A method for generating rare ginsenoside Rg3, composition and application thereof

Abstract

The application discloses a method for generating rare ginsenoside Rg3, which comprises the following steps: adding common diol type ginsenoside to a brazilian aspergillus fermentation liquor for fermentation, sterilizing after the fermentation is completed, and obtaining a fermentation product; and subjecting the fermentation product to enzymolysis by using a beta-glucanase, and deactivating the enzyme after the enzymolysis is completed, so as to obtain the rare ginsenoside Rg3. The application also discloses the rare ginsenoside Rg3 prepared by the method and a composition containing the same. The method of the application is completed by the cooperation of brazilian aspergillus fermentation and beta-glucanase hydrolysis, has high conversion efficiency, is easy to realize large-scale production, and can increase the solubility of the ginsenoside Rg3 in water, and the effect is obvious. The rare ginsenoside Rg3 has excellent skin soothing effect, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioactive ingredient extraction, and particularly relates to a method for generating rare ginsenoside Rg3, a composition and application thereof. BACKGROUND

[0002] Ginsenoside is a kind of tetracyclic triterpene saponin extracted from plants of the Araliaceae family (Panax quinquefolium, Panax ginseng, Panax notoginseng, etc.). The secondary metabolite after transformation of ginsenoside has stronger biological activity. This secondary metabolite is named "rare ginsenoside", and the ginsenoside directly extracted from the Araliaceae family is called "prototype ginsenoside". Rare ginsenoside Rg3 can improve the severe damage of mucosal local immune function caused by the combined action of tumors and cyclophosphamide, increase the area of small intestinal collection lymph nodes, and participate in the regulation of the balance of systemic immune Th1 / Th2 immune response to repair the tumor immune escape mechanism.

[0003] However, the ginsenoside Rg3 extracted by the traditional process has low yield and high cost. For example, although acid hydrolysis or alkali hydrolysis under normal pressure can obtain Rg3 from total ginsenoside, the whole process, including raw material treatment, hydrolysis and subsequent separation and purification, is complicated, and the final saponin yield is not high, which significantly increases the production cost. In addition, Rg3 mainly exists in the form of large-granularity flaky crystals. This crystal form leads to small specific surface area, slow absorption, poor solubility and low bioavailability, thereby affecting the full play of its active efficacy.

[0004] Therefore, there is an urgent need for a method capable of efficiently and quickly converting ordinary ginsenoside into rare ginsenoside Rg3. SUMMARY

[0005] The purpose of the present application is to provide a method capable of efficiently and quickly converting ordinary ginsenoside into rare ginsenoside Rg3 in order to solve the above technical problems.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] In a first aspect, the present application provides a method for generating rare ginsenoside Rg3, comprising the following steps:

[0008] S1. Adding ordinary diol-type ginsenoside to the fermentation broth of Aspergillus brasiliensis for fermentation, sterilizing after the fermentation is completed, and obtaining a fermentation product;

[0009] S2. Enzymatically hydrolyzing the fermentation product with β-glucanase, and deactivating the enzyme after the enzymatic hydrolysis is completed to obtain rare ginsenoside Rg3.

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

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

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

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

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

[0015] Preferably, the preparation method of the Aspergillus brasiliensis fermentation broth is inoculating Aspergillus brasiliensis into a liquid medium, culturing at 25-30℃, and when the bacterial density reaches 1×10 6 CFU / mL, performing solid-liquid separation, and taking the supernatant as the Aspergillus brasiliensis fermentation broth.

[0016] Preferably, in the step S2, the β-glucanase is added in a mass concentration of 0.1-4%, more preferably 1-2%.

[0017] Preferably, in the step S2, the enzymolysis temperature is 50℃.

[0018] Preferably, in the step S2, the pH of the enzymolysis is 5.0.

[0019] Preferably, in the step S2, the stirring speed is 150 rpm.

[0020] Preferably, in the step S2, the enzymolysis time is 12-72 hours, more preferably 24-48 hours.

[0021] In a second aspect, the present application provides a rare ginsenoside Rg3 prepared according to the method.

[0022] In a third aspect, the present application provides a composition comprising the rare ginsenoside Rg3 prepared according to the method, trideceth-9, a polyol, and deionized water.

[0023] Preferably, the polyol comprises any one or more of butanediol, glycerol, and propylene glycol.

[0024] Preferably, the mass ratio of the rare ginsenoside Rg3, trideceth-9, the polyol, and deionized water is 0.3:(3-9):(60-90):(3.7-33.7).

[0025] In a fourth aspect, the present application also provides the use of the composition in skin care products.

[0026] In a fifth aspect, the present application also provides the use of the composition in skin soothing.

[0027] The present application can quickly cut off the two glycosidic bonds at the 20th position of common ginsenoside by the synergistic effect of Aspergillus brasiliensis fermentation and beta-glucanase enzymolysis, thereby efficiently converting the common ginsenoside into the rare ginsenoside Rg3. The conversion efficiency is high, and large-scale production can be easily realized. In addition, the combination of tridecyl alcohol polyether-9 and polyhydric alcohol can significantly improve the solubility and skin soothing effect of ginsenoside Rg3. The raw materials are easy to obtain, the cost is low, the operation is simple and reliable, and the method is easy to realize. The method has good application prospect. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to specific examples. However, the present application can be realized in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0029] In the description of the present application, unless otherwise explicitly defined, the words such as heating, cleaning, weighing, freezing, etc. should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meaning of the above words in the present application in combination with the specific content of the technical solution.

[0030] In the description of the present application, the description of the terms "some embodiments", "examples" and the like means that the specific methods, materials described in combination with the embodiments or examples are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the described specific methods, materials can be combined in any one or more embodiments or examples in a suitable manner.

[0031] 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 the present application belongs. The terms used in the specification of the present application are only for the purpose of describing the specific embodiments and are not intended to limit the present application.

[0032] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0033] In the following examples, the raw materials used are commercially available: Aspergillus brasiliensis (Aspergillus brasiliensis) Aspergillus brasiliensisThe ATCC standard was 16404; ginsenoside Rb1 was purchased from Shaanxi Jinkangtai Biotechnology Co., Ltd.; β-glucanase was purchased from Nanning Pangbo Bioengineering Co., Ltd.; and Sabouraud dextrose liquid medium was purchased from Guangdong Huankai Microbial Technology Co., Ltd.

[0034] 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.

[0035] 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.

[0036] Effect of different microbial (enzyme) compositions and their addition order on the conversion of rare ginsenoside Rg3

[0037] Example 1

[0038] 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 1% w / w β-glucanase was 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. Finally, the reaction was stopped by incubation at 90°C for 30 minutes to obtain the product.

[0039] Extract with an equal volume of n-butanol, evaporate the n-butanol phase to dryness under reduced pressure, redissolve in anhydrous ethanol, and detect the transformation result 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.

[0040] Comparative Example 1

[0041] 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.

[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 2

[0044] Ginsenoside Rb1 was added to an acetate-sodium acetate buffer solution at pH 5.0 to achieve a final concentration of 5 mg / mL. Then, 1% w / w β-glucanase was added for hydrolysis over 48 hours. The hydrolysis temperature was 50°C, 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.

[0045] 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.

[0046] Comparative Example 3

[0047] Ginsenoside Rb1 was added to an acetate-sodium acetate buffer solution at pH 5.0 to achieve a final concentration of 5 mg / mL. Then, 1% w / w β-glucanase was added for hydrolysis over 48 hours. The hydrolysis temperature was 50°C, and the stirring speed was 150 rpm. After hydrolysis, the reaction was stopped by incubation at 90°C for 30 minutes. An equal volume of n-butanol was added for extraction, and the n-butanol phase was evaporated under reduced pressure to obtain the product.

[0048] The above product was added to the Aspergillus brasiliensis fermentation broth to achieve a final concentration of 5 mg / mL. The mixture was incubated at 30°C and 150 rpm with shaking for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes to obtain the product.

[0049] 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.

[0050] Comparative Example 4

[0051] 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 1% w / w xylanase was 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. Finally, 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 yields of rare ginsenoside Rg3 in Example 1 and Comparative Examples 1-4 are shown in Table 1.

[0054] Table 1. Yields of rare ginsenoside Rg3 in Example 1 and Comparative Examples 1-4

[0055]

[0056] Effect of substrate concentration on the conversion of rare ginsenoside Rg3

[0057] According to the concentrations shown in Table 2, ginsenoside Rb1 was 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, then allowed to return to room temperature before adding 1% w / w β-glucanase for 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 reaction was stopped by incubation at 90°C for 30 minutes to obtain the product.

[0058] 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.

[0059] The yields of rare ginsenoside Rg3 with different substrate concentrations are shown in Table 2 below.

[0060] Table 2. Yields of rare ginsenoside Rg3 at different substrate concentrations

[0061]

[0062] Effect of fermentation temperature on the conversion of rare ginsenoside Rg3

[0063] Ginsenoside Rb1 (5 mg / mL) was added to the Aspergillus brasiliensis fermentation broth. The mixture was cultured at 150 rpm with shaking at different temperatures for 10 days. After fermentation, the mixture was sterilized at 100°C for 15 minutes, then allowed to return to room temperature before adding 1% β-glucanase 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.

[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 yields of rare ginsenoside Rg3 at different fermentation temperatures are shown in Table 3 below.

[0066] Table 3. Yield of rare ginsenoside Rg3 at different fermentation temperatures

[0067]

[0068] Effect of fermentation time on the conversion of rare ginsenoside Rg3

[0069] 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, then allowed to return to room temperature before adding 1% β-glucanase 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.

[0070] 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.

[0071] The yields of rare ginsenoside Rg3 at different fermentation times are shown in Table 4 below.

[0072] Table 4. Yield of rare ginsenoside Rg3 at different fermentation times

[0073]

[0074] Effect of β-glucanase concentration on the conversion of rare ginsenoside Rg3

[0075] 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, then allowed to return to room temperature. Different concentrations of β-glucanase (as shown in Table 5) were added sequentially 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. The reaction was then stopped by incubation at 90°C for 30 minutes to 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 yields of rare ginsenoside Rg3 with different β-glucanase concentrations are shown in Table 5 below.

[0078] Table 5. Yield of rare ginsenoside Rg3 at different β-glucanase concentrations

[0079]

[0080] Effect of β-glucanase enzymolysis time on the conversion of rare ginsenoside Rg3

[0081] 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, then allowed to return to room temperature before adding 1% β-glucanase for enzymatic hydrolysis (hydrolysis times are shown in Table 6). The hydrolysis temperature was 50°C, the pH was 5.0, and the stirring speed was 150 rpm. The reaction was then stopped by incubating at 90°C for 30 minutes to obtain the product.

[0082] 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.

[0083] The yields of rare ginsenoside Rg3 at different enzymatic hydrolysis times are shown in Table 6 below.

[0084] Table 6. Yield of rare ginsenoside Rg3 at different enzymatic hydrolysis times

[0085]

[0086] Efficacy verification experiment

[0087] 1. Skin soothing efficacy verification experiment

[0088] Add 2 mL of RAW264.7 cell suspension to each 6-well plate, with 5 × 10⁶ cells per well. 5 Each sample was placed in a 37°C, 5% CO2 cell culture incubator and cultured for 6 hours. Different concentrations of the analyte were added to each well of the culture plate.

[0089] Groups: Blank group (without LPS); LPS control group; LPS experimental group. Triple replicates were performed. The blank group and LPS control group were added with 1% w / w acetate / sodium acetate buffer, while the LPS experimental group was added with 0.5% w / w from Example 1. The culture plates were incubated at 37°C in a cell culture incubator with 5% CO2 and 100% humidity for 24 hours. 1 µg / mL LPS was added to each well of the culture plate (except for the blank group), and the plates were incubated at 37°C in a 5% CO2 cell culture incubator for 16 hours. The culture medium was removed, and the cells were washed three times with phosphate-buffered saline. Cell samples were extracted using 1 mL of RNAiso Plus (TaKaRa) and transferred to 1.5 mL centrifuge tubes. Total RNA extraction and reverse transcription were performed according to the kit instructions, and quantitative PCR was used to detect the inflammatory factor TNF-α.

[0090] The results are shown in Table 7 below.

[0091] Table 7. Relative expression levels of TNF-α

[0092]

[0093] The above results indicate that the rare ginsenoside Rg3 prepared by the method of the present invention can effectively relieve inflammation and soothe the skin.

[0094] 2. Stability experiment

[0095] Compositions for improving the solubility of rare ginsenoside Rg3

[0096] Prepare the composition according to Table 8 below. Weigh component 1 and add it to component 3, stir at 50°C for 30 minutes, then add component 2 and component 4, stir until homogeneous, and conduct a stability study. The amounts of each component are by weight percentage.

[0097] Table 8. Composition Formulation

[0098]

[0099] Stability test

[0100] The stability of 5% aqueous solutions (mass concentration) of Examples 20-22 and Comparative Examples 15-23, as well as the solutions of Examples 20-22 and Comparative Examples 15-23, was investigated by placing them in a constant temperature incubator at 50°C for one month. The results are shown in Table 9.

[0101] Table 9. Stability Results

[0102]

[0103] The ginsenoside Rg3 obtained in Example 1 was prepared according to Table 10 below. Rg3 was weighed and added to butanediol, stirred at 50°C for 30 minutes, and then tridecyl alcohol polyether-9 and deionized water were added and stirred evenly. Stability was then investigated. The amounts of each component are expressed as a percentage by weight.

[0104] Table 10. Composition Formulation

[0105]

[0106] The stability of 1%, 3%, and 5% aqueous solutions (mass concentration) of Examples 20, 23-27, and Comparative Examples 24-26, as well as the solutions of Examples 20, 23-27, and Comparative Examples 24-26, was investigated by placing them in a 50°C incubator for one month. The results are shown in Table 11.

[0107] Table 11. Stability Results

[0108]

[0109] The experimental results above show that tridecanoyl alcohol polyether-9 and polyols play a crucial role in the stability of ginsenoside Rg3 raw material and the stability of Rg3 in the formulation.

[0110] 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 Rg3, 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 was hydrolyzed with β-glucanase. After hydrolysis, the enzyme was inactivated to obtain rare ginsenoside Rg3. In step S1, the common diol-type ginsenoside is ginsenoside Rb1, with an addition amount of 0.1-10 mg / mL, a stirring speed of 150 rpm, a fermentation temperature of 25-35℃, and a fermentation time of 5-15 days. In step S2, the mass concentration of β-glucanase added is 0.1-4%, the enzymatic hydrolysis temperature is 50℃, the pH of the enzymatic hydrolysis is 5.0, the stirring speed is 150 rpm, and the enzymatic hydrolysis time is 12-72 hours.

2. 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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