Ginseng extract rich in rare saponins, preparation method thereof and application in anti-aging

Abstract

The present invention discloses a ginseng extract rich in rare saponins, its preparation and anti-aging application, and belongs to the technical field of skin care products. The ginseng extract is obtained by fermenting ginseng powder with Lactobacillus plantarum, and the deposit number of Lactobacillus plantarum is GDMCC No. 64374. The ginseng extract rich in rare saponins of the present invention is fermented by a specific Lactobacillus plantarum, and the ginsenosides are efficiently converted into CK and F1, and various natural plant components such as ginseng polysaccharides are retained, resulting in a unique synergistic effect. It has been verified that the prepared ginseng extract has high bioavailability and can more effectively inhibit cell inflammation, more effectively restore the mitochondrial function of aging cells and inhibit cell aging. The preparation method provided by the present invention is highly operable and easy to standardize and intelligentize. The fermentation method adopted is more environmentally friendly and efficient than the traditional extraction and purification method, and can achieve large-scale production in industrial production.

Description

Technical Field

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

[0002] The skin, the largest organ of the body exposed to the external environment, is susceptible to aging due to both internal and external factors. The main characteristics of skin aging are sagging and wrinkling, which not only affect appearance but also can impact a person's mental health and social interactions. In recent years, anti-aging health and skincare products have emerged, but they all suffer from unsatisfactory anti-aging effects and difficulties in achieving the effects of their active ingredients. Therefore, there is an urgent need to develop a safe, non-irritating, and effective anti-aging raw material.

[0003] Existing research has found that ginsenosides can promote collagen synthesis and improve skin elasticity and radiance, offering broad potential applications in healthcare products, cosmetics and skincare, supplementary foods, and daily necessities. However, conventionally extracted ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) account for over 80% of the total ginsenosides, while secondary saponins F1 and CK contribute significantly less. These saponins have relatively low bioavailability and poor bioactivity. In contrast, secondary saponins are better absorbed and utilized. Therefore, developing suitable processing techniques to convert ginsenosides into relatively bioavailable secondary saponins and aglycones while preserving ginseng's other naturally active components, thereby maximizing the bioavailability and bioactivity of ginseng's active ingredients, is crucial for ginseng deep processing. Summary of the Invention

[0004] In order to overcome the shortcomings of the existing technology, the present invention provides a ginseng extract rich in rare saponins, its preparation and application in anti-aging, which can convert most of the saponin prototype components into secondary saponins, and the conversion product has higher bioavailability and lower cytotoxicity.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] In a first aspect, the present application provides a ginseng extract rich in rare saponins, obtained by fermenting ginseng powder with Lactobacillus plantarum, the Lactobacillus plantarum having a deposit number of GDMCC No. 64374. This Lactobacillus plantarum is deposited with the Guangdong Provincial Microbiological Culture Collection, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, under the deposit number GDMCC No. 64374 and a deposit date of February 5, 2024.

[0007] The ginseng extract provided by this application not only contains higher concentrations of rare ginsenosides F1 and CK, but the various plant components contained in the ginseng extract can also produce stronger synergistic effects. Compared with single ingredients of ginseng polysaccharides, F1, CK or a mixture of the three, the ginseng extract fermented in this application has higher bioavailability and lower cytotoxicity, can more effectively inhibit cellular inflammation, more effectively restore the mitochondrial function of aging cells and inhibit cell aging.

[0008] Furthermore, based on mass, the rare saponin content is 0.2%~2%, and the ginseng polysaccharide content is 10%~30%.

[0009] Furthermore, the rare saponin includes CK and F1, the total amount of the CK and the F1 accounts for more than 99 wt % of the rare saponin, and the mass ratio of the CK to the F1 is 1:1-5.

[0010] In a second aspect, the present application provides a method for preparing a ginseng extract rich in rare saponins, wherein ginseng powder is fermented with Lactobacillus plantarum, the deposit number of which is GDMCC No. 64374.

[0011] Using a specific Lactobacillus plantarum fermentation method, ginseng prototype saponins can be efficiently converted into rare saponins while retaining other active ingredients of ginseng, ensuring the high bioavailability and low cytotoxicity of the extract, making it suitable for the development of anti-aging products.

[0012] The steps include:

[0013] freeze-drying fresh ginseng and grinding it into powder to obtain the ginseng powder;

[0014] Dispersing the ginseng powder in water, sterilizing it, and cooling it to 37° C. to obtain a fermentation substrate;

[0015] The seed liquid of the Lactobacillus plantarum is inoculated into the fermentation substrate, fermented and cultured, sterilized after monitoring the reaction to be complete, and the supernatant is collected to obtain the ginseng extract rich in rare saponins.

[0016] Furthermore, in the step of dispersing the ginseng powder in water, the mass ratio of the ginseng powder to the water is 1-5:100.

[0017] Controlling the ratio of ginseng powder to water ensures the appropriate concentration of the fermentation substrate, which not only guarantees an adequate supply of nutrients during the fermentation process, but also avoids the effects of too high or too low concentration on fermentation efficiency and product quality.

[0018] Furthermore, 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.

[0019] Controlling the concentration of the seed liquid and the inoculation ratio ensures the activity and fermentation efficiency of the bacteria during the fermentation process, and avoids problems such as incomplete fermentation or unstable product quality caused by too many or too few bacteria.

[0020] Furthermore, the fermentation culture temperature is 30°C to 50°C, and the fermentation time is 8h to 16h.

[0021] Controlling the fermentation temperature and culture time ensures that Lactobacillus plantarum ferments under optimal conditions, maximizing the conversion of ginseng prototype saponins into rare saponins, while avoiding the degradation of active ingredients due to excessive time.

[0022] Furthermore, 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.

[0023] Controlling the sterilization temperature and time ensures the sterility of the fermentation product, while avoiding the loss of active ingredients caused by high temperature or long-term sterilization, and ensuring the stability and safety of the extract.

[0024] In a third aspect, the present application provides the use of the ginseng extract rich in rare saponins described in the first aspect for preparing skin care products with anti-aging function.

[0025] The beneficial effects of the present invention are as follows: the ginseng extract rich in rare saponins of the present invention is fermented by a specific Lactobacillus plantarum, efficiently converting ginsenosides into CK and F1, while also retaining various natural plant components such as ginseng polysaccharides, resulting in a unique synergistic effect. It has been verified that the obtained ginseng extract has high bioavailability and can more effectively inhibit cellular inflammation, more effectively restore mitochondrial function of senescent cells, and inhibit cell aging. The preparation method provided by the present invention is highly operational and easy to standardize and intelligentize. The fermentation method used is more environmentally friendly and efficient than traditional extraction and purification methods, and can achieve large-scale production in industrial production.

[0026] Other features and advantages of the present application will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present application. The purpose and other advantages of the present application can be realized and obtained through the written description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a comparison chart of the saponin composition of ginseng extract before and after fermentation.

[0028] Figure 2 It is a comparison chart of bioavailability of various embodiments and comparative examples.

[0029] Figure 3 It is a comparison chart of the toxicity test results of various embodiments and comparative examples on RAW264.7 cells.

[0030] Figure 4 It is a comparison chart of the anti-inflammatory activity experimental results of various examples and comparative examples on RAW264.7 cells.

[0031] Figure 5 It is a comparison chart of the toxicity test results of each embodiment and comparative example on Hacat cells.

[0032] Figure 6 It is a comparison chart of the statistical results of mitochondrial membrane potential of Hacat cells in various embodiments and comparative examples.

[0033] Figure 7 It is a comparison diagram of the mitochondrial membrane potential fluorescence staining results of Hacat cells in various embodiments and comparative examples.

[0034] Figure 8 It is a comparison chart of the toxicity test results of various embodiments and comparative examples on HSF cells.

[0035] Figure 9 It is a comparison chart of the statistical results of the blue staining rate of HSF cells in various examples and comparative examples.

[0036] Figure 10 It is a comparison chart of the β-galactosidase staining results of HSF cells in various examples and comparative examples. DETAILED DESCRIPTION

[0037] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions of the embodiments of the present disclosure are clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] It should be understood that, under the premise of no conflict, any and all embodiments of the present invention can be combined with the technical features in any other embodiment or multiple other embodiments to obtain additional embodiments. The present invention includes such combinations to obtain additional embodiments.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the standard meanings in the art to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0040] The present invention discloses a ginseng extract rich in rare saponins, which is prepared by the following steps:

[0041] S1: freeze-drying fresh ginseng and grinding it into powder to obtain ginseng powder.

[0042] S2: Dispersing ginseng powder in water, sterilizing, and cooling to 37° C. to obtain a fermentation substrate.

[0043] S3: inoculating the seed liquid of Lactobacillus plantarum into the fermentation substrate, fermenting and culturing, monitoring the reaction to be complete and then sterilizing, collecting the supernatant, and obtaining a ginseng extract rich in rare saponins.

[0044] Specifically, in step S1, the powder 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 may be to place it at 85°C for 30 minutes. In step S3, the plant lactobacillus used is stored in the Guangdong Provincial Microbial Culture Collection Center, GDMCC No. 64374; the concentration of the seed liquid is OD 600 = 0.8-2.0, the volume ratio of seed liquid to fermentation substrate is 1:20-100; the fermentation temperature is 30°C-50°C, and the fermentation time is 8-16 hours; the reaction can be monitored by HPLC (high performance liquid chromatography); after monitoring the reaction completion, the sterilization temperature is 85°C-110°C, and the time is 15-30 minutes. Preferably, the supernatant collected in step S3 can be further embedded, filtered, and dried.

[0045] The obtained ginseng extract is rich in rare saponins, with a rare saponin content of 0.2%~2% and a ginseng polysaccharide content of 10%~30%. The rare saponins include CK and F1, the total amount of CK and F1 accounts for more than 99wt% of the rare saponins, and the mass ratio of CK to F1 is 1:1~5.

[0046] This ginseng extract, rich in rare saponins, can be used to make skin care products with anti-aging properties. These products include, but are not limited to, facial cleansers, eye creams, facial masks, lotions, and face creams. The ginseng extract can be added to these skin care products at a ratio of 0.2% to 2%.

[0047] Unless otherwise specified, the raw materials, equipment, etc. used in the following examples can be obtained by purchase.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0049] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0050] The ginseng in the following examples is commercially available.

[0051] The fermentation bacteria in the following examples are Lactobacillus plantarum subsp. plantarum, which was independently screened and deposited in Guangdong Provincial Microbiological Culture Collection Center with GDMCC No. 64374 and a preservation date of February 5, 2024. Example 1

[0052] Fresh ginseng (Panax ginseng) was freeze-dried, powdered, and sieved to obtain ginseng powder (20 mesh). 10g of ginseng powder was added to 500g of water at a mass ratio of 2:100. The mixture was then sterilized to obtain a fermentation substrate. 10.2mL of Lactobacillus plantarum seed solution was inoculated into 510g of the fermentation substrate at a volume ratio of 1:50. The fermentation system was then placed in a shaker at 45°C for 12 hours to obtain a fermentation product. The fermentation product was then sterilized at 85°C for 30 minutes to inactivate the bacteria. The mixture was then filtered, the precipitate discarded, and the supernatant collected to obtain the ginseng extract. Example 2

[0053] The difference between Example 2 and Example 1 is that the mass ratio of ginseng powder to water is 1:100, the volume ratio of Lactobacillus plantarum seed liquid to fermentation substrate is 1:100, and the other conditions are the same as those of Example 1. Example 3

[0054] The difference between Example 3 and Example 1 is that the mass ratio of ginseng powder to water is 5:100, the volume ratio of Lactobacillus plantarum seed liquid to fermentation substrate is 1:20, and the other conditions are the same as those in Example 1.

[0055] Comparative Example 1

[0056] Fresh ginseng was freeze-dried, pulverized, and sieved to obtain ginseng powder (20 mesh). 10g of ginseng powder was added to 500g of water at a mass ratio of 2:100. After mixing thoroughly, the mixture was sterilized at 85°C for 30 minutes, then immersed in a shaker at 45°C for 12 hours. The mixture was filtered, the precipitate discarded, and the supernatant collected to obtain the unfermented ginseng extract.

[0057] Liquid chromatography was performed on the ginseng extracts prepared in Examples 1, 2, 3, and Comparative Example 1 to determine the content of the substances in the ginseng extracts. The liquid chromatography conditions were: Agilent 1100 Series; Diamonsil® C18 column (250 mm × 4.6 mm × 5 μm); DAD detector at a detection wavelength of 370 nm; flow rate of 1 mL / min; column temperature of 30°C; injection volume of 10 μL; mobile phase of 0.5% aqueous acetic acid:acetonitrile (73:27).

[0058] Test results such as Figure 1 and as shown in Table 1. Figure 1 A is comparative example 1, which shows the saponin composition of ginseng extract before fermentation, and B is example 1, which shows the saponin composition of ginseng extract after fermentation. The results show that after fermentation by Lactobacillus plantarum, ginsenosides Rb1, Rb2, Rc, Rd, Re and Rg1 ( Figure 1 Re / Rg1 represents Re and Rg1. Liquid chromatography analysis under the above conditions cannot separate Re and Rg1 alone, but they are essentially converted into ginsenosides CK and F1. This demonstrates that fermentation with a specific Lactobacillus plantarum strain can selectively convert ginsenosides in ginseng into rare saponins with higher biological activity. The rare saponins obtained in this invention contain only CK and F1.

[0059] Table 1: Results of determination of rare saponins and ginseng polysaccharides in Examples 1 to 3

[0060] As shown in Table 1, the rare saponin content of the ginseng extract of Example 1 is 1%, the mass ratio of CK to F1 is 1:3 (CK content is 0.25%, F1 content is 0.75%), and the ginseng polysaccharide content is 15%; the rare ginsenoside content of Example 2 is 0.2%, the mass ratio of CK to F1 is about 1:5 (CK content is 0.03%, F1 content is 0.17%), and the ginseng polysaccharide content is 10%; the rare saponin content of the ginseng extract of Example 3 is 2%, the mass ratio of CK to F1 is 1:1 (CK content is 1%, F1 content is 1%), and the ginseng polysaccharide content is 30%.

[0061] Next, a bioavailability test was performed, and the specific implementation method was as follows.

[0062] Comparative Example 2

[0063] Prepare a 1% CK monomer solution (Aladdin, G170096).

[0064] Comparative Example 3

[0065] Prepare a 1% F1 monomer solution (Aladdin, G170877).

[0066] Comparative Example 4

[0067] A 15% ginseng polysaccharide solution (Guchen Biotechnology, GC51908) was prepared.

[0068] Comparative Example 5

[0069] A compound solution was prepared, which contained 0.25% CK monomer (Aladdin, G170096), 0.75% F1 monomer (Aladdin, G170877), and 15% ginseng polysaccharide solution (Guchen Bio, GC51908).

[0070] When Caco-2 cells (ATCC cell bank) were grown and confluent to 80-90%, the cells were digested, mixed by gentle pipetting, and then counted. 0.5 mL of cell suspension (2x10 5 ) onto the filter wells of a 12-well transwell plate. Add 1.5 mL of culture medium to the lower chamber of the transwell. The culture medium composition is DMEM (Gibco, USA): fetal bovine serum (Gibco, USA): double-stranded antibody (Guangzhou Ruishu Biological Co., Ltd.) at a ratio of 9:1:0.1.

[0071] Fresh culture medium was replaced daily to obtain differentiated cell monolayers. Caco-2 cell monolayer integrity was measured by epithelial electrical resistance (TEER) using Millicell-ERS electrodes on days 11, 13, 15, 17, 19, and 21.

[0072] When the TEER value reached 400Ω / cm on the 21st day 2 When the above values ​​are obtained, further transport experiments were performed using single-layer membranes.

[0073] The filter holder was washed twice with HBSS (pH 6.8, 37°C) to remove residual culture medium. Samples (ginseng extracts from Examples 1 to 3, Comparative Example 1, and solutions prepared in Comparative Examples 2 to 5) were dissolved in HBSS at a 4% concentration and added to the upper chamber. 1 mL of HBSS (37°C) was added to the basolateral chamber.

[0074] At 30, 60, 90, and 120 min, 0.6 mL of sample was collected from the lower chamber, and the receiving chamber was supplemented with the same volume (0.6 mL) of HBSS.

[0075] Liquid chromatography was used to detect the content of samples permeated into the lower chamber at different time periods, and the bioavailability was evaluated by permeability.

[0076] The results are as follows Figure 2 As shown, the bioavailability of the ginseng extracts prepared in Example 1, Example 2, and Example 3 were 36.26%, 30.74%, and 35.32%, respectively, which were all higher than the bioavailabilities of Comparative Examples 1 to 5, indicating that the ginseng extracts fermented by specific bacteria have unique advantages in bioavailability that cannot be simulated.

[0077] The cytotoxicity of the ginseng extracts obtained in the examples and comparative examples on RAW264.7 macrophages (ATCC cell bank) was evaluated. The specific implementation scheme is as follows:

[0078] Prepare fresh complete culture medium: DMEM (Gibco, USA): fetal bovine serum (Gibco, USA): double antibody (Guangzhou Ruishu Biological Co., Ltd.) = 9:1:0.1. When RAW264.7 cells grow and become 80-90% confluent, digest the cells, gently pipette to mix, and then count. 4 Cells were seeded at a density of 100 cells / well in a 96-well plate, with 0.1 mL per well. After 4 hours of cell attachment, the culture supernatant was discarded and complete culture medium containing various concentrations of the sample (2%, 4%, 6%, 8%, and 10%) was added for a further 24 hours of incubation. Cell viability was assessed according to the CCK-8 kit instructions.

[0079] The results are as follows Figure 3 As shown, Figure 3 Five clusters of bar graphs are presented, corresponding to five different sample concentrations: 2%, 4%, 6%, 8%, and 10%. The eight adjacent bars in each cluster, from left to right, represent Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5. The results indicate that the ginseng extracts prepared in Examples 1 through 3 exhibited no toxicity to RAW264.7 cells within the 10% concentration range. Compared to Comparative Examples 1 through 5, the toxicity was minimal at most concentrations, demonstrating the safety of ginseng extracts fermented with specific bacteria. A 4% concentration was selected for subsequent experiments.

[0080] The inflammatory inhibition rate of the ginseng extracts obtained in the examples and comparative examples on RAW264.7 macrophages (ATCC cell bank) was evaluated. The specific implementation scheme is as follows:

[0081] According to 5×10 4 Cells were seeded at a density of 100 μg / well in a 96-well plate, with 0.1 mL per well. Groups were divided into a blank control group, an LPS model group, and groups with different concentrations of each sample, with five replicates per group. After cell attachment, all groups except the blank control group were treated with fresh complete cell culture medium containing 1 μg / mL LPS (Sigma, USA) for 2 hours to establish an inflammatory model. The supernatant was then discarded, and each sample group was treated with fresh complete culture medium containing 4% of the sample. The DXM positive control group was treated with fresh complete culture medium containing 10 μg / mL DXM. The LPS model group and the blank control group were treated with an equal volume of fresh complete culture medium and continued to culture. After 24 hours of incubation, the effect of each sample on nitric oxide production in RAW264.7 cells was determined according to the Griess reagent manufacturer's instructions, and the inflammation inhibition rate was calculated.

[0082] The results of the effect of ginseng extract on LPS-induced NO production in RAW264.7 macrophages are shown in Figure 4The inflammation inhibition rates of the ginseng extracts prepared in Examples 1 to 3 were 93.85%, 80.43%, and 89.47%, respectively. The ginseng extract prepared in Example 1 had the best anti-inflammatory effect. 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 superior to those of Comparative Examples 1 to 5. The results of Examples 1 to 3 compared with Comparative Example 1 show that the anti-inflammatory effect of the ginseng extracts fermented by the present invention has been significantly improved. The results of Comparative Examples 2 to 5 show that the anti-inflammatory effect of the ginseng extracts prepared in the examples is superior to that of single components ginseng polysaccharides, CK, F1, or a mixture of the three in the same proportion as in Example 1, indicating that the fermented ginseng extracts of the present invention have better synergistic effects. The project team speculates that the fermentation process may not only convert ginsenosides into rare ginsenosides, but also produce some undetected active factors, the effects of which cannot be simulated by a single component or compound.

[0083] The toxicity of ginseng extract to Hacat cells (ATCC cell bank) was tested. The specific implementation plan is as follows:

[0084] Prepare fresh complete culture medium: DMEM (Gibco, USA): fetal bovine serum (Gibco, USA): double antibody (Guangzhou Ruishu Biological Co., Ltd.) = 9:1:0.1. When Hacat cells grow and become 80-90% confluent, digest the cells, gently pipette to mix, and then count. 4 Cells were seeded at a density of 100 cells / well in a 96-well plate, with 0.1 mL per well. After 24 hours of cell attachment, the culture supernatant was discarded and complete culture medium containing various sample concentrations (2%, 4%, 6%, 8%, and 10%) was added for a further 24 hours of incubation. Cell viability was then assessed according to the CCK-8 kit instructions.

[0085] like Figure 5 As shown, Figure 5 Five clusters of bar graphs are shown according to the five different sample concentrations of 2%, 4%, 6%, 8%, and 10%. The eight adjacent columns in each cluster of bar graphs are Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5 from left to right. 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 low 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 components F1 and CK is relatively large, and the ginseng extracts and ginseng polysaccharides prepared in Examples 1 to 3 are less toxic to cells. The subsequent addition amount of 4% was selected for experiments.

[0086] The effect of ginseng extract on mitochondrial membrane potential of aging Hacat cells was tested. The specific implementation plan is as follows:

[0087] When Hacat cells grow and become 80-90% confluent, digest the cells, mix them by gently pipetting, and then count them. 4 The cells were seeded at a density of 1000 μg / well in a 96-well plate, with 0.1 mL per well. The groups were divided into blank control group, H2O2 model group and sample group, with 3 replicates in each group. After the cells adhered to the wall, fresh complete cell culture medium containing 4% concentrations of different samples was added to the other groups except the blank control group and model group for pretreatment for 2 hours, and then H2O2 (1000 μM) was added to induce cell senescence. After 4 hours, the culture medium of each well was removed, and the cells were washed three times with PBS, and then stained according to the instructions of the JC-10 kit (McLean). The blue fluorescence and green fluorescence were counted respectively, and the relative mitochondrial membrane potential (relative mitochondrial membrane potential = ).

[0088] Oxidative damage can cause a decrease in mitochondrial membrane potential, reduce mitochondrial energy production, and cause skin aging. When the mitochondrial membrane potential is high (mitochondrial energy is strong), it appears red; when the mitochondrial membrane potential is low (mitochondrial energy is insufficient), it appears green. Figure 6 (Comparison between sample and model group: **** (P < 0.0001) has extremely significant difference; *** (P < 0.001) has significant difference; ** (P < 0.01) has significant difference; * (P < 0. 05) has significant difference. Comparison between model group and blank group: #### (P < 0.0001) has extremely significant difference; ### (P < 0.001) has significant difference; ## (P < 0.01) has significant difference; # (P < 0.05) has significant difference) and Figure 7 As shown, after H2O2 damage, the mitochondrial membrane potential dropped significantly to 65.6%, and the relative mitochondrial membrane potential of Examples 1 to 3 recovered 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 potential of Comparative Examples 1 to 5 recovered to 70.1%, 73.2%, 79.1%, 68.5%, and 85.1%, respectively, at the same concentration. The effects of Examples 1 to 3 are better than those of Comparative Examples 1 to 5, which shows that the ginseng extract obtained by fermentation of the present invention has been significantly improved in terms of mitochondrial anti-aging effect. The effects of single-ingredient ginseng polysaccharides, F1, and CK on mitochondrial anti-aging are general. After the polysaccharide is mixed with ginseng rare saponins F1 and CK, although the activity has been improved to a certain extent, the effect is still not as good as the ginseng extract obtained by fermentation of specific bacteria, indicating that the ginseng extract obtained by the embodiment has an unsimulatable advantage in the mitochondrial anti-aging effect.

[0089] The toxicity of ginseng extract to HSF cells was tested. The specific implementation plan is as follows:

[0090] When human skin fibroblasts HSF (ATCC cell bank) were grown and confluent to 80-90%, the cells were digested, gently pipetted to mix, and then counted. 4 Cells were seeded at a density of 100 cells / well in a 96-well plate, with 0.1 mL per well. After 24 hours of cell attachment, the culture supernatant was discarded and HSF-specific culture medium (Guangzhou Huatuo Biotechnology Co., Ltd.) containing different concentrations of the sample (2%, 4%, 6%, 8%, and 10%) was added. The cells were incubated for another 24 hours and cell viability was measured according to the CCK-8 kit instructions.

[0091] like Figure 8 As shown, Figure 8 Five clusters of bar graphs are presented based on five different sample concentrations: 2%, 4%, 6%, 8%, and 10%. The eight adjacent bars in each cluster, from left to right, represent 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 maximum safe concentration for Examples 1 through 3 is 8%, while that for Comparative Examples 1 through 5 is 4%, 4%, 4%, 8%, and 6%, respectively. The Examples were less toxic to HSF cells than Comparative Examples 1 through 5, and 4% was selected for subsequent experiments.

[0092] The anti-aging effect of ginseng extract on HSF cells was tested. The specific implementation plan is as follows:

[0093] When HSF cells grow and become 80-90% confluent, digest the cells, mix them by gently pipetting, and then count them. 4 Cells were seeded at a density of 100 μL / well in a 96-well plate, with 0.1 mL per well. The plates were divided into blank control, H2O2 model, and sample groups, with triplicate wells per group. After cell attachment, all groups, except the blank control and model groups, were pretreated with fresh complete cell culture medium containing 4% of the various samples for 2 hours. Cell senescence was then induced by the addition of H2O2 (200 μM). After 4 hours, the medium was removed from each well, and the plates were washed once with PBS. 100 μL of fixative solution was then added to each well, fixed at room temperature for 15 minutes, and washed three times with PBS (3 minutes each). Then, 100 μL of staining solution was added to each well, sealed, and incubated overnight at 37°C (no CO2). Randomly selected fields of view were photographed under a 200x microscope, and the blue staining rate was calculated.

[0094] like Figure 9 and Figure 10As shown, the blue staining rate of the normal group was 15.45%, while the cells in the hydrogen peroxide group were obviously aged and shrunken, and 84.83% of the cells were stained blue-green, indicating a high degree of aging. 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 ginseng extract before fermentation, the single-ingredient ginseng polysaccharide, F1, and CK all had average cell anti-aging effects. After the polysaccharide was mixed with the rare ginsenosides F1 and CK, although the activity was improved to a certain extent, the effect was still not as good as the ginseng extract obtained by fermentation with specific bacteria, indicating that the ginseng extract prepared by the present invention has an unsimilable advantage in cell anti-aging effects.

[0095] The ginseng extract of the present invention uses fermentation technology to ferment ginseng using Lactobacillus plantarum (independently screened and preserved in the Guangdong Provincial Microbial Culture Collection Center, GDMCC No. 64374), so that the prototype ginsenosides of ginseng are converted into rare saponins CK and F1, with the content ratio of CK to F1 being 1:1-5, and retaining natural plant components such as ginseng polysaccharides. The ginseng extract prepared by the present invention not only has higher bioavailability, but also has unique advantages in biological activity that cannot be simulated. It can more effectively inhibit cell inflammation, more effectively restore the mitochondrial function of aging cells and inhibit cell aging, and can be used to prepare skin care products.

[0096] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications are also considered to be within the scope of protection 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, the deposit number of which is GDMCC No.64374; The method for preparing the ginseng extract rich in rare saponins comprises the following steps: freeze-drying fresh ginseng and grinding it into powder to obtain the ginseng powder; Dispersing the ginseng powder in water at a mass ratio of 1 to 5:100, sterilizing, and cooling to 37° C. to obtain a fermentation substrate; inoculating the seed liquid of the Lactobacillus plantarum into the fermentation substrate, fermenting and culturing, sterilizing after monitoring the reaction to be complete, and collecting the supernatant to obtain the ginseng extract rich in rare saponins; The concentration of the seed solution is OD 600 =0.8~2.0, the volume ratio of the seed liquid to the fermentation substrate is 1:20~100; The fermentation temperature is 30° C. to 50° C., and the fermentation time is 8 h to 16 h.

2. The ginseng extract rich in rare saponins according to claim 1, characterized in that By mass, the rare saponin content is 0.2%~2%, and the ginseng polysaccharide content 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. The method for preparing a ginseng extract rich in rare saponins according to any one of claims 1 to 3, wherein: The ginseng powder is fermented with Lactobacillus plantarum, and the preservation number of the Lactobacillus plantarum is GDMCC No.64374.

5. The method for preparing the ginseng extract rich in rare saponins according to claim 4, 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.

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

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