Method for generating rare ginsenoside Rh2 and application of rare ginsenoside Rh2

Abstract

The invention discloses a method for generating rare ginsenoside Rh2, which comprises the following steps: adding common glycol type ginsenoside into aspergillus brazilis fermentation liquor for fermentation, and sterilizing after fermentation to obtain a fermentation product; and carrying out enzymolysis by using beta-glucanase and pectinase, and carrying out enzyme deactivation after the enzymolysis is finished, so as to obtain the rare ginsenoside Rh2. The invention also provides the rare ginsenoside Rh2 prepared by the method and application of the rare ginsenoside Rh2. The common glycol type ginsenoside is efficiently and quickly converted into the rare ginsenoside Rh2 through the synergistic interaction of the aspergillus brazilis, the beta-glucanase and the pectinase; the rare ginsenoside Rh2 has the advantages of being simple in preparation method, easy to implement, low in cost, environmentally friendly, free of pollution and the like, and the generated rare ginsenoside Rh2 shows excellent skin care and repairing effects and has wide market prospects when applied to cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of plant active ingredient extraction technology, specifically relating to a method for generating rare ginsenoside Rh2 and its application. Background Technology

[0002] Ginsenosides, also known as ginsenoside derivatives, are tetracyclic triterpenoid saponins extracted from plants of the Araliaceae family (American ginseng, ginseng, Panax notoginseng, etc.). Ginsenosides possess activities such as relieving fatigue, improving memory, anti-aging, scavenging free radicals, regulating the central nervous system, and anti-cancer properties. Research has found that secondary metabolic derivatives of ginsenosides, after transformation, exhibit even stronger biological activity. These secondary metabolic derivatives are named "rare ginsenosides," while ginsenosides extracted directly from Araliaceae plants are called "prototype ginsenosides." Currently, more than 60 rare ginsenosides have been discovered, including CK, F2, Rk2, Rh2, and Rg5, among others, each possessing different biological activities.

[0003] Ginsenoside Rh2, a rare saponin extracted from red ginseng, has made initial progress in anti-cancer research in recent years. Ginsenoside Rh2 can reduce intracellular calcium ion concentration and inhibit PKCα-mediated proliferation signaling. Simultaneously, ginsenoside Rh2 can also inhibit the proliferation of liver cancer cells, possibly by blocking the IGFIR proliferation signaling pathway in liver cancer cells and reducing its expression. Furthermore, ginsenoside Rh2 can regulate the Caspase and Bcl-2 signaling pathways, inducing apoptosis to achieve anti-tumor effects. Ginsenoside Rh2 can effectively inhibit telomerase activity in tumor cells, reversing the transcriptional level of telomerase on transcriptase mRNA, causing tumor cells to enter a senescent phase. Ginsenoside Rh2 can block the synthesis of certain important components in tumor cells or inhibit their expression, such as mediating changes in JNK1 to induce apoptosis, and inducing p21 protein expression and reducing cyclin D levels to inhibit MCF-7 cell proliferation. These preliminary results have made ginsenoside Rh2 a rising star in cancer research, and many cancer patients and health care groups have begun to pay attention to its potential therapeutic effects.

[0004] Traditional extraction methods yield ginsenoside Rh2 in extremely low quantities and at very high costs. Therefore, there is an urgent need for a simple, high-yield, and low-cost method for extracting ginsenoside Rh2. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned technical problems by providing a simple, high-yield, and low-cost method for generating rare ginsenoside Rh2.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for generating rare ginsenoside Rh2, which includes the following steps: S1. Add common diol-type ginsenosides to the Aspergillus brasiliensis fermentation broth for fermentation, sterilize after fermentation, and obtain the fermentation product. 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.

[0007] Preferably, in step S1, the common diol-type ginsenoside is ginsenoside Rb1.

[0008] Preferably, in step S1, the amount of ordinary diol-type ginsenoside added is 0.1-20 mg / mL, more preferably 1-10 mg / mL.

[0009] Preferably, in step S1, the stirring speed is 150 rpm.

[0010] Preferably, in step S1, the fermentation temperature is 25-35℃, more preferably 25-30℃.

[0011] Preferably, in step S1, the fermentation time is 5-15 days, more preferably 10-15 days.

[0012] 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.

[0013] Preferably, the liquid culture medium is Sabouraud dextrose liquid culture medium.

[0014] Preferably, in step S2, the mass concentration of β-glucanase added is 0.1-4%, more preferably 1-4%.

[0015] Preferably, in step S2, the mass concentration of pectinase added is 0.1-2%, more preferably 0.5-2%.

[0016] Preferably, in step S2, the enzymatic hydrolysis temperature is 50°C.

[0017] Preferably, in step S2, the pH of the enzymatic hydrolysis is 5.0.

[0018] Preferably, in step S2, the stirring speed is 150 rpm.

[0019] Preferably, in step S2, the enzymatic hydrolysis time is 1-6 days, more preferably 2-4 days.

[0020] Secondly, the present invention also provides rare ginsenoside Rh2 prepared according to the method.

[0021] Thirdly, the present invention also provides the application of the rare ginsenoside Rh2 in skin care products.

[0022] Thirdly, the present invention also provides the application of the rare ginsenoside Rh2 in skin repair.

[0023] 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

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] Liquid culture medium: Weigh 30g of Sabouraud dextrose liquid medium, add 1L of deionized water, stir and heat to boiling until completely dissolved, and sterilize at 121℃ for 15 minutes.

[0031] 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.

[0032] The effects of different combinations of microorganisms (enzymes) on the transformation of rare ginsenoside Rh2.

[0033] Example 1 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.

[0034] 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.

[0035] Comparative Example 1 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.

[0036] 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.

[0037] Comparative Example 2 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.

[0038] 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.

[0039] Comparative Example 3 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.

[0040] 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.

[0041] Comparative Example 4 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.

[0042] 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.

[0043] Comparative Example 5 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.

[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 6 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.

[0046] 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.

[0047] Comparative Example 7 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.

[0048] 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.

[0049] Comparative Example 8 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.

[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 9 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.

[0052] 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.

[0053] The yield results of rare ginsenoside Rh2 in Example 1 and Comparative Examples 1-9 are shown in Table 1 below.

[0054] Table 1. Yield of rare ginsenoside Rh2 in Example 1 and Comparative Examples 1-9

[0055] Comparative Example 10 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.

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

[0058] Table 2. Yield of rare ginsenoside Rh2 in Comparative Example 10

[0059] Effect of substrate concentration on the conversion of rare ginsenoside Rh2 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.

[0060] 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.

[0061] The results are shown in Table 3 below.

[0062] Table 3. Concentrations of rare ginsenoside Rh2 corresponding to different substrate concentrations

[0063] Effect of fermentation temperature on the conversion of rare ginsenoside Rh2 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.

[0064] 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.

[0065] The results are shown in Table 4 below.

[0066] Table 4. Concentration of rare ginsenoside Rh2 corresponding to different fermentation temperatures

[0067] Effect of fermentation time on the conversion of rare ginsenoside Rh2 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.

[0068] 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.

[0069] The results are shown in Table 5 below.

[0070] Table 5. Concentration of rare ginsenoside Rh2 corresponding to different fermentation times

[0071] Effect of β-glucanase concentration on the conversion of rare ginsenoside Rh2 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.

[0072] 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.

[0073] The results are shown in Table 6 below.

[0074] Table 6. Concentrations of rare ginsenoside Rh2 corresponding to different β-glucanase concentrations

[0075] Effect of pectinase concentration on the conversion of rare ginsenoside Rh2 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.

[0076] 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.

[0077] The results are shown in Table 7 below.

[0078] Table 7. Concentrations of rare ginsenoside Rh2 corresponding to different pectinase concentrations

[0079] Effects of β-glucanase and pectinase hydrolysis time on the conversion of rare ginsenoside Rh2 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.

[0080] 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.

[0081] The results are shown in Table 8 below.

[0082] Table 8. Concentrations of rare ginsenoside Rh2 corresponding to different pectinase hydrolysis times

[0083] Skin repair efficacy verification experiment 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.

[0084] 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.

[0085] Cells were divided into a blank group (no UVB irradiation), a UVB irradiation control group, and a UVB irradiation experimental group.

[0086] 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.

[0087] Relative ATP content = (A1 - B0) / (B1 - B0) * 100% 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.

[0088] The test results are shown in Table 9 below.

[0089] Table 9. Results of the Skin Repair Efficacy Verification Experiment

[0090] 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.

[0091] 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 generating rare ginsenoside Rh2, characterized in that, Includes the following steps: S1. Add common diol-type ginsenosides to the Aspergillus brasiliensis fermentation broth for fermentation, sterilize after fermentation, and obtain the fermentation product. S2. The fermentation product is hydrolyzed with β-glucanase and pectinase. After hydrolysis, the enzymes are inactivated to obtain rare ginsenoside Rh2.

2. The method according to claim 1, characterized in that, In step S1, the common diol-type ginsenoside is ginsenoside Rb1.

3. The method according to claim 1, characterized in that, In step S1, the amount of ordinary diol-type ginsenoside added is 0.1-20 mg / mL.

4. The method according to claim 1, characterized in that, In step S1, the stirring speed is 150 rpm, the fermentation temperature is 25-35℃, and the fermentation time is 5-15 days.

5. The method according to claim 1, characterized in that, In step S2, the mass concentration of β-glucanase added is 0.1-4%, and the mass concentration of pectinase added is 0.1-2%.

6. The method according to claim 1, characterized in that, The enzymatic hydrolysis temperature is 50℃, the pH is 5.0, the stirring speed is 150 rpm, and the enzymatic hydrolysis time is 1-6 days.

7. The method according to claim 1, characterized in that, The method for preparing the Aspergillus brasiliensis fermentation broth is as follows: Aspergillus brasiliensis is inoculated into a liquid culture medium and cultured at 25-30℃ 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.

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