A method for generating rare ginsenoside Rh2 and its application

Abstract

This invention discloses a method for generating rare ginsenoside Rh2, comprising the following steps: adding common diol-type ginsenosides to Aspergillus brasiliensis fermentation broth for fermentation, sterilizing after fermentation to obtain the fermentation product; then enzymatically hydrolyzing with β-glucanase and pectinase, and inactivating the enzymes after hydrolysis to obtain rare ginsenoside Rh2. This invention also provides the rare ginsenoside Rh2 obtained by this method and its applications. This invention utilizes the synergistic effect of Aspergillus brasiliensis, β-glucanase, and pectinase to efficiently and rapidly convert common diol-type ginsenosides into rare ginsenoside Rh2. It has the advantages of simple preparation method, ease of implementation, low cost, and environmental friendliness. The generated rare ginsenoside Rh2 exhibits excellent skin-care and repairing effects and has broad market prospects for application in cosmetics.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant active ingredient extraction, and particularly relates to a method for generating rare ginsenoside Rh2 and application thereof. BACKGROUND

[0002] Ginsenoside, also known as ginsenoside, is a kind of tetracyclic triterpenoid saponin extracted from plants of the Araliaceae family (Panax quinquefolium, Panax ginseng, Panax notoginseng, etc.). Ginsenoside has the activities of relieving fatigue, improving memory, resisting aging, eliminating free radicals in the body, regulating the central nervous system of the brain, resisting cancer, etc. Research has found that secondary metabolite derivatives of ginsenoside have stronger biological activity. Such secondary metabolite derivatives are named "rare ginsenoside", and ginsenoside directly extracted from plants of the Araliaceae family is called "prototype ginsenoside". At present, more than 60 kinds of rare ginsenosides have been found, including CK, F2, Rk2, Rh2, Rg5 and other rare ginsenosides with different biological activities.

[0003] Ginsenoside Rh2 is a rare saponin extracted from red ginseng, and has made preliminary progress in the research of resisting cancer in recent years. Ginsenoside Rh2 can reduce the intracellular calcium ion concentration and hinder the PKCα-mediated proliferation signal transduction process. Meanwhile, ginsenoside Rh2 can also inhibit the proliferation of hepatocarcinoma cells, and it can block the proliferation signal transduction pathway of IGFIR in hepatocarcinoma cells and reduce its expression in hepatocarcinoma cells. In addition, ginsenoside Rh2 can regulate the signal pathways of Caspase and Bcl-2, induce apoptosis to achieve the effect of resisting tumors. Ginsenoside Rh2 can effectively inhibit the activity of telomerase in tumor cells, reverse the transcription level of telomerase on the mRNA of transcription enzyme, and make tumor cells enter the aging period. Ginsenoside Rh2 can block some important components in tumor cells and inhibit or suppress their expression, such as mediating the change of JNK1 to promote the apoptosis of tumor cells, inducing the expression of p21 protein and reducing the level of cyclin D to inhibit the proliferation of MCF-7 cells. These preliminary results make ginsenoside Rh2 a new star in the field of cancer research, and many tumor patients and health care groups have also begun to pay attention to its potential efficacy.

[0004] The yield of ginsenoside Rh2 obtained by traditional process extraction is extremely low, and the cost is very high. Therefore, there is an urgent need for a ginsenoside Rh2 extraction method with simple preparation method, high yield and low cost. SUMMARY

[0005] The purpose of the present application is to solve the above technical problems, and provide a method for generating rare ginsenoside Rh2 with simple preparation method, high yield and low cost.

[0006] To achieve the above application purpose, the present application provides the following technical solutions:

[0007] In a first aspect, the present invention provides a method for generating rare ginsenoside Rh2, which includes the following steps:

[0008] S1. Add common diol-type ginsenosides to the Aspergillus brasiliensis fermentation broth for fermentation, sterilize after fermentation, and obtain the fermentation product.

[0009] S2. The fermentation product is enzymatically hydrolyzed with β-glucanase and pectinase. After the enzymatic hydrolysis is completed, the enzymes are inactivated to obtain rare ginsenoside Rh2.

[0010] Preferably, in step S1, the common diol-type ginsenoside is ginsenoside Rb1.

[0011] Preferably, in step S1, the amount of ordinary diol-type ginsenoside added is 0.1-20 mg / mL, more preferably 1-10 mg / mL.

[0012] Preferably, in step S1, the stirring speed is 150 rpm.

[0013] Preferably, in step S1, the fermentation temperature is 25-35℃, more preferably 25-30℃.

[0014] Preferably, in step S1, the fermentation time is 5-15 days, more preferably 10-15 days.

[0015] Preferably, the method for preparing the Aspergillus brasiliensis fermentation broth is as follows: Aspergillus brasiliensis inoculates the strain into a liquid culture medium and incubates at 25-30°C until the bacterial density reaches 1×10⁻⁶. 6 When the concentration of CFU / mL is reached, solid-liquid separation is performed, and the supernatant is used as the fermentation broth for Aspergillus brasiliensis.

[0016] Preferably, the liquid culture medium is Sabouraud dextrose liquid culture medium.

[0017] Preferably, in step S2, the mass concentration of β-glucanase added is 0.1-4%, more preferably 1-4%.

[0018] Preferably, in step S2, the mass concentration of pectinase added is 0.1-2%, more preferably 0.5-2%.

[0019] Preferably, in step S2, the enzymatic hydrolysis temperature is 50°C.

[0020] Preferably, in step S2, the pH of the enzymatic hydrolysis is 5.0.

[0021] Preferably, in step S2, the stirring speed is 150 rpm.

[0022] Preferably, in step S2, the enzymatic hydrolysis time is 1-6 days, more preferably 2-4 days.

[0023] Secondly, the present invention also provides rare ginsenoside Rh2 prepared according to the method.

[0024] Thirdly, the present invention also provides the application of the rare ginsenoside Rh2 in skin care products.

[0025] Thirdly, the present invention also provides the application of the rare ginsenoside Rh2 in skin repair.

[0026] This invention utilizes the synergistic effect of *Aspergillus brasiliensis*, β-glucanase, and pectinase to efficiently and rapidly convert common diol-type ginsenosides (such as ginsenoside Rb1) into rare ginsenoside Rh2. The method is simple, easy to implement, low-cost, and environmentally friendly. This rare ginsenoside Rh2 exhibits excellent skin-care and repairing effects, and has broad market prospects for application in cosmetics. Detailed Implementation

[0027] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0028] In the description of this invention, unless otherwise explicitly defined, terms such as heating, cleaning, weighing, and freezing should be interpreted broadly. Those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0029] In the description of this invention, references to terms such as "some embodiments" and "examples" indicate that the specific methods or materials described in connection with that embodiment or example are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the specific methods and materials described may be combined in any suitable manner in one or more embodiments or examples.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0032] In the following examples, all raw materials used are commercially available products: Aspergillus brasiliensis ( Aspergillus brasiliensis The ATCC standard was 16404; ginsenoside Rb1 was purchased from Shaanxi Jinkangtai Biotechnology Co., Ltd.; β-glucanase and pectinase were purchased from Nanning Pangbo Bioengineering Co., Ltd.; Sabouraud dextrose liquid medium was purchased from Guangdong Huankai Microbial Technology Co., Ltd.

[0033] Liquid culture medium: Weigh 30g of Sabouraud dextrose liquid medium, add 1 L of deionized water, stir and heat to boiling until completely dissolved, and sterilize at 121℃ for 15 minutes.

[0034] Preparation of Aspergillus brasiliensis fermentation broth: Inoculate Aspergillus brasiliensis into liquid culture medium and incubate at 28℃ (culture temperature range can be 25-30℃) with shaking at 150 rpm for 72 hours. Add sterilized glass beads and shake at 200 rpm for 10 min until the bacterial density reaches 1×10⁻⁶. 6 When the concentration is CFU / mL, filter with sterile gauze and collect the filtrate, which is the Aspergillus brasiliensis fermentation broth.

[0035] Effect of different microbial (enzyme) combinations on the transformation of rare ginsenoside Rh2

[0036] Example 1

[0037] Ginsenoside Rb1 was added to the Aspergillus brasiliensis fermentation broth to achieve a final concentration of 5 mg / mL. The mixture was incubated at 30°C with shaking at 150 rpm for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes. After returning to room temperature, 1% w / w β-glucanase and 1% w / w pectinase were added for further enzymatic hydrolysis for 48 hours. The hydrolysis temperature was 50°C, the pH was 5.0, and the stirring speed was 150 rpm. After hydrolysis, the reaction was stopped by incubation at 90°C for 30 minutes to obtain the product.

[0038] Extraction was performed using an equal volume of n-butanol to the final reaction solution. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. HPLC detection method: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase (phase A: acetonitrile; phase B: 0.05% w / v phosphoric acid aqueous solution), gradient elution (0–10 min 50% A, 10–25 min 70% A), column temperature 25℃, detection wavelength 203 nm, flow rate 1 mL / min, injection volume 20 μL.

[0039] Comparative Example 1

[0040] Ginsenoside Rb1 was added to the Aspergillus brasiliensis fermentation broth to achieve a final concentration of 5 mg / mL. The mixture was incubated at 30°C with shaking at 150 rpm for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes to obtain the product.

[0041] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0042] Comparative Example 2

[0043] Ginsenoside Rb1 was dissolved in 0.05M acetate-sodium acetate buffer (pH 5.0) to a final concentration of 5 mg / mL, and then 1% w / w β-glucanase was added for enzymatic hydrolysis for 48 hours. The hydrolysis temperature was 50℃ and the stirring speed was 150 rpm. After the hydrolysis was completed, the reaction was stopped by incubation at 90℃ for 30 min to obtain the product.

[0044] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0045] Comparative Example 3

[0046] Ginsenoside Rb1 was dissolved in 0.05M acetate-sodium acetate buffer (pH 5.0) to a final concentration of 5 mg / mL, and then 1% w / w pectinase was added for enzymatic hydrolysis for 48 hours. The hydrolysis temperature was 50℃ and the stirring speed was 150 rpm. After the hydrolysis was completed, the reaction was stopped by incubation at 90℃ for 30 min to obtain the product.

[0047] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0048] Comparative Example 4

[0049] Ginsenoside Rb1 was added to the Aspergillus brasiliensis fermentation broth to achieve a final concentration of 5 mg / mL. The mixture was incubated at 30°C with shaking at 150 rpm for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes, allowed to return to room temperature, and then enzymatically hydrolyzed for 48 hours with 1% w / w β-glucanase. The hydrolysis temperature was 50°C, the pH was 5.0, and the stirring speed was 150 rpm. After hydrolysis, the reaction was stopped by incubation at 90°C for 30 minutes to obtain the product.

[0050] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0051] Comparative Example 5

[0052] Ginsenoside Rb1 was added to the Aspergillus brasiliensis fermentation broth to achieve a final concentration of 5 mg / mL. The mixture was incubated at 30°C with shaking at 150 rpm for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes, allowed to return to room temperature, and then enzymatically hydrolyzed with 1% w / w pectinase for 48 hours. The hydrolysis temperature was 50°C, the pH was 5.0, and the stirring speed was 150 rpm. After hydrolysis, the reaction was stopped by incubation at 90°C for 30 minutes to obtain the product.

[0053] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0054] Comparative Example 6

[0055] Ginsenoside Rb1 was dissolved in 0.05M acetate-sodium acetate buffer (pH 5.0) to a final concentration of 5 mg / mL. Then, 1% w / w β-glucanase and 1% w / w pectinase were added simultaneously for enzymatic hydrolysis for 48 hours. The hydrolysis temperature was 50℃, and the stirring speed was 150 rpm. After hydrolysis, the reaction was stopped by incubation at 90℃ for 30 minutes to obtain the product.

[0056] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0057] Comparative Example 7

[0058] Ginsenoside Rb1 was added to the Aspergillus brasiliensis fermentation broth to achieve a final concentration of 5 mg / mL. The mixture was incubated at 30°C with shaking at 150 rpm for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes. After returning to room temperature, 1% w / w β-glucanase was added for further enzymatic hydrolysis for 48 hours at 50°C and pH 5.0, with a stirring speed of 150 rpm. After hydrolysis, the reaction was stopped by incubation at 90°C for 30 minutes. After returning to room temperature, 1% w / w pectinase was added for further hydrolysis for 48 hours at 50°C and pH 5.0, with a stirring speed of 150 rpm. After hydrolysis, the reaction was stopped by incubation at 90°C for 30 minutes, yielding the product.

[0059] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0060] Comparative Example 8

[0061] Ginsenoside Rb1 was added to the Aspergillus brasiliensis fermentation broth to achieve a final concentration of 5 mg / mL. The mixture was incubated at 30°C with shaking at 150 rpm for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes, allowed to return to room temperature, and then further enzymatically hydrolyzed for 48 hours with 1% w / w cellulase and 1% w / w pectinase. The hydrolysis temperature was 50°C, the pH was 5.0, and the stirring speed was 150 rpm. After hydrolysis, the reaction was stopped by incubation at 90°C for 30 minutes to obtain the product.

[0062] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0063] Comparative Example 9

[0064] Ginsenoside Rb1 was added to the Aspergillus brasiliensis fermentation broth to achieve a final concentration of 5 mg / mL. The mixture was incubated at 30°C with shaking at 150 rpm for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes, allowed to return to room temperature, and then further enzymatically hydrolyzed for 48 hours with 1% w / w β-glucanase and 1% w / w xylanase. The hydrolysis temperature was 50°C, the pH was 5.0, and the stirring speed was 150 rpm. After hydrolysis, the reaction was stopped by incubation at 90°C for 30 minutes to obtain the product.

[0065] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0066] The yield results of rare ginsenoside Rh2 in Example 1 and Comparative Examples 1-9 are shown in Table 1 below.

[0067] Table 1. Yield of rare ginsenoside Rh2 in Example 1 and Comparative Examples 1-9

[0068]

[0069] Comparative Example 10

[0070] Ginsenoside Rg3 was suspended in 0.05M acetate-sodium acetate buffer (pH 5.0) to a final concentration of 5 mg / mL. Then, 1% w / w β-glucanase and 1% w / w pectinase were added simultaneously for enzymatic hydrolysis for 48 hours. The hydrolysis temperature was 50℃, and the stirring speed was 150 rpm. After hydrolysis, the reaction was stopped by incubation at 90℃ for 30 minutes to obtain the product.

[0071] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0072] The yield results of rare ginsenoside Rh2 in Comparative Example 10 are shown in Table 2 below.

[0073] Table 2. Yield of rare ginsenoside Rh2 in Comparative Example 10

[0074]

[0075] Effect of substrate concentration on the transformation of rare ginsenoside Rh2

[0076] Different concentrations (0.05, 0.1, 1, 10, 20, and 30 mg / mL) of the substrate ginsenoside Rb1 were added to the Aspergillus brasiliensis fermentation broth. The mixture was cultured at 30°C and 150 rpm with shaking for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes. After returning to room temperature, 1% w / w β-glucanase and 1% w / w pectinase were added simultaneously for further enzymatic hydrolysis for 48 hours. The hydrolysis temperature was 50°C, the hydrolysis pH was 5.0, and the stirring speed was 150 rpm. After hydrolysis, the mixture was incubated at 90°C for 30 minutes to stop the reaction and obtain the product.

[0077] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0078] The results are shown in Table 3 below.

[0079] Table 3. Concentrations of rare ginsenoside Rh2 corresponding to different substrate concentrations

[0080]

[0081] Effect of fermentation temperature on the transformation of rare ginsenoside Rh2

[0082] Ginsenoside Rb1 (5 mg / mL) was added to the Aspergillus brasiliensis fermentation broth. The mixture was cultured for 10 days at different temperatures (20, 25, 30, 35, 40 °C) with shaking at 150 rpm. After fermentation, the mixture was sterilized at 100 °C for 15 minutes, then allowed to return to room temperature. Simultaneously, 1% w / w β-glucanase and 1% w / w pectinase were added for further enzymatic hydrolysis for 48 hours. The hydrolysis temperature was 50 °C, the hydrolysis pH was 5.0, and the stirring speed was 150 rpm. After hydrolysis, the mixture was incubated at 90 °C for 30 minutes to stop the reaction and obtain the product.

[0083] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0084] The results are shown in Table 4 below.

[0085] Table 4. Concentration of rare ginsenoside Rh2 corresponding to different fermentation temperatures

[0086]

[0087] Effect of fermentation time on the transformation of rare ginsenoside Rh2

[0088] Ginsenoside Rb1 (5 mg / mL) was added to the Aspergillus brasiliensis fermentation broth. The mixture was incubated at 30°C with shaking at 150 rpm. After fermentation, the mixture was sterilized at 100°C for 15 minutes. After returning to room temperature, 1% w / w β-glucanase and 1% w / w pectinase were added simultaneously for further enzymatic hydrolysis for 48 hours. The hydrolysis temperature was 50°C, the pH was 5.0, and the stirring speed was 150 rpm. After hydrolysis, the mixture was incubated at 90°C for 30 minutes to stop the reaction and obtain the product.

[0089] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0090] The results are shown in Table 5 below.

[0091] Table 5. Concentration of rare ginsenoside Rh2 corresponding to different fermentation times

[0092]

[0093] Effect of β-glucanase concentration on the transformation of rare ginsenoside Rh2

[0094] Ginsenoside Rb1 (5 mg / mL) was added to the Aspergillus brasiliensis fermentation broth. The mixture was cultured at 30°C with shaking at 150 rpm for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes. After returning to room temperature, different concentrations (0.05%, 0.1%, 1%, 2%, 4%, 6%) of β-glucanase (as shown in Table 6) and 1% pectinase were added simultaneously for further enzymatic hydrolysis for 48 hours. The hydrolysis temperature was 50°C, the hydrolysis pH was 5.0, and the stirring speed was 150 rpm. After hydrolysis, the mixture was incubated at 90°C for 30 minutes to stop the reaction and obtain the product.

[0095] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0096] The results are shown in Table 6 below.

[0097] Table 6. Concentrations of rare ginsenoside Rh2 corresponding to different β-glucanase concentrations

[0098]

[0099] Effect of pectinase concentration on the transformation of rare ginsenoside Rh2

[0100] Ginsenoside Rb1 (5 mg / mL) was added to the Aspergillus brasiliensis fermentation broth. The mixture was cultured at 30°C with shaking at 150 rpm for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes. After returning to room temperature, 1% β-glucanase and different concentrations (0.05%, 0.1%, 0.5%, 1%, 2%, 3%) of pectinase (as shown in Table 7) were added simultaneously, and enzymatic hydrolysis was continued for 48 hours. The hydrolysis temperature was 50°C, the hydrolysis pH was 5.0, and the stirring speed was 150 rpm. After the hydrolysis was completed, the mixture was incubated at 90°C for 30 minutes to stop the reaction and obtain the product.

[0101] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0102] The results are shown in Table 7 below.

[0103] Table 7. Concentrations of rare ginsenoside Rh2 corresponding to different pectinase concentrations

[0104]

[0105] Effect of β-glucanase and pectinase enzymolysis time on the transformation of rare ginsenoside Rh2

[0106] Add 5 mg / mL of ginsenoside Rb1, the substrate, to the Aspergillus brasiliensis fermentation broth. Incubate at 30°C with shaking at 150 rpm for 10 days. After fermentation, sterilize at 100°C for 15 minutes, allow to return to room temperature, and then simultaneously add 1% w / w β-glucanase and 1% w / w pectinase (enzymatic hydrolysis times are shown in Table 8). The hydrolysis temperature is 50°C, the hydrolysis pH is 5.0, and the stirring speed is 150 rpm. After hydrolysis, incubate at 90°C for 30 minutes to stop the reaction and obtain the product.

[0107] An equal volume of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC. The HPLC detection method was the same as in Example 1.

[0108] The results are shown in Table 8 below.

[0109] Table 8. Concentrations of rare ginsenoside Rh2 corresponding to different pectinase hydrolysis times

[0110]

[0111] Skin repair efficacy verification experiment

[0112] ATP is the "energy" factor for skin cells, providing the energy source for cell repair. The ATP content in aging skin cells is 50% lower than that in young cells. Multiple internal and external factors, such as aging, environmental pollution, and ultraviolet radiation, lead to a significant decrease in ATP, making the skin more prone to aging problems such as dryness, dullness, and wrinkles.

[0113] Experimental method: Human skin fibroblasts were seeded into 6-well culture plates at a cell density of 50,000 cells / well. The culture medium was DMEM cell culture medium containing 10% fetal bovine serum. Each group had 3 replicates and the cells were cultured in an incubator at 37°C, 5% CO2, and 95% humidity for 48 hours.

[0114] Cells were divided into a blank group (no UVB irradiation), a UVB irradiation control group, and a UVB irradiation experimental group.

[0115] Two hours before irradiation, the blank control and irradiation control groups were given 1% (w / w) acetate / sodium acetate buffer, while the irradiation experimental groups were given 0.5% (w / w) of Example 1 and Comparative Example 10, respectively. After incubation for 2 hours, the supernatant of all groups was discarded, and the cells were washed with PBS. Then, an appropriate amount of PBS was added to cover the cells for irradiation. The blank control group was covered with aluminum foil. After irradiation, cells were further incubated with 1% (w / w) acetate / sodium acetate buffer or 1% (w / w) of Example 1 and Comparative Example 10. After 24 hours of incubation, the cells were analyzed according to the kit instructions (Beyotime CellTiter-Lumi). TM The luminescent cell viability assay kit (product number C0065S) is used to detect ATP content.

[0116] Relative ATP content = (A1 - B0) / (B1 - B0) * 100%

[0117] Where A1 represents the detection signal value of the irradiation control group or the irradiation experimental group, B1 represents the detection signal value of the blank group, and B0 represents the detection signal value of the solvent background well.

[0118] The test results are shown in Table 9 below.

[0119] Table 9. Results of the Skin Repair Efficacy Verification Experiment

[0120]

[0121] The experimental results above show that the rare ginsenoside Rh2, generated after the prototype ginsenosides are treated with Aspergillus brasiliensis, β-glucanase and pectinase, can alleviate the damage of ultraviolet rays to cell mitochondria, maintain the normal physiological function of mitochondria, and enhance the self-repair ability of the whole cell through the generation of ATP.

[0122] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for producing the rare ginsenoside Rh2, characterized by, The method comprises the following steps: S1, adding common glycosides to the fermentation liquor of Aspergillus brasiliensis for fermentation, sterilizing after the fermentation is completed, and obtaining a fermentation product; S2, using β-glucanase and pectinase to hydrolyze the fermentation product, and deactivating the enzymes after the hydrolysis is completed, to obtain rare ginsenoside Rh2; In the step S1, the common glycosides are ginsenoside Rb1; In the step S1, the addition amount of the common glycosides is 0.1-20 mg / mL; In the step S1, the stirring speed is 150 rpm, the fermentation temperature is 25-35℃, and the fermentation time is 5-15 days; In the step S2, the mass concentration of the β-glucanase is 0.1-4%, and the mass concentration of the pectinase is 0.1-2%; The hydrolysis temperature is 50℃, the pH is 5.0, the stirring speed is 150 rpm, and the hydrolysis time is 1-6 days.

2. The method of claim 1, wherein, The preparation method of the Aspergillus brasiliensis fermentation liquor is as follows: Aspergillus brasiliensis spores are inoculated into a liquid culture medium, and cultured at 25-30°C; when the bacterial density reaches 1×10 6 CFU / mL, solid-liquid separation is performed, and the supernatant is taken as the Aspergillus brasiliensis fermentation liquor.

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