Anti-acne radix stachyuri probiotic ferment, and preparation method and application thereof

Abstract

The application discloses anti-acne stemona sessiliflora probiotic fermentation and a preparation method and application thereof, and belongs to the technical field of medicines, wherein the preparation method of the stemona sessiliflora probiotic fermentation comprises the following steps: preparing stemona sessiliflora into a suspension and adding an enzyme preparation for enzymolysis to obtain an enzymolysis product; adding the enzymolysis product, a probiotic bacterial liquid and a glucose solution into a culture medium to obtain a fermentation mixture; performing fermentation treatment on the fermentation mixture to obtain a fermentation product; performing material-liquid separation on the fermentation product to obtain stemona sessiliflora residue and a fermentation liquid; extracting the stemona sessiliflora residue with ethanol to obtain an ethanol extract; mixing the fermentation liquid and the ethanol extract, and performing alcohol precipitation, centrifugation, suction filtration and concentration treatment to obtain the stemona sessiliflora probiotic fermentation. The preparation method can decompose large molecules in stemona sessiliflora into small molecule compounds which are more easily absorbed by the body, and produce antibacterial and anti-inflammatory components, thereby increasing the bioavailability and treatment effect of stemona sessiliflora.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an anti-acne probiotic ferment of Stemona japonica, its preparation method, and its application. Background Technology

[0002] Acne, a common chronic inflammatory skin disease of the pilosebaceous unit, is primarily caused by infection with Propionibacterium acnes. Clinical manifestations include comedones, papules, pustules, and even cysts. Currently, Western medicine treatment for acne mainly relies on antibiotics (such as erythromycin and clindamycin), retinoids, and anti-androgens. However, long-term use can easily lead to bacterial resistance, skin irritation, and other adverse reactions. With the development of modern medicine, treatments for acne are increasingly diverse, but topical medications remain the primary therapy. Acne is not just a common dermatological disease. Commonly used topical medications include antibiotics, anti-androgens, and retinoids. However, the use of the antibacterial drug minocycline can lead to a granulocytopenia. Taking anti-androgens can cause menstrual irregularities, nausea, and dizziness, while in male patients it can cause breast enlargement. Vitamin A derivatives have significant adverse reactions, such as strong irritation, which may lead to deformities. Studies show that Artemisia annua and its active ingredients, such as artemisinin, artemether, artesunate, artesunate, and dihydroartemisinin, can inhibit pathogenic bacteria like Propionibacterium acnes and Staphylococcus aureus, demonstrating therapeutic effects on infectious skin diseases. Meanwhile, oral administration of traditional Chinese medicine containing Artemisia annua has been proven to significantly alleviate clinical symptoms of post-pubertal acne in women. Therefore, finding safe and effective topical acne treatments is particularly important. Researching natural plant extracts as alternative or adjunctive therapies has significant scientific implications.

[0003] Radix Stemonae, a traditional Chinese medicine, has gained widespread popularity among skin disease patients due to its all-natural properties, minimal toxicity, significant efficacy, and non-addictive nature. However, traditional Chinese medicine suffers from limitations such as outdated usage methods, potential derivative formation, complex formulations with many components unclear, difficulties in qualitative and quantitative analysis, and inconvenience in portability, all of which hinder its development. Given its potential antibacterial and anti-inflammatory activities, Radix Stemonae has become a research hotspot.

[0004] Current research on Stemona japonica primarily focuses on the analysis of its chemical composition and the exploration of its pharmacological activities. Studies have revealed that the main active components of Stemona japonica include alkaloids (such as Stemonine and Isostemine), flavonoids, and steroidal saponins. These components have been proven to possess significant antibacterial and anti-inflammatory activities. Furthermore, Stemona japonica extract can reduce the expression of pro-inflammatory factors such as TNF-α and IL-6 by inhibiting the NF-κB signaling pathway, providing a theoretical basis for its application in acne treatment. However, research on the anti-acne effects of Stemona japonica extract is relatively limited, and most studies are limited to in vitro antibacterial experiments, lacking systematic in vivo efficacy evaluation and mechanistic exploration. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a fermented product of Stemona japonica for treating acne, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] A method for preparing an anti-acne probiotic ferment of Stemona japonica includes the following steps:

[0008] The Stemona japonica was prepared into a suspension and an enzyme preparation was added for enzymatic hydrolysis to obtain the hydrolysate; the enzyme preparation was pectinase and / or cellulase, and the amount added was 0.1%-0.3% of the mass of Stemona japonica; the enzymatic hydrolysis temperature was 50-70℃.

[0009] The enzymatic hydrolysate, probiotic culture, and glucose solution were added to the culture medium to obtain the fermentation mixture; the probiotic culture was Lactobacillus rhamnosus culture.

[0010] The mixture to be fermented is subjected to fermentation treatment to obtain fermentation products;

[0011] The fermentation products were separated into solid and liquid components to obtain the residue of Stemona japonica and the fermentation liquid.

[0012] The residue of Stemona japonica was extracted with ethanol to obtain an alcohol extract;

[0013] The fermentation broth and alcohol extract were mixed and subjected to alcohol precipitation. After the alcohol precipitation was completed, the mixture was centrifuged, the precipitate was discarded, and then the mixture was filtered and concentrated to obtain the fermented product of Stemona japonica probiotic.

[0014] Preferably, the Lactobacillus rhamnosus is Lactobacillus rhamnosus strain GG, with accession number CGMCC 1.3724.

[0015] Preferably, the absorbance of the probiotic liquid is 2-4, and its addition amount is 10%-40% of the volume of the mixture to be fermented.

[0016] Preferably, the fermentation treatment temperature is 35-39℃ and the time is 2-4 days.

[0017] Preferably, the concentration of the glucose solution is 0.8-1.2 g / mL; the amount of glucose solution added is 20%-40% of the volume of the mixture to be fermented.

[0018] Preferably, the ratio of the Stemona japonica to the fermentation mixture is 1g:(10-25)mL.

[0019] Preferably, the step of extracting the residue of Stemona japonica with ethanol to obtain an ethanol extract specifically includes:

[0020] Mix a portion of the Stemona japonica residue with an ethanol solution of 50%-70% by volume, heat and reflux, then filter. Repeat this step several times to extract the extract, and combine the filtered liquids to obtain the ethanol extract.

[0021] Another object of the present invention is to provide a Stemona japonica probiotic ferment obtained by the above preparation method.

[0022] Another object of the present invention is to provide an application of the above-mentioned Stemona japonica probiotic ferment in the preparation of anti-acne drugs or skin care products.

[0023] Another object of the present invention is to provide an anti-acne facial mask comprising the following components by mass fraction:

[0024] Phase A: Glycerin 0.5%-0.8%; Hyaluronic acid 0.4%-0.6%; Sodium hyaluronate 0.4%-0.6%; 1,2-Butanediol 0.8%-1.2%; Xanthan gum 0.1%-0.5%; Salicylic acid 1%-1.5%; Water 43%-47%;

[0025] Phase B: Nicotinamide 1.5%-2%; Arbutin 1%-1.5%;

[0026] Phase C: PEG-40 hydrogenated castor oil 0.05%-0.15%; L-ascorbic acid 0.1%-0.5%; tea polyphenols 0.1%-0.5%; Centella asiatica extract 1%-3%; the above-mentioned Stemona japonica probiotic ferment 1%-3%; Ganoderma lucidum extract 0.1%-1%; Calendula officinalis extract 0.1%-1%; sodium bicarbonate 0.1%-0.3%; mandelic acid 0.8%-1.2%; balance: water.

[0027] This invention improves the enzymatic hydrolysis process of Stemona japonica and utilizes Lactobacillus rhamnosus fermentation of Stemona japonica to break down the original large molecules in Stemona japonica into smaller molecules that are more easily absorbed by the body, and to produce antibacterial and anti-inflammatory components, thereby increasing the bioavailability and therapeutic effect of Stemona japonica. Furthermore, by first enzymatically hydrolyzing Stemona japonica, this invention promotes the growth of Lactobacillus rhamnosus, which in turn promotes the absorption and utilization of the active ingredients in Stemona japonica. The two complement each other and have a synergistic effect. The Stemona japonica probiotic ferment obtained by this invention has high in vitro antibacterial activity and good anti-acne efficacy. Attached Figure Description

[0028] Figure 1 A comparison chart of total flavonoid and total alkaloid content in fermented Stemona japonica products prepared at different fermentation times.

[0029] Figure 2 A comparison of the total flavonoid and total alkaloid content in the fermented products of Stemona japonica prepared with different amounts of glucose solution.

[0030] Figure 3 A comparison chart of total flavonoid and total alkaloid content in fermented products of Stemona japonica prepared with different amounts of probiotic bacterial solution.

[0031] Figure 4 A comparison chart showing the total flavonoid and total alkaloid content in fermented Stemona japonica products prepared with different material-to-liquid ratios.

[0032] Figure 5 A comparison chart showing the total flavonoid content in fermented Stemona japonica products prepared with different added enzymes.

[0033] Figure 6 A comparison of the total flavonoid and total alkaloid content in fermented Stemona japonica products prepared with different amounts of mixed enzymes.

[0034] Figure 7 A comparison chart of total flavonoid and total alkaloid content in fermented Stemona japonica products prepared at different enzymatic hydrolysis temperatures.

[0035] Figure 8 Figure 1 shows the results of HE staining observation in different groups of mice. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1: This example provides a method for preparing an anti-acne probiotic ferment of Stemona japonica, including the following steps:

[0038] S1. Prepare a suspension of 2g of Stemona japonica and add an enzyme preparation. Perform enzymatic hydrolysis in a constant temperature water bath at 60℃ for 60min. Then, perform high pressure treatment at 121℃ for 15min and cool to obtain the hydrolysate. The enzyme preparation is a mixture of pectinase and cellulase in equal mass ratio, and its addition amount is 0.2% of the mass of Stemona japonica.

[0039] S2. Add the above enzymatic hydrolysate, 20 mL of probiotic culture, and 15 mL of glucose solution with a concentration of 1 g / mL to the MRS medium. Make up the MRS medium to 50 mL to obtain the fermentation mixture. The probiotic culture is Lactobacillus rhamnosus culture.

[0040] MRS culture medium was prepared by mixing 50g of distilled water and 2.622g of MRS. 2.622g of MRS was accurately weighed using an electronic balance and slowly added to 50g of distilled water that was being heated. At the same time, the mixture was stirred until the MRS was completely dissolved. The mixture was then placed in an autoclave for autoclaving (121℃, 15min) for later use.

[0041] The preparation method of Lactobacillus rhamnosus (LGG) bacterial suspension is as follows: The known Lactobacillus rhamnosus GG strain with accession number CGMCC 1.3724 is inoculated into sterilized MRS medium at a 1% inoculation rate. After thorough mixing, it is incubated at 37°C for 24 hours to obtain the first-generation bacterial suspension. The first-generation bacterial suspension is then inoculated into a new sterile test tube at a 1% inoculation rate and incubated for another 24 hours to obtain the second-generation bacterial suspension. Similar to the preparation of the second-generation bacterial suspension, the second-generation bacterial suspension is mixed with MRS medium at a 1% inoculation rate and incubated for 20 hours. The absorbance of the bacterial suspension is then adjusted to 3 to obtain the Lactobacillus rhamnosus bacterial suspension.

[0042] The preparation method of glucose solution (GLU) is as follows: Weigh 30g of glucose, add distilled water, and make up to 30 mL with distilled water. While adding water, stir until the glucose is completely dissolved. Then put the dissolved solution into an autoclave for autoclaving (121℃, 15 min) to obtain glucose solution for later use.

[0043] S3. Place the above-mentioned mixture to be fermented in a constant temperature incubator at 37°C for 3 days to obtain the fermentation product.

[0044] S4. Separate the fermentation products to obtain the residue of Stemona japonica and the fermentation liquid. Place the fermentation liquid in a 1000mL volumetric flask for later use.

[0045] S5. Place the above-mentioned residue of Stemona japonica in a round-bottom flask, add 36 mL of 50% ethanol solution, heat under reflux for 2 hours, filter with filter paper, and extract twice more in the same way. Collect the liquid obtained from the three filtrations to obtain the ethanol extract.

[0046] S6. Mix the above fermentation broth and alcohol extract, and prepare an 85% volume ethanol solution with anhydrous ethanol for overnight alcohol precipitation. After the alcohol precipitation is completed, centrifuge at 4000 r / min for 15 min, discard the precipitate, connect a Buchner funnel to a vacuum pump, filter the filtrate, and concentrate the clarified part to 2 mL by rotary evaporation to obtain the Stemona japonica probiotic fermentation product.

[0047] In addition, this embodiment also provides an anti-acne face mask comprising the following components by mass fraction:

[0048] Phase A: Glycerin 0.65%; Hyaluronic acid 0.5%; Sodium hyaluronate 0.5%; 1,2-Butanediol 1%; Xanthan gum 0.3%; Salicylic acid 1.15%; Water 45%;

[0049] Phase B: Nicotinamide 1.75%; Arbutin 1.25%;

[0050] Phase C: PEG-40 hydrogenated castor oil 0.1%; L-ascorbic acid 0.25%; tea polyphenols 0.25%; Centella asiatica extract 2%; the above-mentioned Stemona japonica probiotic ferment 2%; Ganoderma lucidum extract 0.5%; Calendula officinalis extract 0.5%; sodium bicarbonate 0.15%; mandelic acid 1%; balance water.

[0051] The preparation method for the above-mentioned anti-acne mask is as follows:

[0052] Step 1: Place phase A in a water bath and heat it to 85°C. Stop heating when the colloidal substances are completely dispersed, and then keep it at this temperature for 20 minutes while stirring until completely dissolved.

[0053] Step 2: Transfer to a 60℃ water bath, add phase B, stir well, then transfer to a 45℃ water bath, add phase C, stir well, and then use a 0.22μm sterile filter membrane to filter and sterilize the membrane solution.

[0054] Step 3: Place the sheet mask into the mask liquid and stir manually to make it evenly moistened. After stirring, gently squeeze to keep it at an appropriate level of moisture. Fold the soaked mask into a rectangular shape and package it. Store it in a refrigerator at 4°C.

[0055] Example 2: This example provides a method for preparing an anti-acne probiotic ferment of Stemona japonica, including the following steps:

[0056] S1. Prepare a suspension of 2g of Stemona japonica and add an enzyme preparation. Perform enzymatic hydrolysis in a constant temperature water bath at 50℃ for 30min. Then, perform high pressure treatment at 121℃ for 15min and let it cool to obtain the hydrolysate. The enzyme preparation is pectinase, and the amount added is 0.1% of the mass of Stemona japonica.

[0057] S2. Add the above enzymatic hydrolysate, 5 mL of probiotic culture, and 10 mL of 0.8 g / mL glucose solution to MRS medium. Make up the MRS medium to 50 mL to obtain the fermentation mixture. The probiotic culture is Lactobacillus rhamnosus culture.

[0058] MRS culture medium was prepared by mixing 50g of distilled water and 2.622g of MRS. 2.622g of MRS was accurately weighed using an electronic balance and slowly added to 50g of distilled water that was being heated. At the same time, the mixture was stirred until the MRS was completely dissolved. The mixture was then placed in an autoclave for autoclaving (121℃, 15 min) for later use.

[0059] The preparation method of Lactobacillus rhamnosus (LGG) bacterial suspension is as follows: The known Lactobacillus rhamnosus GG strain with accession number CGMCC 1.3724 is inoculated into sterilized MRS medium at a 1% inoculation rate. After thorough mixing, it is incubated at 37°C for 24 hours to obtain the first-generation bacterial suspension. The first-generation bacterial suspension is then inoculated into a new sterile test tube at a 1% inoculation rate and incubated for another 24 hours to obtain the second-generation bacterial suspension. Similar to the preparation of the second-generation bacterial suspension, the second-generation bacterial suspension is mixed with MRS medium at a 1% inoculation rate and incubated for 20 hours. The absorbance of the bacterial suspension is then adjusted to 2 to obtain the Lactobacillus rhamnosus bacterial suspension.

[0060] The preparation method of glucose solution (GLU) is as follows: Weigh 30g of glucose, add distilled water, and make up to 30 mL with distilled water. While adding water, stir until the glucose is completely dissolved. Then put the dissolved solution into an autoclave for autoclaving (121℃, 15 min) to obtain glucose solution for later use.

[0061] S3. Place the above-mentioned mixture to be fermented in a constant temperature incubator at 35℃ for 2 days to obtain the fermentation product.

[0062] S4. Separate the fermentation products to obtain the residue of Stemona japonica and the fermentation liquid. Place the fermentation liquid in a 1000mL volumetric flask for later use.

[0063] S5. Place the above-mentioned residue of Stemona japonica in a round-bottom flask, add 36 mL of 60% ethanol solution, heat under reflux for 2 hours, filter with filter paper, and extract twice more in the same way. Collect the liquid obtained from the three filtrations to obtain the ethanol extract.

[0064] S6. Mix the above fermentation broth and alcohol extract, and prepare an 80% volume ethanol solution with anhydrous ethanol for overnight alcohol precipitation. After the alcohol precipitation is completed, centrifuge at 4000 r / min for 15 min, discard the precipitate, connect a Buchner funnel to a vacuum pump, filter the filtrate, and concentrate the clear part to 2 mL by rotary evaporation to obtain the Stemona japonica probiotic fermentation product.

[0065] In addition, this embodiment also provides an anti-acne face mask comprising the following components by mass fraction:

[0066] Phase A: Glycerin 0.5%; Hyaluronic acid 0.4%; Sodium hyaluronate 0.4%; 1,2-Butanediol 0.8%; Xanthan gum 0.1%; Salicylic acid 1%; Water 47%;

[0067] Phase B: Nicotinamide 1.5%; Arbutin 1%;

[0068] Phase C: PEG-40 hydrogenated castor oil 0.05%; L-ascorbic acid 0.1%; tea polyphenols 0.1%; Centella asiatica extract 1%; the above-mentioned Stemona japonica probiotic ferment 3%; Ganoderma lucidum extract 0.1%; Calendula officinalis extract 0.1%; sodium bicarbonate 0.1%; mandelic acid 0.8%; balance is water.

[0069] The preparation method for the above-mentioned anti-acne mask is as follows:

[0070] Step 1: Place phase A in a water bath and heat it to 85°C. Stop heating when the colloidal substances are completely dispersed, and then keep it at this temperature for 20 minutes while stirring until completely dissolved.

[0071] Step 2: Transfer to a 60℃ water bath, add phase B, stir well, then transfer to a 45℃ water bath, add phase C, stir well, and then use a 0.22μm sterile filter membrane to filter and sterilize the membrane solution.

[0072] Step 3: Place the sheet mask into the mask liquid and stir manually to make it evenly moistened. After stirring, gently squeeze to keep it at an appropriate level of moisture. Fold the soaked mask into a rectangular shape and package it. Store it in a refrigerator at 4°C.

[0073] Example 3: This example provides a method for preparing an anti-acne probiotic ferment of Stemona japonica, including the following steps:

[0074] S1. Prepare a suspension of 2g of Stemona japonica and add an enzyme preparation. Perform enzymatic hydrolysis in a constant temperature water bath at 65℃ for 90min. Then, perform high pressure treatment at 121℃ for 15min and cool to obtain the hydrolysate. The enzyme preparation is cellulase, and the amount added is 0.3% of the mass of Stemona japonica.

[0075] S2. Add the above enzymatic hydrolysate, 15 mL of probiotic culture, and 20 mL of glucose solution with a concentration of 1.2 g / mL to the MRS medium. Make up the MRS medium to 50 mL to obtain the fermentation mixture. The probiotic culture is Lactobacillus rhamnosus culture.

[0076] MRS culture medium was prepared by mixing 50g of distilled water and 2.622g of MRS. 2.622g of MRS was accurately weighed using an electronic balance and slowly added to 50g of distilled water that was being heated. At the same time, the mixture was stirred until the MRS was completely dissolved. The mixture was then placed in an autoclave for autoclaving (121℃, 15 min) for later use.

[0077] The preparation method of Lactobacillus rhamnosus (LGG) bacterial suspension is as follows: The known Lactobacillus rhamnosus GG strain with accession number CGMCC 1.3724 is inoculated into sterilized MRS medium at a 1% inoculation rate. After thorough mixing, it is incubated at 37°C for 24 hours to obtain the first-generation bacterial suspension. The first-generation bacterial suspension is then inoculated into a new sterile test tube at a 1% inoculation rate and incubated for another 24 hours to obtain the second-generation bacterial suspension. Similar to the preparation of the second-generation bacterial suspension, the second-generation bacterial suspension is mixed with MRS medium at a 1% inoculation rate and incubated for 20 hours. The absorbance of the bacterial suspension is then adjusted to 4 to obtain the Lactobacillus rhamnosus bacterial suspension.

[0078] The preparation method of glucose solution (GLU) is as follows: Weigh 30g of glucose, add distilled water, and make up to 30 mL with distilled water. While adding water, stir until the glucose is completely dissolved. Then put the dissolved solution into an autoclave for autoclaving (121℃, 15 min) to obtain glucose solution for later use.

[0079] S3. Place the above-mentioned mixture to be fermented in a constant temperature incubator at 39℃ for 4 days to obtain the fermentation product.

[0080] S4. Separate the fermentation products to obtain the residue of Stemona japonica and the fermentation liquid. Place the fermentation liquid in a 1000mL volumetric flask for later use.

[0081] S5. Place the above-mentioned residue of Stemona japonica in a round-bottom flask, add 36 mL of 70% ethanol solution, heat under reflux for 2 hours, filter with filter paper, and extract twice more in the same way. Collect the liquid obtained from the three filtrations to obtain the alcohol extract.

[0082] S6. Mix the above fermentation broth and alcohol extract, and prepare an ethanol solution with anhydrous ethanol to a volume concentration of 90% for overnight alcohol precipitation. After the alcohol precipitation is completed, centrifuge at a rate of 4000 r / min for 15 min, discard the precipitate, connect a Buchner funnel to a vacuum pump, filter the filtrate, and concentrate the clarified part to 2 mL by rotary evaporation to obtain the Stemona japonica probiotic fermentation product.

[0083] In addition, this embodiment also provides an anti-acne face mask comprising the following components by mass fraction:

[0084] Phase A: Glycerin 0.8%; Hyaluronic acid 0.6%; Sodium hyaluronate 0.6%; 1,2-Butanediol 1.2%; Xanthan gum 0.5%; Salicylic acid 1.5%; Water 43%;

[0085] Phase B: Nicotinamide 2%; Arbutin 1.5%;

[0086] Phase C: PEG-40 hydrogenated castor oil 0.15%; L-ascorbic acid 0.5%; tea polyphenols 0.5%; Centella asiatica extract 3%; the above-mentioned Stemona japonica probiotic ferment 1%; Ganoderma lucidum extract 1%; Calendula officinalis extract 1%; sodium bicarbonate 0.3%; mandelic acid 1.2%; balance water.

[0087] The preparation method for the above-mentioned anti-acne mask is as follows:

[0088] Step 1: Place phase A in a water bath and heat it to 85°C. Stop heating when the colloidal substances are completely dispersed, and then keep it at this temperature for 20 minutes while stirring until completely dissolved.

[0089] Step 2: Transfer to a 60℃ water bath, add phase B, stir well, then transfer to a 45℃ water bath, add phase C, stir well, and then use a 0.22μm sterile filter membrane to filter and sterilize the membrane solution.

[0090] Step 3: Place the sheet mask into the mask liquid and stir manually to make it evenly moistened. After stirring, gently squeeze to keep it at an appropriate level of moisture. Fold the soaked mask into a rectangular shape and package it. Store it in a refrigerator at 4°C.

[0091] Experimental Example 1: Referring to the preparation method provided in Example 1, several factors that may affect the fermentation effect were selected, including fermentation time (2d, 3d, 4d, 5d, 6d), glucose solution addition amount (0, 10%, 20%, 30%, 40%), probiotic liquid addition amount (5%, 10%, 20%, 30%, 40%), material-liquid ratio (1g:2.5mL, 1g:5mL, 1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL), enzyme preparation (including pectinase, cellulase, glucosidase, papain, and a mixture of pectinase and cellulase), enzyme preparation addition amount (0.1%, 0.2%, 0.3%, 0.4%, 0.5%), and enzymatic hydrolysis temperature (50℃, 55℃, 60℃, 65℃, 70℃). Single-factor experiments were conducted with the total flavonoid and total alkaloid extraction rates as response values.

[0092] The method for detecting total flavonoids is as follows: Accurately measure 200 μL of rutin standard solution (0.2 g / mL) and the sample solution to be tested (fermentation product of Stemona japonica probiotics). Add 400 μL of 70% ethanol and 40 μL of 5% sodium nitrite solution to the tube in sequence, mix thoroughly, and let stand for 6 min. Then add 40 μL of 10% aluminum nitrate, mix again, and let stand for 6 min. Finally, add 80 μL of 4 mol / L sodium hydroxide solution and 240 μL of 70% ethanol, shake well, and let stand for 15 minutes. Measure the absorbance at a wavelength of 510 nm. Calculate the total flavonoid content based on the absorbance. The specific calculation method can be found in existing technologies and will not be elaborated here.

[0093] The method for detecting total alkaloids is as follows: The pH of the *Stemona japonica* probiotic fermentation product was adjusted to 2-3 with hydrochloric acid, and the volume was brought to 2.5 mL. 1 mL of freshly prepared 2% Reichelka salt saturated solution was added. After standing in ice water for 1 hour, the mixture was centrifuged at 3000 rpm and 4°C for 10 minutes. The supernatant was discarded, and the mixture was washed with an ice-water mixture, centrifuged, and repeated until the Reichelka salt precipitate was colorless. All operations were performed on ice. Acetone can dissolve the filtered and washed Reichelka salt precipitate. Finally, the absorbance of the sample was measured at the maximum absorption wavelength of 523 nm. Acetone was used as a blank control. The total alkaloid content of *Stemona japonica* was calculated to determine the content of alkaloids in the *Stemona japonica* alkaloids (C...). 22 H 33 For the calculation of O4N, the specific calculation method can be found in existing technology and will not be elaborated here.

[0094] The experimental results are as follows: Under the conditions of a material-to-liquid ratio of 1:20, a glucose solution addition of 30%, and a probiotic culture addition of 30%, the effect of fermentation time on the total flavonoid and total alkaloid content in the fermented product of Stemona japonica probiotic was investigated. The results are as follows: Figure 1 As shown, the yields of total flavonoids and total alkaloids initially increased and then decreased with increasing fermentation time, with higher yields observed at a fermentation time of 3 days.

[0095] Under the conditions of a material-to-liquid ratio of 1:20, a fermentation time of 3 days, and a probiotic culture concentration of 30%, the effect of glucose solution addition on the total flavonoid and total alkaloid content in the fermented product of Stemona japonica was investigated. The results are as follows: Figure 2 As shown, the total flavonoid yield was higher when the glucose solution concentration was 20%. The total alkaloid yield was higher when the glucose solution concentration was 30%.

[0096] Under the conditions of a material-to-liquid ratio of 1:20, a fermentation time of 3 days, and a glucose solution addition of 30%, the effect of probiotic bacterial solution addition on the total flavonoid and total alkaloid content in the fermented product of Stemona japonica was investigated. The results are as follows: Figure 3 As shown, the total flavonoid yield was higher when the probiotic liquid content was 10%. The total alkaloid yield was higher when the probiotic liquid content was 30%.

[0097] The effect of the material-to-liquid ratio on the total flavonoid and total alkaloid content in the fermented product of Stemona japonica was investigated under the conditions of 3 days of fermentation, 30% probiotic solution addition, and 30% glucose solution addition. The results are as follows: Figure 4 As shown, the yield of total flavonoids first increases and then decreases with the increase of the material-to-liquid ratio, reaching its highest level at 1:10. The yield of alkaloids increases with the increase of the material-to-liquid ratio, with a relatively high yield at a material-to-liquid ratio of around 1:25.

[0098] Under otherwise unchanged conditions, the content of active ingredients was extracted after fermentation with and without the addition of enzymes, and the results are as follows: Figure 5 As shown in the figure, the total flavonoid and total alkaloid content in the Stemona japonica probiotic ferment is highest when a mixture of pectinase and cellulase is added.

[0099] Under otherwise unchanged conditions, different amounts of a mixture of pectinase and cellulase were added, and the active ingredient content was extracted after fermentation. The results are as follows: Figure 6 As shown in the figure, the total alkaloid content in the Stemona japonica probiotic ferment begins to decrease when the addition of pectinase and cellulase exceeds 0.2%, while the total flavonoids decrease when the addition of mixed enzymes exceeds 0.3%. This indicates that the higher the amount of mixed enzymes added, the more significant the inhibition of enzymatic hydrolysis.

[0100] Under otherwise unchanged conditions, the same amount of pectinase and cellulase mixture was added, and the active ingredient content was extracted after fermentation at different enzymatic hydrolysis temperatures. The results are as follows: Figure 7As shown in the figure, the content of active ingredients in the fermented product of Stemona japonica showed a trend of first increasing and then decreasing with the increase of enzymatic hydrolysis temperature. Furthermore, the content of active ingredients in the fermented product of Stemona japonica decreased rapidly after 60℃ and then tended to level off, indicating that the temperature after 60℃ has an inhibitory effect on the precipitation of Stemona japonica enzymatic hydrolysis products.

[0101] Experiment Example 2: Testing the Probiotic-Promoting Effect of Stemona japonica Fermentation on Probiotics:

[0102] Using 96-well microplate culture, 250 μL of ethanol extract of Stemona japonica, 1750 μL of MRS medium, and 10 μL of Lactobacillus rhamnosus (LGG) bacterial suspension (OD) were prepared according to the method provided in Example 1. 600 =3) Add to one well, seal the reaction well with sterile sealing film, and use a multi-functional microplate reader to detect the real-time OD value at 600nm. After shaking for 10s, perform static measurement, automatically record data every 30min, and continue monitoring for 24 hours to obtain a complete growth curve. Determine the maximum biomass in the Stemona japonica-LGG co-culture fermentation model to be 0.03125g / mL, and the LGG addition amount to be 1% (OD... 600 =3), thus establishing the optimal concentration of Stemona japonica-LGG co-culture fermentation system. At this concentration, the OD of LGG... 600 The value reached 2.6, while the blank control group was 0.9, indicating that Stemona japonica can promote the growth of LGG.

[0103] The concentration of plant soluble polysaccharides was determined using the Solarbio BC0030 plant soluble polysaccharide assay kit with anthrone colorimetric method. The concentration of extracellular polysaccharides increased by 30.2% in Stemona japonica-LGG co-culture (0.1092 mg / mL) compared to LGG culture alone (0.0839 mg / mL).

[0104] L-lactic acid was measured using a one-step sandwich enzyme-linked immunosorbent assay (ELISA) with double antibodies. The concentration of L-lactic acid increased by 181.1% when the culture of Stemona japonica and LGG co-cultured (531.9712 ng / mL) was compared with that of LGG culture alone (189.2514 ng / mL).

[0105] Folic acid content was determined using a one-step sandwich enzyme-linked immunosorbent assay (ELISA) with double antibodies. The folic acid concentration was increased by 217.3% in the co-culture of Stemona japonica and LGG (754.2683 ng / mL) compared to LGG culture alone (237.7178 ng / mL).

[0106] Experimental Example 3: The anti-acne mask prepared in Example 1 was tested as a finished product, and the results are as follows:

[0107] (1) The mask is light brown in color, smooth in texture, and slightly shiny. It has a light and elegant medicinal fragrance, a single incense stick, and no irritating smell.

[0108] (2) Physicochemical test results show that the pH value of the mask liquid is 5-6, and the heat resistance, cold resistance and centrifugal stability test results show no stratification.

[0109] (3) Microbial test results showed that the total number of colonies in the mask liquid was 640 CFU / mL, with no Staphylococcus aureus or thermotolerant coliforms detected.

[0110] (4) The total flavonoid concentration of the mask is 1.009 mg / mL, which is not lower than the total flavonoid concentration of the Stemona japonica extract used as the raw material, and the active ingredients are effectively preserved. The total alkaloid concentration of the mask is 1.620 mg / mL, which is not lower than the total alkaloid concentration of the Stemona japonica extract used as the raw material.

[0111] Example 4: The product prepared in Example 1 was subjected to in vitro antibacterial and anti-acne tests. Specifically, 28 healthy female SPF-grade ICR mice aged 6-8 weeks and weighing 36-45 g were selected. These mice were purchased from Changchun Yisi Biotechnology Co., Ltd. After one week of free access to food and water at an indoor temperature of 20-26℃, the mice were randomly divided into several groups, with 7 mice in each group. The mice were divided into a normal group, a negative group (model group), a positive group, an unfermented extract group, and a *Stemona japonica* probiotic fermentation group. The concentration of *Propionibacterium acnes* was adjusted to (1×10⁻⁶) using PBS buffer. 8 Mice in each group were anesthetized with sodium pentobarbital anesthetic via intraperitoneal injection of a bacterial suspension containing CFU / 20μL, and then marked on their backs.

[0112] A mouse acne model was constructed. Except for the normal control group, all other groups received an intradermal injection of 20 μL of *Propionibacterium acnes* bacterial solution into the auricle of mice using a 100 μL syringe, followed by normal feeding. Successful modeling was indicated by the appearance of small bumps on the auricle accompanied by inflammation and redness. After successful modeling, mice in the normal control group received no special treatment throughout the experiment. Mice in the negative control group received 20 μL of PBS buffer twice daily on acne lesions. Mice in the positive control group received 20 μL of erythromycin twice daily on acne lesions. Mice in the unfermented extract group received 20 μL of unfermented *Stemona japonica* extract (prepared according to the method provided in Example 1) twice daily on acne lesions. Mice in the *Stemona japonica* probiotic fermentation group received 20 μL of the *Stemona japonica* probiotic fermentation prepared in Example 1 twice daily on acne lesions. This treatment lasted for 7 days to simulate a therapeutic process. At the end of the experiment, mice were anesthetized intraperitoneally with pentobarbital, and then their ear lesions and serum were collected for study. These ear tissues were subsequently fixed with formaldehyde, and the mice were finally euthanized by cervical dislocation to ensure the experiment's success.

[0113] The experimental results are as follows: The minimum inhibitory concentration (MIC) of the fermented product of Stemona japonica against Propionibacterium acnes was 31.25 mg / mL, the minimum bactericidal concentration (MBC) was 0.5 g / mL, and the MBC / MIC ratio was ≤4. The in vitro MIC values ​​of the fermented product of Stemona japonica against Escherichia coli and Staphylococcus aureus were 0.0625 g / mL, and the MIC value of Propionibacterium acnes was 0.0625 g / mL.

[0114] In the group treated with fermented Stemona japonica probiotics, the redness and swelling of the mouse ear gradually subsided, and the ear thickness decreased by 0.71±0.03 mm (compared to a decrease of 0.633±0.05 mm in the unfermented extract group). HE staining revealed a significant reduction in inflammatory cells, weakened vasodilation, and improved edema in the mice. (Specific details are as follows...) Figure 8 As shown.

[0115] In the group of mice with fermented Bai Bu probiotics, the levels of TNF-α, IL-8, and IL-6 in the auricular skin lesions decreased by 36.71%, 17.64%, and 26.83%, respectively (compared to 25.45%, 15.08%, and 23.62% in the unfermented extract group). The levels of TNF-α, IL-8, and IL-6 in the blood decreased by 35.56%, 40.84%, and 33.76%, respectively (compared to 23.42%, 38.30%, and 30.93% in the unfermented extract group). The number of immune cells producing TNF-α and IL-6 in the spleen decreased by 59% and 65%, respectively (compared to 46% and 60% in the unfermented extract group).

[0116] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A method for preparing an anti-acne probiotic ferment of Stemona japonica, characterized in that, Includes the following steps: The Stemona japonica was prepared into a suspension and an enzyme preparation was added for enzymatic hydrolysis to obtain the hydrolysate; the enzyme preparation was pectinase and / or cellulase, and the amount added was 0.1%-0.3% of the mass of Stemona japonica; the enzymatic hydrolysis temperature was 50-70℃. The enzymatic hydrolysate, probiotic culture, and glucose solution were added to the culture medium to obtain the fermentation mixture; the probiotic culture was Lactobacillus rhamnosus culture. The mixture to be fermented is fermented to obtain the fermentation product; The fermentation products are separated into solid and liquid components to obtain the residue of Stemona japonica and the fermentation liquid. The residue of Stemona japonica was extracted with ethanol to obtain an alcohol extract; The fermentation broth and alcohol extract were mixed and subjected to alcohol precipitation. After the alcohol precipitation was completed, the mixture was centrifuged, the precipitate was discarded, and then the mixture was filtered and concentrated to obtain the fermented product of Stemona japonica probiotic.

2. The method for preparing the anti-acne Stemona japonica probiotic ferment according to claim 1, characterized in that, The Lactobacillus rhamnosus strain mentioned is Lactobacillus rhamnosus GG strain, with accession number CGMCC 1.3724.

3. The method for preparing the anti-acne Stemona japonica probiotic ferment according to claim 1 or 2, characterized in that, The absorbance of the probiotic liquid is 2-4, and its addition amount is 10%-40% of the volume of the mixture to be fermented.

4. The method for preparing the anti-acne Stemona japonica probiotic ferment according to claim 1, characterized in that, The fermentation process is carried out at a temperature of 35-39℃ for 2-4 days.

5. The method for preparing the anti-acne Stemona japonica probiotic ferment according to claim 1, characterized in that, The concentration of the glucose solution is 0.8-1.2 g / mL; the amount of glucose solution added is 20%-40% of the volume of the mixture to be fermented.

6. The method for preparing the anti-acne Stemona japonica probiotic ferment according to claim 1, characterized in that, The ratio of the solids to the fermentation mixture is 1g:(10-25)mL.

7. The method for preparing the anti-acne Stemona japonica probiotic ferment according to claim 1, characterized in that, The steps of extracting the residue of Stemona japonica with ethanol to obtain the ethanol extract include: Mix a portion of the Stemona japonica residue with an ethanol solution of 50%-70% by volume, heat and reflux, then filter. Repeat this step several times to extract the extract, and combine the filtered liquids to obtain the ethanol extract.

8. A fermented product of Stemona japonica prepared by any one of claims 1-7.

9. The use of the Stemona japonica probiotic ferment as described in claim 8 in the preparation of anti-acne drugs or skin care products.

10. An anti-acne facial mask, characterized in that, Includes the following components by mass fraction: Phase A: Glycerin 0.5%-0.8%; Hyaluronic acid 0.4%-0.6%; Sodium hyaluronate 0.4%-0.6%; 1,2-Butanediol 0.8%-1.2%; Xanthan gum 0.1%-0.5%; Salicylic acid 1%-1.5%; Water 43%-47%; Phase B: Nicotinamide 1.5%-2%; Arbutin 1%-1.5%; Phase C: PEG-40 hydrogenated castor oil 0.05%-0.15%; L-ascorbic acid 0.1%-0.5%; tea polyphenols 0.1%-0.5%; Centella asiatica extract 1%-3%; Stemona japonica probiotic ferment as described in claim 8 1%-3%; Ganoderma lucidum extract 0.1%-1%; Calendula officinalis extract 0.1%-1%; sodium bicarbonate 0.1%-0.3%; mandelic acid 0.8%-1.2%; balance: water.

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