A method for converting rare ginsenoside F2

Abstract

The present application provides a kind of rare ginsenoside F2 conversion method.The present application is first with brazilian aspergillus to the prototype ginsenoside Rb1 fermentation, then hydrolysis is carried out using complex enzyme, i.e.can obtain the rare ginsenoside F2;Wherein, complex enzyme is mass ratio 0.8~1.2:0.8~1.2 of alpha-amylase and xylanase.The present application uses easily accessible prototype ginsenoside Rb1 as raw material, by brazilian aspergillus, alpha-amylase, xylanase are step by step biotransformation, significantly improve the conversion rate and purity of rare ginsenoside F2, and then play better anti-shedding effect, it is conducive to the further application of rare ginsenoside F2 in the field of cosmetics, drugs and the like.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and cosmetics technology. More specifically, it relates to a method for converting rare ginsenoside F2. Background Technology

[0002] Ginsenosides, also known as ginsenoside derivatives, are tetracyclic triterpenoid saponins extracted from plants of the Araliaceae family (American ginseng, Chinese ginseng, Korean ginseng, etc.). They have various effects, such as enhancing immunity and delaying aging. Ginsenosides extracted directly from Araliaceae plants are called "proto-ginsenosides" (such as Rb1, Rc, Rd, etc.), while their secondary metabolic derivatives after transformation are called "rare ginsenosides" (such as F2, Rk2, Rg3, etc.).

[0003] Rare ginsenosides have shown great potential in the pharmaceutical and cosmetic fields, playing an important role in improving the body's health and anti-aging. Among the many rare ginsenosides, rare ginsenoside F2 stands out due to its diverse biological activities. Its application value has been fully demonstrated in the pharmaceutical and cosmetic fields. For example, its effects in the pharmaceutical field include: (1) metabolic regulation: it can improve insulin resistance and regulate blood lipid levels; (2) anti-tumor: it can inhibit tumor cell proliferation, induce tumor cell apoptosis, and inhibit tumor cell invasion and metastasis; (3) antioxidant: it can scavenge free radicals and inhibit oxidative stress; (4) anti-inflammatory: it can inhibit the release of inflammatory factors and reduce inflammatory reactions; (5) immune Immune regulation: can enhance the activity of immune cells and regulate immune balance, etc.; (6) Cardiovascular protection: can dilate blood vessels and protect myocardial tissue, etc.; (7) Neuroprotection: can promote nerve cell repair and improve cognitive function, etc.; For example, in the field of cosmetics, it has the following effects: (1) Anti-aging: can neutralize skin free radicals, delay the process of photoaging, and achieve anti-aging, etc.; (2) Sensitive skin repair: can inhibit the release of inflammatory factors and reduce skin inflammation, etc.; (3) Whitening and brightening: can regulate pigment metabolism, inhibit oxidation and dullness, and create a clear and bright skin condition, etc.

[0004] Because rare ginsenoside F2 is present in extremely low or even non-existent amounts in Araliaceae plants, it is difficult to obtain it from these plants through simple extraction and separation methods. It typically requires conversion using chemical or biological methods. Chemical methods have significant drawbacks, including difficulty in controlling reaction conditions, the production of byproducts, and environmental pollution. Biological methods, on the other hand, stand out due to their mild reaction conditions, high selectivity, and lack of environmental pollution. However, the conversion capabilities and products of different microorganisms vary significantly. Therefore, finding a highly efficient method for converting rare ginsenoside F2 requires extensive screening of the microorganisms used for conversion and targeted optimization of the conversion process. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a method for converting rare ginsenoside F2. Using readily available prototype ginsenoside Rb1 as raw material, the method involves stepwise bioconversion using Aspergillus brasiliensis, α-amylase, and xylanase, significantly improving the conversion rate and purity of rare ginsenoside F2. This method is beneficial for the further application of rare ginsenoside F2 in cosmetics, pharmaceuticals, and other fields.

[0006] The primary objective of this invention is to provide a method for converting rare ginsenoside F2.

[0007] A second objective of this invention is to provide rare ginsenoside F2 obtained by the above-described method.

[0008] A third objective of this invention is to provide the application of the above-mentioned rare ginsenoside F2 in the preparation of cosmetics and / or pharmaceuticals.

[0009] The fourth objective of this invention is to provide the application of the aforementioned rare ginsenoside F2 in preventing hair loss.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention provides a method for converting rare ginsenoside F2, specifically: first, fermenting the prototype ginsenoside Rb1 with Aspergillus brasiliensis, and then hydrolyzing it with a compound enzyme to obtain the rare ginsenoside F2; wherein, the compound enzyme is α-amylase and xylanase in a mass ratio of 0.8-1.2:0.8-1.2.

[0012] Preferably, the Aspergillus brasiliensis ferments the prototype ginsenoside Rb1 in the form of a seed liquid.

[0013] More preferably, the seed liquid is prepared by inoculating Aspergillus brasiliensis into Sabouraud dextrose liquid medium for expansion culture.

[0014] More preferably, before inoculation, the Sabouraud dextrose liquid culture medium is further diluted, heated, and sterilized in sequence.

[0015] Furthermore, the dilution is performed with water, for example, by adding 1L of water to every 28–32 g of Sabouraud dextrose liquid medium.

[0016] Furthermore, the heating is heating until complete dissolution, for example, heating at 95–100 °C until complete dissolution.

[0017] Furthermore, the sterilization is performed at 120–125 °C for 13–17 min, such as at 121 °C for 15 min.

[0018] More preferably, the temperature for the expanded culture is 25–30 °C.

[0019] More preferably, the endpoint of the expanded culture is when the density of Aspergillus brasiliensis reaches 0.8 × 10⁻⁶. 6 ~1.2×10 6 At CFU / mL, such as 1×10 6 CFU / mL.

[0020] More preferably, the culture is expanded while oscillating, for example, at 120–170 rpm.

[0021] More preferably, after the expanded culture, solid-liquid separation is performed, such as by filtration.

[0022] Preferably, the ratio of Aspergillus brasiliensis to the prototype ginsenoside Rb1 is (5 × 10⁻⁶). 4 ~10 7 CFU: 1 mg.

[0023] Preferably, the fermentation time is 5 to 15 days.

[0024] Preferably, the fermentation temperature is 25–35 °C.

[0025] Preferably, the fermentation is carried out simultaneously with stirring, such as stirring at 120–170 rpm.

[0026] Preferably, after fermentation, sterilization is performed, such as sterilization at 90-100 °C for 10-30 min.

[0027] Preferably, the mass ratio of the prototype ginsenoside Rb1 to the complex enzyme is 1:0.2-4.

[0028] Preferably, the hydrolysis time is 24 to 96 hours.

[0029] Preferably, the hydrolysis temperature is 45–55 °C.

[0030] Preferably, the pH of the hydrolysis is 5 to 6. For example, it can be adjusted using an acetate-sodium acetate buffer solution.

[0031] Preferably, the hydrolysis is performed simultaneously with stirring, such as stirring at 120–170 rpm.

[0032] Preferably, after hydrolysis, extraction is also performed.

[0033] More preferably, before extraction, enzyme inactivation is performed, such as incubation at 80–100 °C for 25–35 min.

[0034] More preferably, the extraction reagent used in the extraction is n-butanol.

[0035] More preferably, after extraction, the material is dried, such as by evaporation at -0.1 to -0.08 MPa.

[0036] The rare ginsenoside F2 obtained by the above method has high purity and can thus exert a better anti-hair loss effect. Therefore, the rare ginsenoside F2 obtained by the above method and its application in the preparation of cosmetics and / or pharmaceuticals and anti-hair loss should be within the protection scope of this invention.

[0037] The present invention has the following beneficial effects:

[0038] This invention uses readily available prototype ginsenoside Rb1 as raw material. By performing stepwise biotransformation with Aspergillus brasiliensis, α-amylase, and xylanase, the conversion rate and purity of rare ginsenoside F2 are significantly improved, thereby exerting better anti-hair loss effects and facilitating the further application of rare ginsenoside F2 in cosmetics, pharmaceuticals, and other fields. Detailed Implementation

[0039] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0040] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0041] Aspergillus brasiliensis, with accession number ATCC 16404.

[0042] The Sabouraud dextrose liquid medium used in the examples was obtained by sequentially diluting, heating and sterilizing. The specific preparation method was as follows: weigh 30 g of commercially available Sabouraud dextrose liquid medium, add 1 L of deionized water, heat at 100 ℃ until completely dissolved, and finally sterilize at 121 ℃ for 15 min.

[0043] Example 1: Transformation of rare ginsenoside F2

[0044] Aspergillus brasiliensis was inoculated into Sabouraud dextrose liquid medium and cultured at 28 °C and 150 rpm until the bacterial density reached 1 × 10⁻⁶. 6 At CFU / mL, the *Aspergillus brasiliensis* seed solution was obtained by filtration. Prototype ginsenoside Rb1 was then added to the *Aspergillus brasiliensis* seed solution (making the ratio of *Aspergillus brasiliensis* to prototype ginsenoside Rb1 2 × 10⁻⁶). 5After fermentation at 30 °C and 150 rpm for 10 days (CFU: 1 mg), the mixture was sterilized at 95 °C for 20 min, and then a compound enzyme (the mass ratio of the original ginsenoside Rb1 to the compound enzyme was 1:2, and the compound enzyme was a 1:1 mass ratio of α-amylase and xylanase) was added. The mixture was hydrolyzed at pH 5.5 (adjusted with acetate-sodium acetate buffer), 50 °C, and 150 rpm for 60 h, and then incubated at 90 °C for 30 min to inactivate the enzyme. Finally, the mixture was extracted with n-butanol and evaporated to dryness at -0.09 MPa to obtain the rare ginsenoside F2.

[0045] Example 2: Transformation of rare ginsenoside F2

[0046] Aspergillus brasiliensis was inoculated into Sabouraud dextrose liquid medium and cultured at 25 °C and 170 rpm until the bacterial density reached 0.8 × 10⁻⁶. 6 At CFU / mL, the *Aspergillus brasiliensis* seed solution was obtained by filtration. Prototype ginsenoside Rb1 was then added to the *Aspergillus brasiliensis* seed solution (making the ratio of *Aspergillus brasiliensis* to prototype ginsenoside Rb1 5 × 10⁻⁶). 4 After fermentation at 35 °C and 170 rpm for 5 days (CFU: 1 mg), the mixture was sterilized at 90 °C for 30 min, and then a compound enzyme (the mass ratio of the original ginsenoside Rb1 to the compound enzyme was 1:4, and the compound enzyme was α-amylase and xylanase with a mass ratio of 0.8:1.2) was added. The mixture was hydrolyzed at pH 6.0 (adjusted with acetate-sodium acetate buffer), 45 °C, and 170 rpm for 96 h, and then incubated at 80 °C for 35 min to inactivate the enzyme. Finally, the mixture was extracted with n-butanol and evaporated to dryness at -0.1 MPa to obtain the rare ginsenoside F2.

[0047] Example 3: Transformation of rare ginsenoside F2

[0048] Aspergillus brasiliensis was inoculated into Sabouraud dextrose liquid medium and cultured at 30 °C and 120 rpm until the bacterial density reached 1.2 × 10⁻⁶. 6 At CFU / mL, the *Aspergillus brasiliensis* seed solution was obtained by filtration. Prototype ginsenoside Rb1 was then added to the *Aspergillus brasiliensis* seed solution (making the ratio of *Aspergillus brasiliensis* to prototype ginsenoside Rb1 1:1). 7After fermentation at 25 °C and 120 rpm for 15 days (CFU: 1 mg), the mixture was sterilized at 100 °C for 10 min. Then, a compound enzyme (the mass ratio of the original ginsenoside Rb1 to the compound enzyme was 1:0.2, and the compound enzyme was α-amylase and xylanase with a mass ratio of 1.2:0.8) was added. The mixture was hydrolyzed at pH 5.0 (adjusted with acetate-sodium acetate buffer), 55 °C, and 120 rpm for 24 h. After incubation at 100 °C for 25 min to inactivate the enzyme, the mixture was finally extracted with n-butanol and evaporated to dryness at -0.08 MPa to obtain the rare ginsenoside F2.

[0049] Comparative Example 1

[0050] Same as Example 1, except that hydrolysis with a complex enzyme is not performed. That is, the specific details of this comparative example are as follows:

[0051] Aspergillus brasiliensis was inoculated into Sabouraud dextrose liquid medium and cultured at 28 °C and 150 rpm until the bacterial density reached 1 × 10⁻⁶. 6 At CFU / mL, the *Aspergillus brasiliensis* seed solution was obtained by filtration. Prototype ginsenoside Rb1 was then added to the *Aspergillus brasiliensis* seed solution (making the ratio of *Aspergillus brasiliensis* to prototype ginsenoside Rb1 2 × 10⁻⁶). 5 CFU (1 mg) was fermented at 30 °C and 150 rpm for 10 days, then sterilized at 95 °C for 20 min. Finally, it was extracted with n-butanol and evaporated to dryness at -0.09 MPa to obtain the rare ginsenoside F2.

[0052] Comparative Example 2

[0053] Same as Example 1, except that fermentation with Aspergillus brasiliensis is not performed. That is, the specific details of this comparative example are as follows:

[0054] The prototype ginsenoside Rb1 was dissolved in 0.05 M acetate-sodium acetate buffer (pH 5.5) to achieve a final concentration of 5 mg / mL. After incubation at 30 °C and 150 rpm for 10 days, the mixture was sterilized at 95 °C for 20 min. Then, a compound enzyme (the mass ratio of prototype ginsenoside Rb1 to compound enzyme was 1:2, and the compound enzyme was a 1:1 mass ratio of α-amylase and xylanase) was added. The mixture was hydrolyzed at pH 5.5 (adjusted with acetate-sodium acetate buffer), 50 °C, and 150 rpm for 60 h. After incubation at 90 °C for 30 min, the enzyme was inactivated. Finally, the mixture was extracted with n-butanol and evaporated to dryness at -0.09 MPa to obtain the rare ginsenoside F2.

[0055] Comparative Example 3

[0056] Similar to Example 1, except that hydrolysis with a complex enzyme is performed first, followed by fermentation with *Aspergillus brasiliensis*. Specifically, this comparative example is as follows:

[0057] The prototype ginsenoside Rb1 was dissolved in 0.05 M acetate-sodium acetate buffer (pH 5.5) to achieve a final concentration of 5 mg / mL. After incubation at 30 °C and 150 rpm for 10 days, the mixture was sterilized at 95 °C for 20 min. Then, a compound enzyme (the mass ratio of prototype ginsenoside Rb1 to compound enzyme was 1:2, and the compound enzyme was a 1:1 mass ratio of α-amylase and xylanase) was added. The mixture was hydrolyzed at pH 5.5 (adjusted with acetate-sodium acetate buffer), 50 °C, and 150 rpm for 60 h. After incubation at 90 °C for 30 min, the enzyme was inactivated. Finally, the mixture was extracted with n-butanol and evaporated to dryness at -0.09 MPa to obtain the intermediate product.

[0058] Aspergillus brasiliensis was inoculated into Sabouraud dextrose liquid medium and cultured at 28 °C and 150 rpm until the bacterial density reached 1 × 10⁻⁶. 6 At CFU / mL, the *Aspergillus brasiliensis* seed culture was obtained by filtration. The intermediate reaction product was then added to the *Aspergillus brasiliensis* seed culture (to achieve a ratio of *Aspergillus brasiliensis* to intermediate reaction product of 2 × 10⁻⁶). 5 CFU (1 mg) was fermented at 30 °C and 150 rpm for 10 days, then sterilized at 95 °C for 20 min. Finally, it was extracted with n-butanol and evaporated to dryness at -0.09 MPa to obtain the rare ginsenoside F2.

[0059] Comparative Example 4

[0060] Same as Example 1, except that the xylanase in the complex enzyme is replaced with an equal mass of α-amylase. That is, this comparative example is as follows:

[0061] Aspergillus brasiliensis was inoculated into Sabouraud dextrose liquid medium and cultured at 28 °C and 150 rpm until the bacterial density reached 1 × 10⁻⁶. 6 At CFU / mL, the *Aspergillus brasiliensis* seed solution was obtained by filtration. Prototype ginsenoside Rb1 was then added to the *Aspergillus brasiliensis* seed solution (making the ratio of *Aspergillus brasiliensis* to prototype ginsenoside Rb1 2 × 10⁻⁶). 5After fermentation at 30 °C and 150 rpm for 10 days (CFU: 1 mg), the mixture was sterilized at 95 °C for 20 min, and then α-amylase was added (to make the mass ratio of the original ginsenoside Rb1 to α-amylase 1:2). The mixture was hydrolyzed at pH 5.5 (adjusted with acetate-sodium acetate buffer), 50 °C, and 150 rpm for 60 h. The mixture was then incubated at 90 °C for 30 min to inactivate the enzyme. Finally, the mixture was extracted with n-butanol and evaporated to dryness at -0.09 MPa to obtain the rare ginsenoside F2.

[0062] Comparative Example 5

[0063] Same as Example 1, except that the α-amylase in the complex enzyme is replaced with an equal mass of xylanase. That is, the specific comparative example is as follows:

[0064] Aspergillus brasiliensis was inoculated into Sabouraud dextrose liquid medium and cultured at 28 °C and 150 rpm until the bacterial density reached 1 × 10⁻⁶. 6 At CFU / mL, the *Aspergillus brasiliensis* seed solution was obtained by filtration. Prototype ginsenoside Rb1 was then added to the *Aspergillus brasiliensis* seed solution (making the ratio of *Aspergillus brasiliensis* to prototype ginsenoside Rb1 2 × 10⁻⁶). 5 After fermentation at 30 °C and 150 rpm for 10 days (CFU: 1 mg), the mixture was sterilized at 95 °C for 20 min, and then xylanase was added (to make the mass ratio of the original ginsenoside Rb1 to xylanase 1:2). The mixture was hydrolyzed at pH 5.5 (adjusted with acetate-sodium acetate buffer), 50 °C, and 150 rpm for 60 h. The mixture was then incubated at 90 °C for 30 min to inactivate the enzyme. Finally, the mixture was extracted with n-butanol and evaporated to dryness at -0.09 MPa to obtain the rare ginsenoside F2.

[0065] Comparative Example 6

[0066] Same as Example 1, except that the complex enzyme is replaced with an equal mass of β-glucosidase. That is, this comparative example is as follows:

[0067] Aspergillus brasiliensis was inoculated into Sabouraud dextrose liquid medium and cultured at 28 °C and 150 rpm until the bacterial density reached 1 × 10⁻⁶. 6 At CFU / mL, the *Aspergillus brasiliensis* seed solution was obtained by filtration. Prototype ginsenoside Rb1 was then added to the *Aspergillus brasiliensis* seed solution (making the ratio of *Aspergillus brasiliensis* to prototype ginsenoside Rb1 2 × 10⁻⁶). 5After fermentation at 30 °C and 150 rpm for 10 days (CFU: 1 mg), the mixture was sterilized at 95 °C for 20 min, and then β-glucosidase was added (to make the mass ratio of the original ginsenoside Rb1 to β-glucosidase 1:2). The mixture was hydrolyzed at pH 5.5 (adjusted with acetate-sodium acetate buffer), 50 °C, and 150 rpm for 60 h. The mixture was then incubated at 90 °C for 30 min to inactivate the enzyme. Finally, the mixture was extracted with n-butanol and evaporated to dryness at -0.09 MPa to obtain the rare ginsenoside F2.

[0068] Comparative Example 7

[0069] Same as Example 1, except that the complex enzyme is replaced with an equal mass of β-galactosidase. That is, this comparative example is as follows:

[0070] Aspergillus brasiliensis was inoculated into Sabouraud dextrose liquid medium and cultured at 28 °C and 150 rpm until the bacterial density reached 1 × 10⁻⁶. 6 At CFU / mL, the *Aspergillus brasiliensis* seed solution was obtained by filtration. Prototype ginsenoside Rb1 was then added to the *Aspergillus brasiliensis* seed solution (making the ratio of *Aspergillus brasiliensis* to prototype ginsenoside Rb1 2 × 10⁻⁶). 5 After fermentation at 30 °C and 150 rpm for 10 days (CFU: 1 mg), the mixture was sterilized at 95 °C for 20 min, and then β-galactosidase was added (to make the mass ratio of the original ginsenoside Rb1 to β-galactosidase 1:2). The mixture was hydrolyzed at pH 5.5 (adjusted with acetate-sodium acetate buffer), 50 °C, and 150 rpm for 60 h. The mixture was then incubated at 90 °C for 30 min to inactivate the enzyme. Finally, the mixture was extracted with n-butanol and evaporated to dryness at -0.09 MPa to obtain the rare ginsenoside F2.

[0071] Comparative Example 8

[0072] Same as Example 1, except that *Aspergillus brasiliensis* is replaced with *Saccharomyces cerevisiae* ATCC 26785 of equivalent activity. That is, this comparative example is as follows:

[0073] Saccharomyces cerevisiae was inoculated into Sabouraud dextrose liquid medium and cultured at 28 °C and 150 rpm until the bacterial density reached 1 × 10⁻⁶. 6 At CFU / mL, the *Saccharomyces cerevisiae* seed culture was obtained by filtration. Prototype ginsenoside Rb1 was then added to the *Saccharomyces cerevisiae* seed culture (making the ratio of *Saccharomyces cerevisiae* to prototype ginsenoside Rb1 2 × 10⁻⁶). 5After fermentation at 30 °C and 150 rpm for 10 days (CFU: 1 mg), the mixture was sterilized at 95 °C for 20 min, and then a compound enzyme (the mass ratio of the original ginsenoside Rb1 to the compound enzyme was 1:2, and the compound enzyme was a 1:1 mass ratio of α-amylase and xylanase) was added. The mixture was hydrolyzed at pH 5.5 (adjusted with acetate-sodium acetate buffer), 50 °C, and 150 rpm for 60 h, and then incubated at 90 °C for 30 min to inactivate the enzyme. Finally, the mixture was extracted with n-butanol and evaporated to dryness at -0.09 MPa to obtain the rare ginsenoside F2.

[0074] Test Example 1: Conversion Rate Determination of Rare Ginsenoside F2

[0075] The products obtained in Examples 1-3 and Comparative Examples 1-8 were dissolved in anhydrous ethanol. The yield of rare ginsenoside F2 in the products was detected by HPLC. The conversion rate of rare ginsenoside F2 was calculated according to the formula "conversion rate (%) = yield of rare ginsenoside F2 / amount of original ginsenoside Rb1 added × 100%". The results are shown in Table 1. The HPLC conditions were: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase A was acetonitrile, and mobile phase B was 0.05% ( v / v Phosphoric acid aqueous solution, gradient elution (0→10 min, 50% ( v / v A; 10→25 min, 70% ( v / v (A) Column temperature 25 ℃, detection wavelength 203 nm, flow rate 1 mL / min, injection volume 20 μL.

[0076] Table 1

[0077]

[0078] As can be seen, the conversion rate of rare ginsenoside F2 in Examples 1-3 was significantly higher than that in Comparative Examples 1-8, indicating that the present invention can significantly improve the conversion rate of rare ginsenoside F2 by stepwise biotransformation of Aspergillus brasiliensis, α-amylase, and xylanase, which is beneficial to the further application of rare ginsenoside F2 in cosmetics, pharmaceuticals and other fields.

[0079] Test Example 2: Anti-hair loss efficacy test of rare ginsenoside F2

[0080] After DHT binds to AR, it enters the cell nucleus and induces the secretion of DKK-1. DKK-1, as a negative regulator of the Wnt pathway, can inhibit cell proliferation by suppressing the Wnt pathway, thus leading to hair loss. Therefore, reducing DKK-1 secretion can play a certain role in preventing hair loss.

[0081] Human dermal papilla cells (HDPs) were seeded into 24-well plates containing slides and cultured at 37 °C for 18 h. After the supernatant was discarded, the cells were divided into blank group, model group, positive control group, and experimental group (Examples 1-3 and Comparative Examples 1-8).

[0082] Blank group: Add 1 mL of 10% ( ) to each well. v / v FBS was incubated in DMEM medium at 37 °C for 24 h.

[0083] Model group: Add 1 mL of 0.0002% ( ) to each well w / v Dihydrotestosterone and 10% ( v / v FBS was incubated in DMEM medium at 37 °C for 24 h.

[0084] Positive control group: Add 1 mL of 0.0002% ( ) to each well. w / v Dihydrotestosterone, 0.0008% w / v finasteride and 10% ( v / v FBS was incubated in DMEM medium at 37 °C for 24 h.

[0085] Experimental group: The products obtained in Examples 1-3 and Comparative Examples 1-8 were dissolved in butanediol to make the final Rb1 equivalent concentration in butanediol 0.1% (w / v), which is the F2 solution. 1 mL of solution containing 0.0002% ( w / v Dihydrotestosterone, 0.5% (w / v) F2 solution and 10% ( v / v FBS was incubated in DMEM medium at 37 °C for 24 h.

[0086] After incubation of the four groups, discard the culture medium, wash three times with PBS (pH=7.4), and then add 10% ( w / v Fix with paraformaldehyde for 20 min, discard the solution, wash 3 times with PBS (pH=7.4), add 0.1% (w / v) Triton X-100, incubate at 4 ℃ for 10 min, discard the solution, wash 3 times with PBS (pH=7.4), add 10% ( w / v Goat serum was used as a pretreatment, followed by blocking at 37 °C for 30 min. The solution was discarded, and primary antibody (rabbit anti-DKK-1) was added. The mixture was incubated at 4 °C for 16 h, discarded, and washed three times with PBS (pH=7.4). FITC-labeled goat anti-rabbit IgG was added, and the mixture was incubated at 37 °C for 1 h, discarded, and washed three times with PBS (pH=7.4). 5 μg / mL DAPI was added and the mixture was allowed to stand in the dark for 10 min, discarded, and washed three times with PBS (pH=7.4). The slides were mounted with anti-quenching fluorescent mounting medium and finally placed under a fluorescence microscope to characterize the expression level of DKK-1 by fluorescence intensity. The results are shown in Table 2.

[0087] Table 2

[0088]

[0089] As can be seen, compared with the blank group, the average fluorescence intensity of DKK-1 in the model group was significantly increased, indicating that the hair loss model was successfully constructed. Compared with the model group, the average fluorescence intensity of DKK-1 in Examples 1-3 and the positive control group was significantly reduced, and even the average fluorescence intensity of DKK-1 in Example 1 was significantly lower than that in the positive control group, indicating that the rare ginsenoside F2 obtained by the method of the present invention has excellent anti-hair loss efficacy. In addition, the average fluorescence intensity of DKK-1 in Examples 1-3 was significantly lower than that in Comparative Examples 1-8, indicating that the present invention can significantly improve the purity of rare ginsenoside F2 by stepwise biotransformation with Aspergillus brasiliensis, α-amylase, and xylanase, thus exerting its better anti-hair loss efficacy and facilitating the further application of rare ginsenoside F2 in cosmetics, pharmaceuticals, and other fields.

[0090] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for converting rare ginsenoside F2, characterized in that, First, the prototype ginsenoside Rb1 is fermented with *Aspergillus brasiliensis*, and then hydrolyzed with a complex enzyme to obtain the rare ginsenoside F2; wherein the ratio of *Aspergillus brasiliensis* to prototype ginsenoside Rb1 is (5 × 10⁻⁶). 4 ~10 7 CFU: 1 mg, wherein the mass ratio of the prototype ginsenoside Rb1 to the complex enzyme is 1:0.2-4, and the complex enzyme is α-amylase and xylanase with a mass ratio of 0.8-1.2:0.8-1.

2.

2. The conversion method according to claim 1, characterized in that, The fermentation time is 5 to 15 days.

3. The conversion method according to claim 1, characterized in that, The fermentation temperature is 25–35 °C.

4. The conversion method according to claim 1, characterized in that, After fermentation, the mixture is sterilized.

5. The conversion method according to claim 1, characterized in that, The hydrolysis time is 24–96 h.

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