco-2 cells (ATCC cell bank) grow and reach 80 - 90% confluence, digest the cells, gently pipette to mix well and then count. Inoculate 0.5 mL of the cell suspension (2x10 5 )onto the transwell filter pores of a 12-well plate, and add 1.5 mL of the culture medium to the lower chamber of the Transwell filter pores. The composition of the culture medium is DMEM (Gibco, USA): fetal bovine serum (Gibco, USA): double antibody (Guangzhou Ruishu Biotechnology Co., Ltd.) = 9:1:0.1.

[0060] Replace the fresh culture medium every day to obtain differentiated monolayer cells. On the 11th, 13th, 15th, 17th, 19th and 21st days, measure the integrity of the Caco-2 cell monolayer by transepithelial electrical resistance (TEER) using a Millicell-ERS electrode.

[0061] When the TEER value reaches 400 Ω / cm 2 or above on the 21st day, use the monolayer membrane for further transport experiments.

[0062] Wash the filter holder twice with HBSS (pH = 6.8, 37°C) to remove the residual culture medium. Dissolve the samples (ginseng extracts obtained from Examples 1 to 3 and Comparative Example 1, and solutions prepared in Comparative Examples 2 to 5) in HBSS, with the sample addition amount being 4%, add to the upper chamber, and add 1 mL of HBSS (37°C) to the basolateral chamber.

[0063] At 30, 60, 90, 120 min, collect 0.6 mL of the sample from the lower chamber, and simultaneously supplement the same volume (0.6 mL) of HBSS in the receiving chamber.

[0064] Detect the content of the sample permeating into the lower chamber at different time periods by liquid chromatography, and evaluate the bioavailability with the permeability rate.

[0065] The results are as Figure 2 shown. The bioavailabilities of the ginseng extracts prepared in Examples 1, 2, and 3 are 36.26%, 30.74%, and 35.32% respectively, all higher than those of Comparative Examples 1 to 5, indicating that the ginseng extracts fermented by specific bacteria have unique and non-imitable advantages in bioavailability.

[0066] Evaluate the cytotoxicity of the ginseng extracts obtained in the examples and comparative examples on RAW264.7 macrophages (ATCC cell bank). The specific implementation scheme is as follows: Prepare fresh complete medium, DMEM (Gibco, USA): fetal bovine serum (Gibco, USA): double antibody (Guangzhou Ruishu Biotechnology Co., Ltd.) = 9:1:0.1. When RAW264.7 cells grow and reach 80 - 90% confluence, digest the cells, gently pipette and mix well, then count the cells. 4 Inoculate the cells at a density of 5×10

[0067] cells / well into a 96-well plate, 0.1 mL per well. After the cells adhere to the wall for 4 h, discard the culture supernatant, and add complete medium containing different concentrations of the sample (2%, 4%, 6%, 8%, 10%) to continue incubation for 24 h. According to the CCK-8 kit instructions, detect the cell viability. Figure 3 As shown Figure 3 in the figure, according to the 5 different sample concentrations of 2%, 4%, 6%, 8%, and 10%, 5 clusters of column charts are shown. In each cluster of the column chart, the 8 adjacent columns from left to right are Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5. The results show that the ginseng extracts prepared in Examples 1 to 3 have no toxicity to RAW264.7 cells within the range of 10%, and have the least toxicity at most concentrations compared with Comparative Examples 1 to 5, indicating that the ginseng extracts fermented by specific bacteria have higher safety. Subsequently, a 4% concentration is selected for subsequent experiments.

[0068] Evaluate the inflammation inhibition rate of the ginseng extracts obtained in the examples and comparative examples on RAW264.7 macrophages (ATCC cell bank). The specific implementation plan is as follows: Inoculate the cells at a density of 5×10 4 cells / well into a 96-well plate, 0.1 mL per well. Set the groups as blank control group, LPS model group, and sample groups with different concentrations. Each group has 5 replicates. After the cells adhere to the wall, except for the blank control group, the other groups are added with fresh cell complete medium containing 1 μg / mL LPS (Sigma, USA) to induce for 2 h to establish an inflammation model. Subsequently, discard the supernatant. The sample groups are added with fresh complete medium containing 4% sample, the DXM positive control group is added with fresh complete medium containing DXM (10 μg / mL), and the LPS model group and the blank control group are added with an equal amount of fresh complete medium to continue culturing. After incubation for 24 h, according to the Griess reagent instructions, measure the effect of each sample on the production of nitric oxide by RAW264.7 cells, and calculate the inflammation inhibition rate.

[0069] The results of the effect of ginseng extract on the production of NO by LPS-induced RAW264.7 macrophages are shown in Figure 4, the inflammation inhibition rates of the ginseng extracts prepared in Examples 1 to 3 were 93.85%, 80.43%, and 89.47% respectively, and the anti-inflammatory effect of the ginseng extract prepared in Example 1 was the best. The inflammation inhibition rates of Comparative Examples 1 to 5 were 50.4%, 62.4%, 72.5%, 40.2%, and 79.5% respectively. The anti-inflammatory effects of Examples 1 to 3 were better than those of Comparative Examples 1 to 5. The results of the comparison between Examples 1 to 3 and Comparative Example 1 showed that the anti-inflammatory effect of the ginseng extract fermented by the present invention was significantly improved. The results of Comparative Examples 2 to 5 showed that the anti-inflammatory effect of the ginseng extract prepared in the Examples was better than that of single-component ginseng polysaccharide, CK, F1, or a mixture of the three in the same proportion as Example 1, indicating that the ginseng extract fermented by the present invention had a better synergistic effect. The project team speculated that the fermentation process might not only convert protopanaxadiol saponins into rare ginseng saponins, but also produce some undetected active factors, whose effects could not be simulated by the current single component or compound.

[0070] Test the toxicity of the ginseng extract on Hacat cells (ATCC cell bank). The specific implementation plan is as follows: Prepare fresh complete medium, DMEM (Gibco, USA): fetal bovine serum (Gibco, USA): double antibody (Guangzhou Ruishu Biotechnology Co., Ltd.) = 9:1:0.1. When the Hacat cells grow and fuse to 80-90%, digest the cells, gently pipette and mix well, and then count. Inoculate the cells in a 96-well plate at a cell density of 1×10 4 cells / well, 0.1 mL per well. After the cells adhere for 24 h, discard the culture supernatant, add complete medium containing samples at different concentrations (2%, 4%, 6%, 8%, 10%) and continue to incubate for 24 h. According to the CCK-8 kit instructions, detect the cell viability.

[0071] As Figure 5 shown, Figure 5 According to the 5 sample concentrations of 2%, 4%, 6%, 8%, and 10%, 5 clusters of column charts are shown. In each cluster of the column charts, the 8 adjacent columns from left to right are Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5 in turn. The results show that the maximum safe concentrations of Examples 1 to 3 are 8%, 10%, and 8% respectively. The low toxicity of Example 2 may be due to the lower solute concentration. The maximum safe concentrations of Comparative Examples 1 to 4 are 4%, 4%, 8%, and 6% respectively. It can be seen that the toxicity of single-component F1 and CK is relatively large. The ginseng extracts and ginseng polysaccharides prepared in Examples 1 to 3 have relatively low toxicity to cells. Subsequently, an addition amount of 4% is selected for the experiment.

[0072] To test the effect of ginseng extract on the mitochondrial membrane potential of senescent Hacat cells, the specific implementation plan is as follows: When Hacat cells grow and reach 80 - 90% confluence, digest the cells, gently pipette to mix well and then count. Inoculate the cells into a 96 - well plate at a cell density of 1×10 4 cells / well, 0.1 mL per well. Set the groups as blank control group, H 2 O 2 model group and sample groups, with 3 replicates in each group. After the cells adhere to the wall, except for the blank control group and the model group, add fresh complete cell medium containing different samples at a concentration of 4% to the other groups for pretreatment for 2 h, and then add H 2 O 2 (1000 μM) to induce cell senescence. After 4 h, remove the medium in each well, wash three times with PBS, and then stain according to the JC - 10 kit (Maclean) instructions. Count the blue fluorescence and green fluorescence respectively, and calculate the relative mitochondrial membrane potential (Relative mitochondrial membrane potential = ).

[0073] Oxidative damage can lead to a decrease in mitochondrial membrane potential, reduce mitochondrial energy production, and cause skin aging. When the mitochondrial membrane potential is high (abundant mitochondrial energy), it shows red at this time; when the mitochondrial membrane potential is low (insufficient mitochondrial energy), it shows green at this time. As Figure 6 (Comparison between the sample group and the model group: **** (P < 0.0001) indicates extremely significant difference; *** (P < 0.001) indicates significant difference; ** (P < 0.01) indicates significant difference; * (P < 0.05) indicates significant difference. Comparison between the model group and the blank group: # (P < 0.0001) indicates extremely significant difference; (P < 0.001) indicates significant difference; ## (P < 0.01) indicates significant difference; # (P < 0.05) indicates significant difference) and Figure 7 shown, H 2 O 2After injury, the mitochondrial membrane potential decreased significantly to 65.6%. The relative mitochondrial membrane potentials of Examples 1 to 3 were restored to 94.2%, 85.5%, and 90.1% respectively. Among them, the ginseng extract described in Example 1 had the best effect. The relative mitochondrial membrane potentials of Comparative Examples 1 to 5 were restored to 70.1%, 73.2%, 79.1%, 68.5%, and 85.1% respectively at the same concentration. The effects of Examples 1 to 3 were all better than those of Comparative Examples 1 to 5. It can be seen that the ginseng extract obtained by fermentation of the present invention has significantly improved the effect on mitochondrial anti-aging. The effects of single components of ginseng polysaccharide, F1, and CK on mitochondrial anti-aging were average. After the polysaccharide was mixed with rare ginseng saponins F1 and CK, although the activity was improved to a certain extent, the effect was still inferior to the ginseng extract obtained by fermentation with specific bacteria, indicating that the ginseng extract prepared in the examples has an irreplaceable advantage in mitochondrial anti-aging effect.

[0074] The toxicity of the ginseng extract to HSF cells was tested, and the specific implementation plan is as follows: When human skin fibroblasts HSF (ATCC cell bank) grew and reached 80-90% confluence, the cells were digested, gently pipetted and mixed evenly, and then counted. Inoculate into a 96-well plate at a cell density of 1×10 4 cells / well, 0.1 mL per well. After the cells adhered for 24 h, discard the culture supernatant, add the HSF special medium (Guangzhou Huatuo Biotechnology Co., Ltd.) containing different concentrations of the sample (2%, 4%, 6%, 8%, 10%), continue to incubate for 24 h, and detect the cell viability according to the CCK-8 kit instructions.

[0075] As Figure 8 shown, Figure 8 According to the 5 different sample concentrations of 2%, 4%, 6%, 8%, and 10%, 5 clusters of column charts are shown. In each cluster of the column chart, the 8 adjacent columns from left to right are Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5 in turn. The results show that the maximum safe concentration of Examples 1 to 3 is 8%, and the maximum safe concentrations of Comparative Examples 1 to 5 are 4%, 4%, 4%, 8%, and 6% respectively. The toxicity of the examples to HSF cells is less than that of Comparative Examples 1 to 5, and 4% is selected for subsequent experiments.

[0076] The anti-aging effect of the ginseng extract on HSF cells was tested, and the specific implementation plan is as follows: When HSF cells grew and reached 80-90% confluence, the cells were digested, gently pipetted and mixed evenly, and then counted. Inoculate into a 96-well plate at a cell density of 1×10 4 cells / well, 0.1 mL per well. Set the grouping as blank control group, H 2 O 2The model group and the sample group were each set with 3 replicate wells. After the cells adhered to the wall, except for the blank control group and the model group, the remaining groups were pretreated with fresh complete cell medium containing 4% of different samples for 2 h, and then H 2 O 2 (200 μM) was added to induce cell senescence. After 4 h, the culture medium in each well was removed, and the cells were washed once with PBS. Then, 100 μL of fixative was added to each well and fixed at room temperature for 15 min. The cells were washed 3 times with PBS solution (3 min each time), and then 100 μL of staining working solution was added to each well. The wells were sealed and incubated overnight at 37 °C (without CO 2 ). The cells were observed and photographed randomly under a microscope at 200× magnification, and the blue staining rate was calculated.

[0077] As Figure 9 and Figure 10 shown, the blue staining rate of the normal group was 15.45%. In the hydrogen peroxide group, the cells were significantly senescent and atrophied, and 84.83% of the cells were stained blue-green, indicating a high degree of senescence. The blue staining rates of the cells in Examples 1 to 3 were reduced to 26.11%, 39.21%, and 30.16%, respectively. Among them, the ginseng extract prepared in Example 1 had the best effect. The blue staining rates of the cells in Comparative Examples 1 to 5 were 69.2%, 60.4%, 50.11%, 75.13%, and 40.83%, respectively. The effects of the examples were significantly better than those of the comparative examples. The cell anti-aging effects of the ginseng extract before fermentation, single-component ginseng polysaccharide, F1, and CK were average. After the polysaccharide was mixed with rare ginseng saponins F1 and CK, although the activity was improved to a certain extent, the effect was still not as good as that of the ginseng extract obtained by fermentation with specific bacteria. This indicates that the ginseng extract prepared by the present invention has an irreplaceable advantage in cell anti-aging effects.

[0078] The ginseng extract of the present invention is prepared by fermenting ginseng with Lactiplantibacillus plantarum (self-screened and preserved in the Guangdong Provincial Microbial Culture Collection Center, GDMCC No. 64374) through fermentation technology, which converts protopanaxadiol saponins in ginseng into rare saponins CK and F1. The content ratio of CK to F1 is 1:1 to 5, and natural plant components such as ginseng polysaccharide are retained. The ginseng extract prepared by the present invention not only has a higher bioavailability but also has an irreplaceable unique advantage in biological activity. It can more effectively inhibit cell inflammation, more effectively restore the mitochondrial function of senescent cells and inhibit cell senescence, and can be used in the preparation of foods and skin care products.

[0079] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. A ginseng extract rich in rare saponins, characterized in that: The product is obtained by fermenting ginseng powder with Lactobacillus plantarum, and the preservation number of the Lactobacillus plantarum is GDMCC No.64374.

2. The ginseng extract rich in rare saponins according to claim 1, characterized in that The content of rare saponins is 0.2%~2%, and the content of ginseng polysaccharides is 10%~30%.

3. The ginseng extract rich in rare saponins according to claim 1, characterized in that: The rare saponins include CK and F1, the total amount of the CK and the F1 accounts for more than 99wt% of the rare saponins, and the mass ratio of the CK to the F1 is 1:1-5.

4. A method for preparing a ginseng extract rich in rare saponins, characterized in that: The ginseng powder is fermented by using Lactobacillus plantarum, and the preservation number of the Lactobacillus plantarum is GDMCC No.64374.

5. The method for preparing a ginseng extract rich in rare saponins according to claim 4, characterized in that the steps include: freeze-drying fresh ginseng and grinding it into powder to obtain the ginseng powder; Dispersing the ginseng powder in water, sterilizing, and cooling to 37° C. to obtain a fermentation substrate; The seed liquid of the Lactobacillus plantarum is inoculated into the fermentation substrate, fermented and cultured, sterilized after monitoring the reaction is complete, and the supernatant is collected to obtain the ginseng extract rich in rare saponins.

6. The method for preparing the ginseng extract rich in rare saponins according to claim 5, characterized in that: In the step of dispersing the ginseng powder in water, the mass ratio of the ginseng powder to the water is 1-5:

100.

7. The method for preparing the ginseng extract rich in rare saponins according to claim 5, characterized in that: The concentration of the seed solution is OD 600 =0.8~2.0, and the volume ratio of the seed liquid to the fermentation substrate is 1:20~100.

8. The method for preparing the ginseng extract rich in rare saponins according to claim 5, characterized in that: The fermentation culture temperature is 30°C to 50°C, and the fermentation culture time is 8h to 16h.

9. The method for preparing the ginseng extract rich in rare saponins according to claim 5, characterized in that: The sterilization temperature for sterilization after the monitoring reaction is complete is 85° C. to 110° C., and the time is 15 min to 30 min.

10. The use of the ginseng extract rich in rare saponins according to any one of claims 1 to 3, characterized in that: Used to make food or skin care products with anti-aging function.

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