Synthetic biological process preparation method and application of rare ginsenoside Rg3

Abstract

The invention provides a preparation method of rare ginsenoside Rg3, which comprises the following steps: by taking common glucose as a raw material, fermenting through a specific yeast strain to directly generate fermentation liquor containing ginsenoside 20 (S)-Rg3, and subsequently adsorbing, washing, extracting, purifying and crystallizing to efficiently obtain the rare ginsenoside Rg3 with higher purity. The ginsenoside Rg3 prepared by the invention shows huge application potential in the aspects of preventing photoaging, repairing damaged barriers and promoting skin health by accurately regulating and controlling a PPAR-alpha core pathway.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the synthetic bioprocess preparation method and application of rare ginsenoside Rg3. Background Technology

[0002] Ginsenosides are important active components of ginseng, belonging to the triterpenoid glycoside class of compounds. Ginsenosides possess various therapeutic effects, including antioxidant, anti-inflammatory, vasodilatory, anti-allergic, and anti-diabetic properties. Ginsenoside 20(S)-Rg3, extracted from ginseng (a plant in the Araliaceae family), is a rare component of ginsenosides, exhibiting significant biological activities and medicinal value, including anti-inflammatory, antioxidant, and anti-tumor effects. Currently, 20(S)-Rg3 is mainly obtained through direct extraction from Araliaceae plants, in addition to indirect extraction methods such as enzymatic catalysis, microbial transformation, and hydrolysis. Extraction methods primarily use plant raw materials, resulting in low yields and susceptibility to seasonal variations. Insufficient raw materials are a major bottleneck limiting the large-scale production of 20(S)-Rg3. Furthermore, 20(S)-Rg3 obtained through plant extraction often has low purity and low yield, making it difficult to meet market demand.

[0003] Therefore, there is an urgent need in this field to develop a bio-fermentation method for producing 20(S)-Rg3 that is highly efficient, green and natural, suitable for large-scale industrialization, and free from environmental damage. Summary of the Invention

[0004] This invention aims to develop a bio-fermentation method for producing 20(S)-Rg3 that is highly efficient, green, natural, suitable for large-scale industrialization, and free from environmental damage. Specifically, it relates to a bio-process preparation method and application for the synthesis of rare ginsenoside Rg3.

[0005] In a first aspect of the present invention, a method for preparing rare ginsenoside Rg3 is provided, the method comprising the steps of:

[0006] (1) Fermentation with yeast strain in the presence of glucose to obtain fermentation broth containing ginsenoside 20(S)-Rg3, wherein the yeast strain has the preservation number CCTCC M 20232647.

[0007] (2) The fermentation broth containing 20(S)-Rg3 obtained in step (1) was heated, then diatomaceous earth was added for adsorption, and then the mixture was separated by filtration.

[0008] (3) Extract the solid product obtained by adsorption in step (2) with ethanol to obtain an organic phase extract;

[0009] (4) After concentrating the organic phase extract obtained in step (3), the extract is purified by chromatography through a macroporous resin column and a silica gel column in sequence. The eluent rich in 20(S)-Rg3 is collected and then recrystallized to obtain the rare ginsenoside Rg3.

[0010] In another preferred embodiment, the fermentation in step (1) is carried out according to the method described in Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts (Wang PP et al., Metabolic engineering. 2015; 29: 97-105).

[0011] In another preferred embodiment, in step (1), the concentration of glucose in the fermentation medium is 5-60 g / L.

[0012] In another preferred embodiment, the fermentation in step (1) is carried out in a fed medium containing glucose and ethanol; preferably, the mass ratio of glucose to ethanol in the fed medium is 1.8-2.2:1, wherein the initial concentration of glucose is 380-420 g / L.

[0013] In another preferred embodiment, in step (1), the concentration of the yeast strain in the fermentation medium is 100-150 g dry weight / L.

[0014] In another preferred embodiment, the heating temperature in step (2) is 55°C-65°C.

[0015] In another preferred embodiment, the heating time in step (2) is 0.8h-1.2h.

[0016] In another preferred embodiment, in step (2), the heating treatment includes heating 5L of fermentation broth containing 20(S)-Rg3 to 55-65°C and maintaining it for 0.5-2 hours.

[0017] In another preferred embodiment, in step (3), the volume ratio of the amount of ethanol used to the volume of the fermentation broth before concentration in step (2) is 4-6:5.

[0018] In another preferred embodiment, in step (4), the filler used for the macroporous resin column is a non-polar or weakly polar macroporous adsorption resin with polystyrene as the skeleton, and the macroporous resin is selected from the following group: D101, AB-8, LX-T83, LX-T28, DM130, Amberlite XAD series, HP series, SP800 series, SP70 series, or SP700 series macroporous resin.

[0019] In another preferred embodiment, the mobile phase described in step (4) is a 0%-90% ethanol-water solution.

[0020] In another preferred embodiment, the purification process further includes: concentrating the organic extract obtained in step (3) by 18-22 times, and then using the concentrate for chromatography purification.

[0021] In another preferred embodiment, the macroporous resin column chromatography purification in step (4) includes: concentrating the organic extract by 18-22 times at 100-150 mbar and 60-70°C, eluting the concentrated organic extract at a column flow rate of 1.2-2 BV / h with 0%-90% ethanol-water as the flow medium, to obtain an eluent rich in 20(S)-Rg3.

[0022] In another preferred embodiment, step (4) of the silica gel column chromatography purification includes using 80-85 v / v ethanol-water as the mobile phase.

[0023] In another preferred embodiment, the silica gel column chromatography purification further includes: concentrating the elution buffer rich in 20(S)-Rg3 under reduced pressure to obtain a macroporous concentrate of 20(S)-Rg3 for use in silica gel column chromatography.

[0024] In another preferred embodiment, the silica gel column chromatography purification in step (4) includes the following steps: after eluting the 20(S)-Rg3-rich eluent obtained by macroporous resin column elution, the eluent is purified and concentrated, the concentrated solution is loaded onto the gel column, and the concentrated solution is eluted at a rate of 2.5-3.5 BV / h with 80-85 v / v% ethanol-water as the mobile phase to obtain a silica gel column eluent rich in 20(S)-Rg3.

[0025] In another preferred embodiment, the mobile phase is isopropyl acetate: 80-85% ethanol-water = 75-80: 20-25.

[0026] In another preferred embodiment, the crystallization step in step (4) includes: evaporating the silica gel column eluent under reduced pressure at 280-320 mbar and 55℃-65℃, then dissolving it in 70-80 v / v% ethanol, and gradually cooling it to 10-20℃ at a rate of 12-18℃ / h to crystallize it.

[0027] In a second aspect of the invention, a composition is provided comprising the rare ginsenoside Rg3 described in the first aspect as an active ingredient.

[0028] In another preferred embodiment, the composition is capable of reducing the number of sunburned cells by >70%.

[0029] In another preferred embodiment, the composition is capable of reducing the content of cyclobutanepyrimidine dimer (CPD), a marker of DNA damage.

[0030] In another preferred embodiment, the composition is able to increase the content of filaggrin (FLG), a core component of the skin barrier.

[0031] In another preferred embodiment, the composition is capable of activating the proliferative activity of basal cells in the skin.

[0032] In another preferred embodiment, the composition is capable of increasing the content of PPAR-α.

[0033] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, detergent, oil, foundation, emulsion foundation, wax foundation, face mask, massage cream, or spray.

[0034] In another preferred embodiment, the concentration of rare ginsenoside Rg3 in the composition is 0.05-0.15 g / L.

[0035] In a third aspect of the invention, there is provided the use of the rare ginsenoside Rg3 as described in the first aspect and the composition as described in the second aspect for preparing cosmetic compositions that promote skin repair, strengthen skin texture, delay skin aging, improve skin wrinkles, or enhance skin elasticity.

[0036] In another preferred embodiment, the cosmetic composition is a sunscreen cosmetic.

[0037] In another preferred embodiment, the cosmetic composition is an anti-aging cosmetic.

[0038] In another preferred embodiment, the cosmetic composition is a soothing and repairing cosmetic.

[0039] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0040] Figure 1 The HPLC chromatogram of the 20(S)-Rg3 product is shown.

[0041] Figure 2 The diagram shows the migration of keratinocytes.

[0042] Figure 3 The bar chart shows the relative migration rate of keratinocytes.

[0043] Figure 4The bar chart shows the number of Sunburn cells.

[0044] Figure 5 The bar chart shows the average relative integrated optical density (IOD) of DNA damage CPD.

[0045] Figure 6 The bar chart shows the Ki67 immunohistochemistry results.

[0046] Figure 7 The bar chart shows the average relative integrated optical density (IOD) of FLG.

[0047] Figure 8 The bar chart shows the average relative integrated optical density (IOD) of PPAR-α.

[0048] Figure 9 The results of the tissue morphology test are shown in the figure.

[0049] Figure 10 Immunohistochemical map of DNA damage CPD is shown.

[0050] Figure 11 The Ki67 immunohistochemistry results are shown.

[0051] Figure 12 The immunofluorescence results of filaggrin (FLG) are shown.

[0052] Figure 13 The results of PPAR-α immunofluorescence are shown. Detailed Implementation

[0053] Through extensive and in-depth research and numerous experimental screenings, the inventors have unexpectedly developed a method for preparing a rare ginsenoside Rg3. This method uses ordinary glucose as a raw material, fermented with a specific yeast strain to directly generate a fermentation broth containing ginsenoside 20(S)-Rg3. Subsequent adsorption and washing, extraction, purification, and crystallization efficiently yield a high-purity rare ginsenoside Rg3. The ginsenoside 20(S)-Rg3 of this invention can significantly reduce sunburned cells by 75% and repair DNA damage caused by ultraviolet radiation. Simultaneously, it can strongly activate cell regeneration, more than doubling the proliferation rate and doubling the filaggrin content, providing fundamental protection and fundamentally rebuilding the skin barrier. Furthermore, the ginsenoside 20(S)-Rg3 of this invention, by precisely regulating the PPAR-α core pathway, promotes lipid synthesis to improve the barrier and enhances cellular energy to support repair, demonstrating its multi-target, synergistic technological advantages in photoaging prevention and skin health promotion that are unmatched by existing products. Based on these findings, the inventors completed this invention.

[0054] Terminology Explanation

[0055] 20(S)-Rare Ginsenoside Rg3 (hereinafter referred to as 20(S)-Rg3) is a natural active ingredient extracted from ginseng. In wild ginseng, this component is present in extremely low concentrations, only about 0.003%, and is considered one of the rare ginsenosides.

[0056] In this invention, "ginsenoside 20(S)-Rg3 of the present invention", "20(S)-Rg3", "rare ginsenoside 20(S)-Rg3", "rare ginsenoside 20(S)-Rg3 of the present invention", and "Rg3 of the present invention" can be used interchangeably and all refer to ginsenoside 20(S)-Rg3 prepared by the fermentation process of the specific yeast strain of the present invention.

[0057] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0058] 1. This invention uses ordinary glucose as raw material and ferments it with a specific yeast strain to directly generate a fermentation broth containing ginsenoside 20(S)-Rg3. This process requires only simple post-processing to efficiently obtain high-purity products and has great application potential for large-scale production.

[0059] 2. The ginsenoside 20(S)-Rg3 of the present invention can significantly reduce sunburned cells by 75% and repair DNA damage caused by ultraviolet rays. At the same time, it can strongly activate cell regeneration, increase the proliferation rate by more than 100%, and double the filaggrin content, providing root protection and fundamentally rebuilding the skin barrier.

[0060] 3. The ginsenoside 20(S)-Rg3 of the present invention promotes lipid synthesis to improve the barrier and enhances cellular energy to support repair by precisely regulating the PPAR-α core pathway, demonstrating that it has multi-target and synergistic technical advantages in the prevention of photoaging and the promotion of skin health that are difficult for existing products to match.

[0061] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0062] Example 1: Preparation of Ginsenoside 20(S)-Rg3

[0063] (1) Fermentation broth containing ginsenoside 20(S)-Rg3: A yeast strain producing ginsenoside 20(S)-Rg3 was constructed according to the literature, with the preservation number CCTCC M 20232647. Fermentation was carried out according to the method provided in the literature to obtain a fermentation broth containing ginsenoside 20(S)-Rg3, with a yield of over 1 g / L. The fermentation broth was prepared according to the method in the literature, Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts (Wang PP et al., Metabolic engineering. 2015; 29:97-105).

[0064] (2) Diatomaceous earth culture: 5L of fermentation broth containing 20(S)-Rg3 was heated to 60℃ and kept for 1h to obtain fermentation broth containing about 5g of 20(S)-Rg3; then 1kg of diatomaceous earth was added to the fermentation broth and stirred at 300rpm for 30min. Then, the aqueous phase and bacterial sludge were separated by vacuum filtration, and the filter cake was washed 3 times with 2.5L of pure water.

[0065] (3) Ethanol extraction: After washing, the filter cake is transferred to the extraction vessel, 4.5L of ethanol is added, and the mixture is stirred at 300rpm for 3h at 25℃. The organic phase is separated by filtration and then rinsed with 1.5L of ethanol.

[0066] (4) Eluting with D101 macroporous resin column: 6L of extract was concentrated under reduced pressure to 300ml at 65℃ under pressure of 100-150mbar. The resulting extract was passed through a D101 macroporous resin column with a column volume of 2.5L at a rate of 1.5BV / h. Then, elution was performed with a gradient of 0% to 90% ethanol-water as the mobile phase. 10L of eluent rich in 20(S)-Rg3 was collected according to the HPLC results.

[0067] (5) Removal of insoluble impurities: Under pressure of 100-150 mbar and temperature of 65℃, 10 L of eluent rich in 20(S)-Rg3 was concentrated to 25 ml under reduced pressure, a small amount of ethanol was added to dissolve and the insoluble impurities were removed by filtration to obtain macroporous concentrate of 20(S)-Rg3.

[0068] (6) Silica gel column elution: The macroporous concentrate was wet-loaded onto a silica gel column with a column volume of 300 ml. Then, a mobile phase of isopropyl acetate: 83% ethanol-water = 78:22 was prepared and eluted at a rate of 3 BV / h for 7 column volumes. The silica gel column eluent rich in 20(S)-Rg3 was collected according to the HPLC results.

[0069] (7) Crystallization: Under pressure of 300 mbar and temperature of 60℃, the silica gel column eluent was evaporated under reduced pressure, and 60 ml of 75% ethanol aqueous solution was added to dissolve it at 60℃. Then, the temperature was gradually reduced to 15℃ at a rate of 15℃ / h, with water added dropwise while stirring to reduce the ethanol aqueous solution to 45%. Crystallization was carried out for 30 min, and the crystals were separated by filtration. The crystallization was repeated twice. Finally, the obtained crystals were vacuum dried at 75℃ for 24 h to obtain 3.5 g of 20(S)-Rg3 (rare ginsenoside Rg3) product. Its HPLC detection is shown in [see figure]. Figure 1 The purity of 20(S)-Rg3 is 98%.

[0070] Example 2: Experiment on the repair efficacy of rare ginsenoside Rg3 1

[0071] The relative migration rate of cells was observed using a keratinocyte assay. After cell resuscitation, when the cell plating rate reached approximately 60%, cells were seeded into 6-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2). Blank and positive controls were also included, and the working solutions of the test substances were prepared according to the test groups in Table 1 below.

[0072] Table 1 Test Groups

[0073]

[0074] According to the test groups, when the cell seeding rate in the 6-well plates reached approximately 70%–80%, the cells were administered to the appropriate groups, with three replicates per group. 2 mL of culture medium was added to each well of the BC group, 2 mL of EGF-containing culture medium was added to each well of the PC group, and 2 mL of culture medium containing the corresponding test sample was added to each well of the sample group. After administration, the 6-well plates were incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours. After incubation, scratching was performed. After scratching, the cells were washed twice with PBS, and cell culture medium was added, followed by incubation in a CO2 incubator (37℃, 5% CO2) for 24 hours. Cell images were taken under a 4x microscope 0 hours after scratching, and again 24 hours after scratching, after washing once with PBS, and taken under a 4x microscope. Cell migration patterns are shown below. Figure 2 The average relative cell migration rate is shown in the figure. Figure 3 The cell migration results are shown in Table 2. The method for calculating the improvement rate is as follows:

[0075]

[0076] Table 2 Summary of Cell Migration Results

[0077]

[0078] Note: When performing statistical analysis using the t-test method, significance compared to group BC is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **. Rare ginsenoside Rg3, also known as 20(S)-Rg3.

[0079] Based on keratinocytes, compared with the control group, the sample At a concentration of 0.0004% (m / v), rare ginsenoside Rg3 (Rg3) significantly increased the relative cell migration rate, with an increase rate of 38.00%, indicating that the sample can improve the relative cell migration rate and has a repair effect at this concentration.

[0080] Example 3: Experiment on the repair efficacy of rare ginsenoside Rg3 2

[0081] 3D epidermal skin model based on UVB irradiation Compared with the control group, the sample At a concentration of 0.1% (m / v), rare ginsenoside Rg3 significantly decreased the number of Sunburn cells and the content of DNA damage cyclobutane pyrimidine dimer (CPD), with inhibition rates of 75.00% and 37.20%, respectively. The contents of PPARα and filaggrin (FLG) significantly increased, with enhancement rates of 129.73% and 100.00%, respectively. The positive expression rate of nuclear proliferation-associated antigen (Ki67) also significantly increased, with an enhancement rate of 102.24%. This indicates that at this concentration, the sample can improve tissue morphology, downregulate the content of DNA damage cyclobutane pyrimidine dimer (CPD), increase the content of PPARα and filaggrin (FLG), and enhance the positive expression rate of nuclear proliferation-associated antigen (Ki67), demonstrating a repairing effect, as detailed below.

[0082] 3.1 Materials and Methods

[0083] 3D epidermal skin model based on UVB irradiation The following tests were performed on the groups listed in the table below to detect tissue morphology, PPARα, DNA damage cyclobutane pyrimidine dimer (CPD), filaggrin (FLG) content, and cell nuclear proliferation-related parameters.

[0084] Table 3 Test Groups

[0085]

[0086]

[0087] Note: PC1 (WY14643) is a positive control for detecting FLG, PPAR-α, and Ki67, and PC2 (VE) is a positive control for detecting tissue morphology and DNA damage CPD.

[0088] According to the test groups, the models were transferred to 6-well plates (with 0.9 mL of EpiGrowth medium added beforehand), and the test group numbers were labeled on the 6-well plates. Groups BC and NC received no treatment, group PC received the appropriate concentration of working solution, and the working solution for the sample groups was evenly distributed on the model surface. Except for groups BC, all other groups underwent UVB irradiation at a dose of 600 mJ / cm². 2 After irradiation, the 6-well plates were placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 hours. The enhancement rate and inhibition rate were calculated using the following formulas:

[0089]

[0090] 3.2 Tissue Morphology Detection

[0091] The model used for testing was fixed with 4% paraformaldehyde for 24 hours, and then subjected to...

[0092] H&E staining was performed, and images were taken and analyzed under a microscope. The number of Sunburn cells is summarized in Table 4, and the tissue morphology test results are as follows: Figure 9 As shown in the figure, the red circle in the figure is labeled Sunburncell.

[0093] Table 4 Summary of Sunburn cell quantity data

[0094]

[0095]

[0096] Note: When performing statistical analysis using the t-test method, significance compared with the BC group is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; significance compared with the NC group is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **, and PC2 is VE.

[0097] As shown in the figure, compared with the BC group, the number of Sunburn cells in the NC group was significantly increased, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the number of Sunburn cells in the PC2 group was significantly decreased, indicating that the positive control in this test was effective. Compared with the NC group, the sample... The number of Sunburn cells was significantly reduced by rare ginsenoside Rg3-0.1%, with an inhibition rate of 75.00%.

[0098] 3.3 Immunohistochemical detection

[0099] The model used for testing was fixed with 4% paraformaldehyde for 24 hours. Immunohistochemical detection was performed, and images were taken and analyzed under a microscope.

[0100] 1) DNA damage cyclobutane pyrimidine dimer (CPD) test results: DNA damage CPD immunohistochemistry results are as follows: Figure 10 As shown in the figure, the results of the DNA damage CPD immunohistochemical analysis are summarized as follows: Figure 5 As shown in Table 5.

[0101] Table 5 Summary of Immunohistochemical Analysis Results of DNA Damage CPD

[0102]

[0103] Note: The average relative integrated optical density (IOD) reflects the content of DNA damage CPD, and PC2 is VE.

[0104] Compared to the BC group, the DNA damage CPD content in the NC group was significantly increased, indicating that the stimulation conditions in this test were effective. Compared to the NC group, the DNA damage CPD content in the PC group was significantly decreased, indicating that the positive control in this test was effective. Compared to the NC group, the sample... Rare ginsenoside Rg3-0.1% significantly reduced the content of DNA damage CPD.

[0105] 2) Results of positive expression rate test for nuclear proliferation-associated antigen (Ki67): Summary of Ki67 immunohistochemical results as follows Figure 11 As shown in Table 6, the immunohistochemical analysis of Ki67 is presented.

[0106] Table 6 Summary of Ki67 Immunohistochemical Analysis Results

[0107]

[0108] Note: Positive cell rate refers to the percentage of Ki67-positive cells out of the total number of cells; PC1 is WY14643.

[0109] Compared with the BC group, the Ki67-positive cell rate in the NC group was significantly lower, indicating that the stimulation conditions were effective in this test. Compared with the NC group, the Ki67-positive cell rate in the PC group was significantly higher, indicating that the positive control was effective in this test. Compared with the NC group, the sample... The percentage of Ki67 positive cells with rare ginsenoside Rg3-0.1% increased significantly, with an increase rate of 102.24%.

[0110] 3.4 Immunofluorescence assay

[0111] The model used for detection was fixed with 4% paraformaldehyde for 24 hours. Immunofluorescence detection was then performed, and images were taken and analyzed under a fluorescence microscope. The results of filaggrin (FLG) immunofluorescence are as follows: Figure 12 As shown in Table 7, the immunofluorescence analysis results are shown in Table 8; the PPAR-α immunofluorescence results are shown in Table 9. Figure 13 As shown in Table 8, the results of PPAR-α immunofluorescence analysis are shown in Table 8.

[0112] Table 7 Summary of FLG Immunofluorescence Analysis Results

[0113]

[0114] Note: The average relative integrated optical density (IOD) reflects the FLG content. PC1 is WY14643.

[0115] Compared with the BC group, the FLG content in the NC group was significantly lower, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the FLG content in the PC group was significantly higher, indicating that the positive control in this test was effective. Compared with the NC group, the sample... The content of rare ginsenoside Rg3-0.1% FLG increased significantly, with an increase rate of 100.00%.

[0116] Table 8 Summary of PPAR-α Immunofluorescence Analysis Results

[0117]

[0118] Note: The average relative integrated optical density (IOD) reflects the content of PPAR-α. PC1 is WY14643.

[0119] Compared with the BC group, the PPAR-α content in the NC group was significantly lower, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the PPAR-α content in the PC group was significantly higher, indicating that the positive control in this test was effective. Compared with the NC group, the sample... The content of PPAR-α in rare ginsenoside Rg3-0.1% increased significantly, with an increase rate of 129.73%.

[0120] discuss

[0121] In summary, the present invention Rare ginsenoside Rg3 (0.1% concentration) has demonstrated superior technical effects far exceeding those of existing products through multi-dimensional biological indicator verification, especially in skin protection, repair, and barrier function strengthening. Its beneficial effects are not achieved through a single pathway, but rather through a synergistic network, specifically manifested in the following four key aspects:

[0122] UV damage repair and cell protection capabilities: As shown by the experimental data above, the present invention... Rare ginsenoside Rg3 can significantly reduce the number of sunburned cells by 75.00%. Such a high inhibition rate indicates that the rare ginsenoside Rg3 of this invention can extremely effectively protect skin cells from the deadly damage of ultraviolet radiation, reducing skin damage at its source. Furthermore, this invention… Rare ginsenoside Rg3 can also significantly reduce the content of cyclobutane pyrimidine dimer (CPD), a marker of DNA damage, which confirms that its protective effect extends to the gene level and can reduce the risk of gene mutations caused by ultraviolet radiation, providing a fundamental guarantee for achieving long-term skin health.

[0123] Barrier function reconstruction and moisturizing effects: This invention The rare ginsenoside Rg3 exhibits a 100.00% increase in the concentration of filaggrin (FLG), a core component of the skin barrier. Filagrin is fundamental to maintaining skin hydration and the integrity of the physical barrier; doubling its content signifies a fundamental enhancement of the skin's water-locking ability and its resistance to external stimuli. This effect far surpasses that of ordinary moisturizing ingredients, indicating that the rare ginsenoside Rg3 of this invention activates the skin's own barrier repair mechanism at the molecular level.

[0124] Activating cell regeneration and promoting tissue regeneration: Unlike simple protection, this invention... Rare ginsenoside Rg3 can actively promote healthy skin renewal. The above experiments show that it can increase the positive rate of Ki67, a marker of nuclear proliferation, by 102.24%. This means that the proliferative activity of basal cells in the skin is significantly activated, doubling the number of new cells, thereby accelerating the repair and normal turnover of damaged skin, keeping the skin youthful and plump.

[0125] Regulating key biological pathways to achieve synergistic effects: This invention Rare ginsenoside Rg3 enhanced PPAR-α by up to 129.73%, which is one of the core mechanisms by which it achieves multiple benefits. PPAR-α is a key nuclear receptor for cellular energy metabolism, lipid synthesis, and inflammation regulation. Its significant enhancement can optimize the health of skin cells globally, promote lipid synthesis to improve the skin barrier, enhance cellular energy to support repair, and work synergistically with the above effects to jointly build a healthy and resilient skin internal environment.

[0126] In summary, the ginsenoside Rg3 of this invention does not possess a single function; it constructs a complete chain of action from "gene protection (reducing CPD) → cell protection (reducing sunburned cells) → promoting cell regeneration (increasing Ki67) → barrier reconstruction (increasing FLG)," and is globally regulated by the key target PPAR-α. This multi-target, synergistic mechanism makes this invention... Rare ginsenoside Rg3 exhibits comprehensive and powerful technological advantages that are difficult for existing products to match in preventing photoaging, repairing damaged skin barriers, and promoting skin health.

[0127] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing rare ginsenoside Rg3, characterized in that, The method includes the following steps: (1) Fermentation with yeast strain in the presence of glucose to obtain fermentation broth containing ginsenoside 20(S)-Rg3, wherein the yeast strain has the preservation number CCTCC M 20232647. (2) The fermentation broth containing 20(S)-Rg3 obtained in step (1) was heated, then diatomaceous earth was added for adsorption, and then the mixture was separated by filtration. (3) Extract the solid product obtained by adsorption in step (2) with ethanol to obtain an organic phase extract; (4) After concentrating the organic phase extract obtained in step (3), the extract is purified by chromatography through a macroporous resin column and a silica gel column in sequence. The eluent rich in 20(S)-Rg3 is collected and then recrystallized to obtain the rare ginsenoside Rg3.

2. The method as described in claim 1, characterized in that, In step (1), the concentration of glucose in the fermentation medium is 5-60 g / L.

3. The method as described in claim 1, characterized in that, The fermentation described in step (1) is carried out in a fed medium containing glucose and ethanol; preferably, the mass ratio of glucose to ethanol in the fed medium is 1.8-2.2:1, wherein the initial concentration of glucose is 380-420 g / L.

4. The method as described in claim 1, characterized in that, In step (1), the concentration of the yeast strain in the fermentation medium is 100-150 g dry weight / L.

5. The method as described in claim 1, characterized in that, In step (4), the filler used in the macroporous resin column is a non-polar or weakly polar macroporous adsorption resin with polystyrene as the skeleton. The macroporous resin is selected from the following group: D101, AB-8, LX-T83, LX-T28, DM130, Amberlite XAD series, HP series, SP800 series, SP70 series, or SP700 series macroporous resin.

6. The method as described in claim 1, characterized in that, In step (4), the silica gel column chromatography purification includes using 80-85 v / v ethanol-water as the mobile phase.

7. A composition, characterized in that, The composition comprises the rare ginsenoside Rg3 as described in claim 1 as an active ingredient.

8. The composition according to claim 7, characterized in that, The dosage form of the composition is selected from the group consisting of: solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, detergent, oil, foundation, emulsion foundation, wax foundation, face mask, massage cream, or spray.

9. The composition according to claim 8, characterized in that, The concentration of rare ginsenoside Rg3 in the composition is 0.05-0.15 g / L.

10. The use of the rare ginsenoside Rg3 as described in claim 1 and the composition as described in claim 8, characterized in that, Used to prepare cosmetic compositions that promote skin repair, strengthen skin texture, delay skin aging, improve skin wrinkles, or enhance skin elasticity.

Images