A ginseng adventitious root cell strain ccum-re-7, extract thereof, and preparation method and application thereof

Abstract

The application discloses a ginseng adventitious root cell strain CCUM-Re-7, an extract of the ginseng adventitious root cell strain CCUM-Re-7, a preparation method of the ginseng adventitious root cell strain CCUM-Re-7 and application of the ginseng adventitious root cell strain CCUM-Re-7, and belongs to the technical field of biotechnology, and specifically provides a method for extracting an extract rich in ginsenoside Re from ginseng cambium stem cell induced ginseng adventitious roots, and application of the extract in anti-aging and skin whitening.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically referring to a ginseng adventitious root cell line CCUM-Re-7, its extract, preparation method, and application. Background Technology

[0002] In recent years, with the improvement of people's living standards and their pursuit of beauty, anti-aging and skin whitening have become a research hotspot in the cosmetics market. Currently, many anti-aging and skin whitening products on the market are mainly derived from chemically synthesized substances. For example, some common whitening ingredients such as arbutin and kojic acid, while having certain whitening effects, suffer from poor stability, easy decomposition, and potential cytotoxicity. Meanwhile, anti-aging ingredients such as retinol may cause adverse reactions such as skin allergies and irritation.

[0003] Skin aging and pigmentation are common skin problems, primarily caused by oxidative stress and excessive melanin deposition. Melanin is synthesized in melanocytes, pigment cells located in the basal layer of the epidermis. These cells contain various enzymes. Melanin, a dark brown pigment, is formed through an oxidative reaction that polymerizes specific amino acids into substrates. Melanin formed in this way is transferred to keratinocytes to display skin color. Furthermore, melanin forms a cap-like structure around the cell nucleus, protecting genes from UV damage by scavenging free radicals, protecting intracellular proteins, and absorbing or scattering UV rays in the skin to prevent damage. However, if melanin is overexposed to UV radiation or environmental factors such as air pollution, excessive melanin production occurs, leading to deposition and skin darkening. Therefore, it is necessary to intervene in the activity of tyrosinase to prevent skin damage and darkening.

[0004] Tyrosinase is an enzyme that directly participates in melanin formation and effectively prevents oxidative stress that severely affects skin aging and melanin production. For healthy and fair skin, it is necessary to discover natural substances that are harmless to the human body and possess strong antioxidant activity and effectively inhibit melanin formation.

[0005] Plant extracts are gaining increasing attention due to their natural, mild, and diverse bioactive properties. Ginseng, a traditional and precious Chinese medicinal herb, possesses a variety of pharmacological activities. Ginsenosides, the main active ingredient in ginseng, exhibit antioxidant, anti-inflammatory, anti-tumor, and immunomodulatory activities. However, traditionally cultivated ginseng suffers from long growth cycles, high pesticide residues, and excessive heavy metals. Research has found that adventitious roots induced from ginseng stem cells are not limited by seasons, allowing for the extraction of effective components at any time while maintaining consistent quality. This results in a completely pesticide-free, green, and pollution-free health product. Ginseng adventitious roots are ginseng tissue obtained through plant tissue culture technology, containing abundant ginsenosides, polysaccharides, amino acids, and other bioactive components. The preparation process involves slicing ginseng roots aged 5 years or older and inducing adventitious root production. Given that ginseng roots grow in complex soil environments and are highly susceptible to soil microbial contamination, preparing sterile ginseng slices suitable for tissue culture is extremely challenging, resulting in a high slice contamination rate. In addition to hypochlorous acid disinfectant, which is the main traditional disinfectant, there is an urgent need to develop a new type of disinfectant that combines better sterilization efficiency with higher safety.

[0006] Furthermore, current research and application of ginseng adventitious roots in the fields of anti-aging and skin whitening are still insufficient, especially the cultivation and extraction technology of adventitious roots based on ginseng stem cells, which has not yet been fully developed. Given that ginseng stem cells possess stronger differentiation potential and the ability to synthesize bioactive substances, they hold promise for helping to develop more effective anti-aging and skin whitening products. Summary of the Invention

[0007] This invention aims to provide a method for extracting an extract rich in ginsenoside Re from ginseng adventitious roots induced by ginseng cambium stem cells, and the application of this extract in anti-aging and skin whitening. This invention significantly reduces the contamination rate and increases the content of ginsenoside Re by optimizing the sterilization method and culture conditions.

[0008] To solve the above problems, the technical solution adopted by the present invention is as follows: This invention provides a ginseng adventitious root cell line CCUM-Re-7, which was deposited on June 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30712, and classified as follows: Panax ginseng The address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101.

[0009] On one hand, the present invention proposes a method for constructing the ginseng adventitious root cell line CCUM-Re-7, comprising the following steps: Step S1: The ginseng was sterilized by double treatment with sodium hypochlorite and silver nitrate. After slicing, the cambium stem cells were cultured and expanded and then isolated. Step S2: Directly induce the generation of ginseng adventitious roots from isolated cambium stem cells; Step S3: Culture induced ginseng adventitious roots and screen cell lines rich in ginsenoside Re from the induced adventitious roots, such as CCUM-Re-7.

[0010] On the one hand, the present invention also provides a method for preparing an extract from the ginseng adventitious root cell line CCUM-Re-7, comprising: The cell line CCUM-Re-7 obtained in step S3 was cultured on a large scale to extract ginseng adventitious root extract rich in saponins, and this extract was used as the active ingredient to prepare an anti-aging and whitening composition.

[0011] Preferably, the specific steps in step S1 of dual-processing sterilization and disinfection of ginseng, amplification of cambium stem cells, and isolation of cambium stem cells include: S1.1: Take 5-year-old ginseng, wash it and cut it into 5cm pieces. Disinfect it with 2% sodium hypochlorite, then wash it 3 times with sterile distilled water. Cut it a second time at 1cm intervals, disinfect it a second time with silver nitrate, and then wash it with sterile distilled water. S1.2: After disinfection and cleaning, the ginseng slices are cut at 0.5cm intervals and inoculated onto a nutrient-free water agar medium. When the cambium expands and separates well, it is positioned and used as a tissue culture section.

[0012] Preferably, step S2 includes the following steps: cutting a sterile section of water agar medium that has been free from contamination for 10 days and has an easily separable cambium layer into pieces, and inoculating the separated cambium stem cells into 1 / 2 MS medium containing 5 mg / L IBA to induce the production of ginseng adventitious roots.

[0013] Preferably, in step S3, the total content of major saponins in the CCUM-Re-7 cell line is 13.67 mg / g or more, of which ginsenoside Re accounts for 37% or more.

[0014] Preferably, the conditions for the large-scale cultivation are: In a bioreactor; Temperature 21-26℃; The cultivation period is 6-8 weeks; Use 1 / 2 MS medium containing 2 mg / L IBA.

[0015] Preferably, the extraction step further includes: drying the cultured ginseng adventitious roots, extracting them with purified water at 95°C, and concentrating them to obtain an extract rich in ginsenoside Re.

[0016] Preferably, the extraction conditions are: Add 10 times the volume of purified water; Extract twice at 95℃, each time for 6 hours; The extract was concentrated to 60 Brix.

[0017] On the other hand, a ginseng adventitious root extract is prepared by the above method, and the content of ginsenoside Re accounts for 37% or more of the total saponins.

[0018] The ginseng adventitious root extract prepared according to the above method has the following functions: It has the ability to scavenge DPPH free radicals; It has the ability to inhibit the generation of reactive oxygen species; No cytotoxicity; It can inhibit tyrosinase activity; It can inhibit melanin production.

[0019] On the other hand, the present invention provides an anti-aging and whitening composition based on the aforementioned ginseng adventitious root extract as an active ingredient.

[0020] The beneficial effects achieved by the present invention using the above solution are as follows: 1. First, the biggest problem when inducing ginseng adventitious roots from cambium stem cells is contamination. To minimize contamination, in addition to treatment with sodium hypochlorite, silver nitrate can be used for secondary disinfection on top of existing disinfection methods to solve the problem of excessive contamination rate during inoculation.

[0021] 2. Secondly, after sterilization, the contamination rate of sections prepared from cambium stem cells is lower. Inoculating sterilized ginseng sections onto nutrient-free water agar medium facilitates the isolation of stem cells from the sections and further reduces the risk of contamination, thus aiding in the screening of new cell lines.

[0022] 3. Furthermore, by culturing the cambium layer to increase its volume before separation, and then inducing its growth in a special medium containing indolebutyric acid (IBA), adventitious roots of ginseng can be successfully cultivated. Based on this method, cell lines with rapid growth characteristics and rich in ginsenoside Re can be developed, which can then be used to prepare related extracts.

[0023] 4. Furthermore, the ginseng adventitious root CCUM-Re-7 extract, obtained from stem cell cambium induced dermal growth, is rich in ginsenoside Re, exhibiting potent antioxidant activity. It possesses the ability to scavenge DPPH free radicals and inhibit the generation of reactive oxygen species. This extract has no toxic effects on skin cells; it effectively inhibits melanin synthesis in melanocytes and suppresses tyrosinase activity—an enzyme that plays a crucial role in melanin production. Based on these properties, CCUM-Re-7 extract demonstrates excellent skin whitening effects, making it suitable as an active ingredient in skin whitening cosmetics, pharmaceuticals, and foods (such as oral beauty products). Attached Figure Description

[0024] Figure 1 Diagram showing the disinfection treatment and adventitious root induction process of ginseng; Figure 2 A diagram showing the screening and optimization of culture conditions for CCUM-Re-7; Figure 3 It is the DPPH free radical scavenging ability of CCUM-Re-7 extract from ginseng adventitious root rich in ginsenoside Re; Figure 4 The reducing power of CCUM-Re-7 extract from ginseng adventitious roots, which is rich in ginsenoside Re; Figure 5 Cytotoxicity of CCUM-Re-7 extract from adventitious ginseng roots rich in ginsenoside Re against human immortalized keratinocytes (HaCaT cells) (MTT assay). Figure 6 The inhibitory effect of CCUM-Re-7 extract from ginseng adventitious roots rich in ginsenoside Re on free radical production in HaCaT cells; Figure 7 The inhibitory effect of CCUM-Re-7 extract from adventitious ginseng roots rich in ginsenoside Re on tyrosinase activity; Figure 8 The effect of CCUM-Re-7 extract from adventitious ginseng roots rich in ginsenoside Re on the growth of mouse skin melanoma cells (B16F10); Figure 9 The effect of CCUM-Re-7 extract from adventitious ginseng roots rich in ginsenoside Re on melanin production in B16F10 cells; Figure 10 To investigate the inhibitory effect of CCUM-Re-7 extract from adventitious ginseng roots rich in ginsenoside Re on tyrosinase activity in B16F10 cells.

[0025] It should be noted that in the attached diagram: Figure 1Inoculation of ginseng slices treated with sodium hypochlorite and silver nitrate (A), direct induction of ginseng adventitious roots by IBA treatment (B), and growth diagrams of different induced cell lines (C).

[0026] Figure 2 To select different ginseng adventitious roots and their growth rates and saponin contents in IBA-containing media, CCUM-Re-7 (D) was screened out as having the highest ginsenoside Re content. The growth of CCUM-Re-7 in IBA and NAA was investigated, and the results showed that CCUM-Re-7 grew best at 2 mg / L IBA (E). Large-scale cultivation in a bioreactor (F) was performed, and the ginseng adventitious roots harvested for extract preparation are shown in Figure G.

[0027] Figure 3 The horizontal axis represents the concentration of CCUM-Re-7 extract from ginseng adventitious roots rich in ginsenoside Re, in mg / ml. The vertical axis represents the DPPH free radical scavenging capacity.

[0028] Figure 4 The horizontal axis represents the concentration of CCUM-Re-7 extract from ginseng adventitious roots rich in ginsenoside Re, in mg / ml, and the vertical axis represents the absorbance at a wavelength of 700 nm.

[0029] Figure 5 The horizontal axis represents the concentration of CCUM-Re-7 extract from ginseng adventitious roots rich in ginsenoside Re, in mg / ml, and the vertical axis represents the percentage of cell viability.

[0030] Figure 6 The horizontal axis represents the inhibitory effect of vitamin C and ginseng adventitious root CCUM-Re-7 extract rich in ginsenoside Re on free radical production in HaCaT cells at different concentrations, while the vertical axis represents the percentage of reactive oxygen species (ROS) levels.

[0031] Figure 7 The horizontal axis represents the concentration of CCUM-Re-7 extract from ginseng adventitious roots rich in ginsenoside Re, in mg / ml, and the vertical axis represents the percentage of tyrosinase activity.

[0032] Figure 8 The horizontal axis represents the concentration of CCUM-Re-7 extract from ginseng adventitious roots rich in ginsenoside Re, in μg / ml, and the vertical axis represents the percentage of cell viability.

[0033] Figure 9The horizontal axis represents the effect of arbutin and ginsenoside-rich ginseng adventitious root CCUM-Re-7 extract at different concentrations on melanin production in B16F10 cells, and the vertical axis represents the percentage of melanin content.

[0034] Figure 10 The horizontal axis represents the effect of arbutin and ginsenoside-rich ginseng adventitious root CCUM-Re-7 extract at different concentrations on tyrosinase activity, and the vertical axis represents the percentage of tyrosinase activity.

[0035] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] Example 1: Preparation of extract from adventitious ginseng root CCUM-Re-7 rich in ginsenoside Re.

[0038] 1-1 A sterile section preparation method that significantly reduces the contamination rate of ginseng sections with stem cell cambium: The cambium is a layer of meristematic tissue in the stem or root of a plant. It consists of a single layer of cells capable of continuous division and is a key structure for secondary growth in plants. In ginseng tissue culture, the most crucial aspect is obtaining sterile ginseng slices. Because ginseng has a long growth cycle and prefers shade, it is easily contaminated by various microorganisms, resulting in a high contamination rate and making processing difficult.

[0039] In this embodiment, 5-year-old ginseng was washed and cut into 5-cm pieces. It was disinfected with 2% sodium hypochlorite, washed three times with sterile distilled water, cut again at 1cm intervals, disinfected with silver nitrate, and then washed with sterile distilled water (as shown in Table 1 below).

[0040] Table 1. Comparison of contamination rates between the general method for obtaining sterile ginseng cambium sections and the CCUM sterilization method.

[0041] The washed ginseng was finally cut at 0.5 cm intervals and inoculated onto nutrient-free water agar medium. After 10 days, when the cambium had expanded and separated well, it was positioned and used as tissue culture sections, such as... Figure 1 As shown, Figure 1 A is the inoculation of ginseng slices treated with sodium hypochlorite and silver nitrate. Figure 1 B represents the direct induction of ginseng adventitious roots by IBA treatment. Figure 1 C represents the growth diagram of different cell lines induced by the process.

[0042] Sterile sections of ginseng, uncontaminated and with easily separable cambium layers after 10 days on water agar, were cut into pieces and inoculated into 1 / 2 MS medium (3% sugar, 5 mg / L IBA) to induce adventitious roots. To enhance lateral root formation from the induced adventitious roots, various hormone treatments were performed, and the results showed that IBA had the best effect on lateral root formation. Figure 1 ).

[0043] 1-2 Screening and growth conditions of ginseng adventitious roots CCUM-Re-7 rich in ginsenoside Re induced in the cambium: refer to Figure 2 As shown, Figure 2 To select the growth rate and saponin content of different ginseng adventitious roots in a medium containing IBA, the ginseng adventitious root CCUM-Re-7 (D) with the highest ginsenoside Re content was screened. The growth of CCUM-Re-7 in IBA and NAA was examined, and the results showed that CCUM-Re-7 grew best at IBA concentration of 2 mg / L (E). Large-scale cultivation in a bioreactor was performed (F), and the ginseng adventitious roots harvested for extract preparation are shown in Figure G. From adventitious roots 1, 3, 5, and 7 derived from the cambium, root 7, which showed the fastest growth rate and the highest ginsenoside Re content, was selected and named CCUM-Re-7.

[0044] The growth rate and lateral root formation of the selected CCUM-Re-7 on 1 / 2 MS medium and 1 / 2 SH medium were investigated, as shown in Table 2 below. CCUM-Re-7 grew best on IBA 2 mg / L 1 / 2 MS medium. The adventitious roots of ginseng were harvested by large-scale culture in a bioreactor to prepare extracts.

[0045] Table 2. Effects of plant hormones on the growth and lateral root formation of adventitious roots CCUM-Re-7 ginseng.

[0046] Analysis of ginsenoside content in 1-3 ginseng adventitious roots CCUM-Re-7: The ginsenoside content of ginseng adventitious root CCUM-Re-7 was analyzed by high performance liquid chromatography (HPLC). The specific procedure was as follows: 1 g each of CCUM-Re-7 and cultivated ginseng powder were accurately weighed and added to 30 mL of 80% methanol solution. The mixture was refluxed at 70°C for 1 hour. The extracts were filtered, combined, and evaporated to dryness using a rotary evaporator at 45°C. The residue was dissolved in 15 mL of distilled water and extracted twice with 20 mL of water-saturated n-butanol each time. The water-saturated n-butanol extracts were combined, and an appropriate amount of water was added. The mixture was evaporated to dryness using a rotary evaporator at 50°C, then dissolved in 100 mL of HPLC-grade methanol. The solution was filtered through a 0.45 µm membrane filter and then diluted to a 5 mL volumetric flask with HPLC-grade methanol.

[0047] The analysis was performed using an Agilent 1260 high-performance liquid chromatograph (HPLC) with a Phenomenex reversed-phase Kinetex C18 column (4.6 mm × 100 mm, 5 μm particle size). Ginsenosides in the extracts of each sample were separated using a mobile phase composed of deionized water and HPLC-grade acetonitrile at a column temperature of 45°C. The concentration gradient of the two components in the mobile phase ranged from 81:19 to 10:90 (deionized water:acetonitrile, volume ratio). The flow rate was 0.6 mL / min, and detection was performed using a UV-Vis detector at a wavelength of 203 nm. The results were used to quantify 14 ginsenosides using standards, as shown in Table 3 below.

[0048] Table 3. Content of 14 ginsenosides in ginseng adventitious root CCUM-Re-7

[0049] The total content of the main saponins in CCUM-Re-7 is 13.67 mg / g, and the content of saponin Re is 5.17 mg / g, accounting for about 37% of the total saponins.

[0050] 1-4 Preparation of Cambium Stem Cell Extract from Ginseng Adventitious Root CCUM-Re-7: Adventitious ginseng roots (CCUM-Re-7) derived from ginseng cambium stem cells and cultured in large quantities were washed twice with tap water, then once with distilled water, and dried using a hot air dryer at 48-50°C until the moisture content was 13%-14%. The dried CCUM-Re-7 was then added to 10 times its volume of purified water and extracted twice at 95°C for 6 hours each time. The extract was concentrated to 60 brise using a rotary evaporator at 70°C to prepare the final concentrate. The extract yield was 45%-50%.

[0051] Example 2: Determination of antioxidant activity of ginseng adventitious root CCUM-Re-7 extract.

[0052] 2-1 Free radical scavenging and reducing capabilities of CCUM-Re-7: The antioxidant activity of ginseng adventitious root CCUM-Re-7 extract was determined using two methods: DPPH free radical scavenging method and reducing power assay.

[0053] The DPPH free radical scavenging method involves dissolving DPPH in ethanol to prepare a 0.2 mM solution. Then, 20 µl of the extract and 180 µl of the DPPH solution are added to a 96-well plate, and the plate is shaken vigorously in the dark for 30 minutes. The absorbance at 517 nm is then measured.

[0054] One of the standards for measuring antioxidant activity is reducing power. The specific steps are as follows: Dissolve the sample and standard solutions with a concentration of 2.0~10 mg / ml in 1.0 ml of deionized distilled water, then mix with 2.5 ml of phosphate buffer (pH 6.5) and 2.5 ml of potassium ferricyanide solution. React the solution at 50°C for 20 minutes, cool, add 2.5 ml of 10% trichloroacetic acid solution, mix thoroughly, and centrifuge at 3000 rpm for 10 minutes. Take 2.5 ml of the supernatant, add 2.5 ml of distilled water and 0.1% ferric chloride solution, and measure the change in absorbance at 700 nm. The reducing power is demonstrated by measuring ascorbic acid as a control.

[0055] Figure 3 The results show the DPPH free radical scavenging capacity of ginseng adventitious root CCUM-Re-7 extract. Specifically, increasing the concentration of ginseng adventitious root CCUM-Re-7 extract from 1.0 mg / ml to 10 mg / ml resulted in a significant increase in free radical scavenging capacity in a concentration-dependent manner, indicating that ginseng adventitious root CCUM-Re-7 extract possesses high antioxidant activity.

[0056] Figure 4 These are the results of the reducing power determination of ginseng adventitious root CCUM-Re-7 extract. The reducing power, consistent with its free radical scavenging ability, increases in a concentration-dependent manner. Combining these two results reveals that ginseng adventitious root CCUM-Re-7 extract can protect organisms from oxidative stress by effectively scavenging reactive oxygen species and other oxidizing substances produced in the body.

[0057] 2-2 Reactive oxygen species inhibition capacity of CCUM-Re-7: To evaluate the inhibitory effect of ginseng adventitious root CCUM-Re-7 extract on ROS production, cells were pretreated with 500 μmol / L hydrogen peroxide for 2 hours. The supernatant was then aspirated, and cells were reacted with samples of different concentrations (62.5, 125, 250, 500, and 1000 μg / mL) for 24 hours, with ascorbic acid as a positive control. After the reaction, cells were washed twice with PBS, and 20 μM DCFH-DA working solution was added to the cells. The cells were incubated for another 30 minutes, and the supernatant was removed. The fluorescence intensity at excitation wavelength of 480 nm and emission wavelength of 525 nm was measured using a microplate reader.

[0058] Figure 5 This study demonstrates the effect of ginseng adventitious root CCUM-Re-7 extract on HaCaT cell growth. The ginseng adventitious root CCUM-Re-7 extract showed no toxicity at any of the tested concentrations; in fact, it promoted cell growth at concentrations ranging from 10 to 500 μg / ml. This reflects a cell proliferation-promoting effect, with the ginseng adventitious root CCUM-Re-7 extract increasing HaCaT cell growth by 120% to 250%.

[0059] Figure 6 This study demonstrates how ginseng adventitious root CCUM-Re-7 extract inhibits reactive oxygen species (ROS) production in HaCaT cells under oxidative stress. Treatment of HaCaT cells with hydrogen peroxide increased ROS levels by 150%, and treatment with ginseng adventitious root CCUM-Re-7 extract inhibited ROS production in a dose-dependent manner, to a degree comparable to that of vitamin C.

[0060] Based on the aforementioned results of free radical scavenging and reducing capacity measurements, it can be concluded that the ginseng adventitious root CCUM-Re-7 extract has strong antioxidant activity.

[0061] Example 3: Effect of inhibiting melanin formation.

[0062] 3-1 Effect of inhibiting extracellular tyrosinase activity: The effect of ginseng adventitious root CCUM-Re-7 extract on the extracellular activity of tyrosinase, an enzyme that catalyzes melanin production in the skin, was measured using the following methods: Add 40 µl of 0.1 M PBS buffer, 40 µl of 2 mM L-tyrosine, 40 µl of mushroom tyrosinase (125 units / ml), and 2–10 mg / ml of ginseng adventitious root CCUM-Re-7 extract to a 96-well plate to bring the total volume to 120 µl. Stir the mixture at 37 °C for 10 min and measure the absorbance at 475 nm to calculate the degree of inhibition of tyrosinase activity.

[0063] Figure 7 The graph shows the inhibitory effect of ginseng adventitious root CCUM-Re-7 extract on tyrosinase activity, as determined by extracellular assays. At a concentration of 4 mg / ml, the ginseng adventitious root CCUM-Re-7 extract inhibited the enzyme activity by approximately 17%, with the inhibitory activity increasing in a concentration-dependent manner. At a concentration of 10 mg / ml, it effectively inhibited the enzyme activity, with an inhibition rate exceeding 50%. Figure 7 This result indicates that the extract of ginseng adventitious root CCUM-Re-7 can effectively inhibit melanin formation.

[0064] 3-2 Toxicity of melanocytes, inhibition of melanin content and intracellular tyrosinase activity: The inhibitory effect of ginseng adventitious root CCUM-Re-7 extract on intracellular tyrosinase activity in B16F10 cells was determined. Specifically, B16F10 cells were cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2. B16F10 cells were aliquoted into 96-well microplates at 2.5 × 10³ cells / well and cultured at 5% CO2. The cells were cultured in a 37°C incubator. After 24 hours, 100 μl of culture medium was removed from each well and different concentrations of sample and stimulant were added. At this point, the final concentration of the stimulant α-MSH was 1 nM. After processing the samples and stimulants, 150 μl of culture medium from 3 days of culture was transferred to a new 96-well microplate to measure the absorbance at 405 nm. The inhibition rate of melanin production for each sample was calculated by subtracting the absorbance values ​​of cells not stimulated by α-MSH (α-melanocyte stimulating hormone) or protoporphyrin IX (as a control group).

[0065] Figure 8 The effect of ginseng adventitious root CCUM-Re-7 extract on the growth of B16F10 cells was investigated. Even when the extract concentration was increased to 1,000 µg / ml, the cells maintained a good growth status of 90% or more and did not exhibit any cytotoxicity. Generally, when cells maintain a growth rate of 80% or more after treatment, the substance is considered non-cytotoxic.

[0066] Figure 9 The results were obtained in B16F10 cells after stimulating melanin production with α-MSH, followed by treatment with ginseng adventitious root CCUM-Re-7 extract. When cells were stimulated with α-MSH, melanin production increased to 120% of the control group. However, when ginseng root CCUM-Re-7 extract at a concentration of 0.1 mg / ml was added, melanin production decreased to or below the control group level, and its inhibitory effect was more significant than that of cells treated with the positive control (arbutin) (see...). Figure 10These results indicate that ginseng root CCUM-Re-7 extract can strongly inhibit intracellular melanin production.

[0067] Figure 10 This indicates that the CCUM-Re-7 extract from ginseng adventitious roots has an inhibitory effect on the activity of tyrosinase, an enzyme involved in melanin synthesis in melanoma cells. Figure 9 The results were consistent; the inhibitory activity at a concentration of 0.1 mg / ml was higher than that at 0.01 mg / ml, and it was more effective than the positive control arbutin treatment. This clearly demonstrates that ginseng adventitious root CCUM-Re-7 extract reduces melanin production by inhibiting the activity of melanin-producing enzymes.

[0068] Example 4: Effects of ginseng adventitious root CCUM-Re-7 extract on the growth of skin-related cell lines.

[0069] The growth and cytotoxicity of human skin-related cells, namely human dermal fibroblasts (nHDF), HaCaT cells, and B16F10 cells, were determined using the MTT reduction assay.

[0070] Cells were treated with ginseng adventitious root CCUM-Re-7 extract at concentrations ranging from 10 to 1000 µg / ml. After 24 hours of cell culture, cells were placed in wells containing 0.1 mg MTT / (200 µl) and cultured for 3 hours. Each experimental group was configured with three replicates. After culture, the supernatant was removed, and 100 µl of dimethyl sulfoxide was added to dissolve the formazan produced by MTT reduction. The absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader. Cell viability was expressed as a percentage of cells relative to the control group.

[0071] Table 4 presents the results of the growth effect assays on the representative cell types (human nHDF, HaCaT, and B16F10 cells) primarily used in the skin-related tests. At all tested concentrations, the ginseng adventitious root CCUM-Re-7 extract promoted the growth rate of these three skin-related cell types to 90% or higher, indicating that the extract is non-cytotoxic and safe. In particular, at low concentrations, the ginseng adventitious root CCUM-Re-7 extract exhibited a significant growth-promoting effect on HaCaT cells.

[0072] Table 4. Effects of ginseng adventitious root CCUM-Re-7 extract on the growth of human skin-related cells.

[0073] Based on all the results confirmed in the above embodiments, the following conclusions can be drawn: 1) The new ginseng adventitious root CCUM-Re-7 extract has high antioxidant activity; 2) It is completely non-toxic to skin-related cell lines; 3) It can strongly inhibit the tyrosinase activity that forms melanin both inside and outside the cells, thereby inhibiting melanin production and ultimately showing a skin whitening effect.

[0074] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method for preparing an extract from ginseng adventitious root cell line CCUM-Re-7, wherein the ginseng adventitious root cell line CCUM-Re-7 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30712, characterized in that... The preparation methods of its extract include: Step S1: The ginseng is sterilized by double treatment with sodium hypochlorite and silver nitrate. After slicing, the cambium stem cells are cultured and expanded, and then the cambium stem cells are isolated. Step S2: Directly induce the generation of ginseng adventitious roots from isolated cambium stem cells; Step S3: Culture induced ginseng adventitious roots and screen cell lines rich in ginsenoside Re from the induced adventitious roots, such as CCUM-Re-7. Step S4: Large-scale culture of cell line CCUM-Re-7 was carried out to extract and prepare ginseng adventitious root extract rich in saponins, and this extract was used as the active ingredient to prepare an anti-aging and whitening composition.

2. The preparation method according to claim 1, characterized in that, The specific steps in step S1, including the dual treatment and sterilization of ginseng, the amplification of cambium stem cells, and the isolation of cambium stem cells, include: S1.1: Take 5-year-old ginseng, wash it and cut it into 5cm pieces. Disinfect it with 2% sodium hypochlorite, then wash it 3 times with sterile distilled water. Cut it a second time at 1cm intervals, disinfect it a second time with silver nitrate, and then wash it with sterile distilled water. S1.2: After disinfection and cleaning, the ginseng slices are cut at 0.5cm intervals and inoculated onto a nutrient-free water agar medium. When the cambium expands and separates well, it is positioned and used as a tissue culture section.

3. The preparation method according to claim 2, characterized in that, The specific steps of step S2 include: cutting a sterile section of water agar medium that has been free from contamination for 10 days and has an easily separable cambium layer into pieces, and inoculating the separated cambium stem cells into 1 / 2 MS medium containing 5 mg / L IBA to induce the production of ginseng adventitious roots.

4. The preparation method according to claim 1, characterized in that, The conditions for the large-scale cultivation are as follows: In a bioreactor; Temperature 21-26℃; The cultivation period is 6-8 weeks; Use 1 / 2 MS medium containing 2 mg / L IBA.

5. The preparation method according to claim 1, characterized in that, The extraction process also includes: drying the ginseng adventitious roots obtained from large-scale culture, extracting them twice with 10 times the volume of purified water at 95°C for 6 hours each time, combining the extracts and concentrating them to 60 Brix to obtain a ginseng adventitious root extract rich in ginsenoside Re.

6. A ginseng adventitious root extract, characterized in that, Prepared by the method described in any one of claims 1-5, wherein the content of ginsenoside Re accounts for 37% or more of the total saponins.

7. An anti-aging and whitening composition, characterized in that: It contains the ginseng adventitious root extract as described in claim 6 as an active ingredient.

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