Anti-acne radix stemonae probiotic fermented product as well as preparation method and application thereof

Abstract

The invention discloses an anti-acne radix stemonae probiotic fermented product and a preparation method and application thereof, and belongs to the technical field of medicines.The preparation method of the radix stemonae probiotic fermented product comprises the following steps that radix stemonae is prepared into a suspension, an enzyme preparation is added for enzymolysis, and zymolyte is obtained; adding the zymolyte, the probiotic liquid and the glucose solution into the culture medium to obtain a mixed solution to be fermented; performing fermentation treatment on the to-be-fermented mixed solution to obtain a fermentation product; performing material-liquid separation on the fermentation product to obtain radix stemonae material residues and fermentation liquid; extracting the radix stemonae residues with ethanol to obtain an alcohol extract; and mixing the fermentation liquor and the alcohol extract, and carrying out alcohol precipitation, centrifugation, suction filtration and concentration treatment to obtain the radix stemonae probiotic fermentation product. According to the preparation method provided by the invention, original macromolecular substances in the radix stemonae can be decomposed into small molecular compounds which are more easily absorbed by a body, and bacteriostatic and anti-inflammatory components are generated, so that the bioavailability and the treatment effect of the radix stemonae are improved.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a Stemona probiotic fermentate for treating acne, its preparation method and application. Background Art

[0002] Acne, as a common chronic inflammatory skin disease of hair follicles and sebaceous glands, its main pathogenic factor is the infection of Propionibacterium acnes. The clinical manifestations include comedones, papules, pustules and even cysts. At present, the western medicine treatment of acne mainly relies on antibiotics (such as erythromycin, clindamycin), retinoid drugs and anti-androgen therapy. However, long-term use is likely to cause adverse reactions such as bacterial drug resistance and skin irritation. With the development of modern medicine, the treatment of acne is increasing day by day, and topical medication is still the main therapy. Acne is not only a common disease in dermatology. Commonly used topical medications clinically mainly include antibiotics, anti-androgens and retinoids. However, the application of the antibacterial drug minocycline will lead to the risk of granulocytopenia. Taking anti-androgen drugs can cause menstrual disorders, nausea and dizziness, while male patients will have enlarged breasts. Vitamin A drugs have significant adverse reactions, such as strong irritating effects, which may cause deformities. Research shows that Artemisia annua and its active ingredients such as artemisinin, artemether, arteether, artesunate, dihydroartemisinin, etc. can all inhibit pathogenic bacteria such as Propionibacterium acnes and Staphylococcus aureus, and have a therapeutic effect on infectious skin diseases. At the same time, taking traditional Chinese medicine containing Artemisia annua orally has been proven to significantly relieve the clinical symptoms of acne in post-adolescent women. Therefore, it is particularly important to find safe and effective topical medications for treating acne. Studying natural plant extracts as an alternative or adjuvant treatment means has important scientific significance.

[0003] Stemona (Radix Stemonae), as a traditional Chinese medicine, has won wide favor among skin disease patients due to its natural properties, small toxic and side effects, significant curative effects, no dependence and other advantages. However, problems such as backward usage methods of traditional Chinese medicine, possible generation of derivatives, complex preparation components and most components being unclear, difficult to qualitatively and quantitatively determine, and inconvenient to carry have restricted the development of traditional Chinese medicine. In view of the potential antibacterial and anti-inflammatory activities of Stemona, it has become one of the research hotspots.

[0004] Current research on Stemona mainly focuses on the analysis of its chemical components and the exploration of its pharmacological activities. Research has revealed that the main active components of Stemona include alkaloids (such as stemonine and isostemonine), flavonoids, and steroidal saponins, etc. These components have been proven to have significant antibacterial and anti-inflammatory activities. In addition, Stemona extracts can also reduce the expression of pro-inflammatory factors such as TNF-α and IL-6 by inhibiting the NF-κB signaling pathway, which provides a theoretical basis for its application in the treatment of acne. However, the anti-acne research on Stemona extracts is relatively scarce, and most of them are limited to in vitro antibacterial experiments, lacking systematic in vivo efficacy evaluation and mechanism exploration. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of an anti-acne Stemona probiotic ferment, so as to solve the problems proposed in the above background technology.

[0006] To achieve the above purpose, the embodiments of the present invention provide the following technical solutions: A preparation method of an anti-acne Stemona probiotic ferment, comprising the following steps: Prepare Stemona into a suspension and add an enzyme preparation for enzymatic hydrolysis to obtain an enzymatic hydrolysate; the enzyme preparation is pectinase and / or cellulase, and its addition amount is 0.1%-0.3% of the mass of Stemona; the temperature of enzymatic hydrolysis is 50-70°C; Add the enzymatic hydrolysate, probiotic bacterial solution, and glucose solution to a culture medium to obtain a mixed solution to be fermented; the probiotic bacterial solution is a bacterial solution of Lactobacillus rhamnosus. Ferment the mixed solution to be fermented to obtain a fermentation product; Separate the fermentation product into a Stemona residue and a fermentation broth; Extract the Stemona residue with ethanol to obtain an ethanol extract; Mix the fermentation broth and the ethanol extract, and perform ethanol precipitation. After the ethanol precipitation is completed, perform centrifugation, discard the precipitate, and then perform suction filtration and concentration treatment to obtain the Stemona probiotic ferment.

[0007] Preferably, the Lactobacillus rhamnosus is Lactobacillus rhamnosus GG strain, and its preservation number is CGMCC 1.3724.

[0008] Preferably, the absorbance of the probiotic bacterial solution is 2-4, and its addition amount is 10%-40% of the volume of the mixed solution to be fermented.

[0009] Preferably, the temperature of fermentation treatment is 35-39°C, and the time is 2-4 days.

[0010] Preferably, the concentration of the glucose solution is 0.8-1.2 g / mL; the addition amount of the glucose solution is 20%-40% of the volume of the mixed solution to be fermented.

[0011] Preferably, the material-liquid ratio of Stemona sessilifolia Miq. to the liquid to be fermented is 1 g:(10 - 25) mL.

[0012] Preferably, the step of extracting Stemona sessilifolia Miq. residue with ethanol to obtain an ethanol extract specifically includes: Mix a part of Stemona sessilifolia Miq. residue with an ethanol solution having a volume concentration of 50% - 70%, carry out heating under reflux, then filter, repeat this step for extraction several times, and combine the filtered liquids to obtain an ethanol extract.

[0013] Another object of the present invention is to provide a Stemona sessilifolia Miq. probiotic ferment prepared by the above preparation method.

[0014] Another object of the present invention is to provide an application of the above Stemona sessilifolia Miq. probiotic ferment in the preparation of anti-acne drugs or skin care products.

[0015] Another object of the present invention is to provide an anti-acne facial mask, comprising the following components by mass fraction: Phase A: glycerol 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: niacinamide 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%; the above Stemona sessilifolia Miq. 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%; the balance is water.

[0016] By improving the enzymatic hydrolysis process of Stemona sessilifolia Miq. and fermenting Stemona sessilifolia Miq. with Lactobacillus rhamnosus, the present invention can decompose the original macromolecular substances in Stemona sessilifolia Miq. into small molecular compounds that are more easily absorbed by the body, and produce antibacterial and anti-inflammatory components, thereby increasing the bioavailability and therapeutic effect of Stemona sessilifolia Miq. In addition, by first performing enzymatic hydrolysis on Stemona sessilifolia Miq., the growth of Lactobacillus rhamnosus can be promoted, and Lactobacillus rhamnosus can promote the absorption and utilization of the active ingredients of Stemona sessilifolia Miq., and the two complement each other and synergistically enhance the effect; the Stemona sessilifolia Miq. probiotic ferment prepared by the present invention has high in vitro antibacterial activity and good anti-acne efficacy. Description of the Drawings

[0017] Figure 1 It is a comparison chart of the total flavonoid and total alkaloid contents in the Stemona sessilifolia Miq. probiotic ferment prepared at different fermentation times.

[0018] Figure 2 Comparison chart of the contents of total flavonoids and total alkaloids in Stemona sessilifolia probiotic fermentates prepared with different addition amounts of glucose solution.

[0019] Figure 3 Comparison chart of the contents of total flavonoids and total alkaloids in Stemona sessilifolia probiotic fermentates prepared with different addition amounts of probiotic bacterial liquid.

[0020] Figure 4 Comparison chart of the contents of total flavonoids and total alkaloids in Stemona sessilifolia probiotic fermentates prepared with different solid-liquid ratios.

[0021] Figure 5 Comparison chart of the total flavonoid content in Stemona sessilifolia probiotic fermentates prepared with different enzyme preparations added.

[0022] Figure 6 Comparison chart of the contents of total flavonoids and total alkaloids in Stemona sessilifolia probiotic fermentates prepared with different addition amounts of mixed enzymes.

[0023] Figure 7 Comparison chart of the contents of total flavonoids and total alkaloids in Stemona sessilifolia probiotic fermentates prepared at different enzymatic hydrolysis temperatures.

[0024] Figure 8 Observation result chart of HE staining of different groups of mice. Specific implementation manners

[0025] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0026] Embodiment 1: This embodiment provides a preparation method of an anti-acne Stemona sessilifolia probiotic fermentate, including the following steps: S1. Prepare 2 g of Stemona sessilifolia into a suspension, add an enzyme preparation, carry out enzymatic hydrolysis at 60 °C under a constant water bath condition for 60 min, then place it under high pressure at 121 °C for 15 min and cool it to obtain an enzymolysis product; wherein, the enzyme preparation is an equimass ratio mixture of pectinase and cellulase, and its addition amount is 0.2% of the mass of Stemona sessilifolia.

[0027] S2. Add the above enzymolysis product, 20 mL of probiotic bacterial liquid, and 15 mL of glucose solution with a concentration of 1 g / mL to MRS medium, and make up MRS medium to 50 mL to obtain a mixed liquid to be fermented; wherein, the probiotic bacterial liquid is a bacterial liquid of Lactobacillus rhamnosus. The MRS medium was prepared with 50 g of distilled water and 2.622 g of MRS. 2.622 g of MRS was accurately weighed using an electronic balance and slowly added to the 50 g of distilled water that was being heated. At the same time, it was stirred until the MRS was completely dissolved, and then placed in an autoclave for autoclaving (121 °C, 15 min) for later use.

[0028] The preparation method of the bacterial liquid of Lactobacillus rhamnosus (LGG) is as follows: The Lactobacillus rhamnosus GG strain with the existing publicly known deposit number of CGMCC 1.3724 was inoculated into the sterilized MRS medium at an inoculation amount of 1%. After thorough mixing, it was statically cultured at 37 °C for 24 h to obtain the first-generation bacterial liquid for later use; then, the first-generation bacterial liquid was inoculated into the MRS medium in a new sterile test tube at an inoculation ratio of 1%, and after thorough mixing, it was continuously statically cultured for 24 h to obtain the second-generation bacterial liquid for later use; similar to the preparation of the second-generation bacterial liquid, the second-generation bacterial liquid was aspirated at an inoculation ratio of 1% and mixed with the MRS medium, and cultured for 20 h. The absorbance of the bacterial liquid was adjusted to 3 to obtain the bacterial liquid of Lactobacillus rhamnosus.

[0029] The preparation method of the glucose solution (GLU) is as follows: Weigh 30 g of glucose, add distilled water, and make up the volume to 30 mL with distilled water. Stir while adding water until the glucose is completely dissolved. Then, place the dissolved solution in an autoclave for autoclaving (121 °C, 15 min) to obtain the glucose solution for later use.

[0030] S3. Place the above-mentioned mixed liquid to be fermented in a constant temperature incubator at 37 °C for fermentation treatment for 3 d to obtain the fermentation product.

[0031] S4. Separate the fermentation product into the Stemonae residue and the fermentation broth. The fermentation broth was placed in a 1000 mL volumetric flask for later use.

[0032] S5. Place the above-mentioned Stemonae residue in a round-bottom flask, add 36 mL of ethanol solution with a volume concentration of 50%, heat under reflux for 2 h, then filter with filter paper, and extract twice more in the same way. Collect the liquids obtained from the three filtrations to obtain the ethanol extract.

[0033] S6. Mix the above-mentioned fermentation broth and ethanol extract, and adjust it to an ethanol solution with a volume concentration of 85% with absolute ethanol for overnight ethanol precipitation. After the ethanol 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, and filter the filtrate. The clarified part was rotary evaporated and concentrated to 2 mL to obtain the Stemonae probiotic ferment.

[0034] In addition, this example also provides an anti-acne facial mask, which includes the following components by mass fraction: 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%; Phase B: Niacinamide 1.75%; Arbutin 1.25%; Phase C: PEG-40 Hydrogenated castor oil 0.1%; L-Ascorbic acid 0.25%; Tea polyphenols 0.25%; Centella asiatica extract 2%; The above Stemona japonica probiotic ferment 2%; Ganoderma lucidum extract 0.5%; Calendula officinalis extract 0.5%; Sodium bicarbonate 0.15%; Mandelic acid 1%; The balance is water.

[0035] The preparation method of the above anti-acne facial mask is as follows: Step 1: Put Phase A into a water bath, heat up to 85°C, stop heating when the colloidal substances are completely dispersed, then keep warm for 20 minutes and stir until completely dissolved; Step 2: Transfer to a 60°C water bath, add Phase B, stir evenly, then transfer to a 45°C water bath, add Phase C, stir evenly, and use a 0.22μm sterile filter membrane to filter and sterilize the facial mask liquid.

[0036] Step 3: Put the paper mask into the facial mask liquid, stir manually to make it evenly moist, after stirring, gently squeeze to keep it moderately moist; Fold the soaked facial mask into a rectangle and package it, then store it in a 4°C refrigerator.

[0037] Example 2: This example provides a preparation method of a Stemona japonica probiotic ferment for anti-acne, including the following steps: S1. Prepare a suspension of 2g of Stemona japonica, add an enzyme preparation, and perform enzymatic hydrolysis at 50°C in a constant temperature water bath for 30 minutes, then place it under high pressure at 121°C for 15 minutes and let it cool to obtain an enzymatic hydrolysate; Among them, the enzyme preparation is pectinase, and its addition amount is 0.1% of the mass of Stemona japonica.

[0038] S2. Add the above enzymatic hydrolysate, 5mL of probiotic bacterial liquid, and 10mL of glucose solution with a concentration of 0.8g / mL to MRS medium, and make up the MRS medium to 50mL to obtain a mixed liquid to be fermented; Among them, the probiotic bacterial liquid is the bacterial liquid of Lactobacillus rhamnosus; The MRS medium is prepared with 50g of distilled water and 2.622g of MRS. Weigh 2.622g of MRS accurately with an electronic balance, and slowly add it to the 50g of distilled water that is being heated, and at the same time stir until the MRS is completely dissolved, then put it into an autoclave for autoclaving (121°C, 15 minutes) for standby.

[0039] The preparation method of the bacterial liquid of Lactobacillus rhamnosus (LGG) is as follows: The Lactobacillus rhamnosus GG strain with the existing publicly known preservation number of CGMCC 1.3724 is inoculated into the sterilized MRS medium at an inoculation amount of 1%, and after thorough mixing, it is statically cultured at 37°C for 24 h to obtain the first-generation bacterial liquid for standby; then, the first-generation bacterial liquid is continuously inoculated into the MRS medium in a new sterile test tube at an inoculation ratio of 1%, and after thorough mixing, it is continuously statically cultured for 24 h to obtain the second-generation bacterial liquid for standby; similar to the preparation of the second-generation bacterial liquid, the second-generation bacterial liquid is aspirated at an inoculation ratio of 1% and mixed with the MRS medium, and cultured for 20 h, and the absorbance of the bacterial liquid is adjusted to 2 to obtain the bacterial liquid of Lactobacillus rhamnosus.

[0040] The preparation method of the glucose solution (GLU) is as follows: Weigh 30 g of glucose, add distilled water, make up the volume to 30 mL with distilled water, stir while adding water until the glucose is completely dissolved, and then put the dissolved solution into an autoclave for autoclaving (121°C, 15 min) to obtain the glucose solution for standby.

[0041] S3. Place the above-mentioned mixed liquid to be fermented in a constant temperature incubator at 35°C for fermentation treatment for 2 d to obtain the fermentation product.

[0042] S4. Separate the liquid and solid of the fermentation product to obtain the Stemonae residue and the fermentation broth, and place the fermentation broth in a 1000 mL volumetric flask for standby.

[0043] S5. Place the above-mentioned Stemonae residue in a round-bottom flask, add 36 mL of ethanol solution with a volume concentration of 60%, heat under reflux for 2 h, filter with filter paper, extract twice more in the same way, collect the liquid obtained from the three filtrations to obtain the ethanol extract.

[0044] S6. Mix the above-mentioned fermentation broth and ethanol extract, and adjust it to an ethanol solution with a volume concentration of 80% with anhydrous ethanol for overnight ethanol precipitation. After the ethanol 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 by rotary evaporation to 2 mL to obtain the Stemonae probiotic ferment.

[0045] In addition, this example also provides an anti-acne facial mask, which includes the following components by mass fraction: Phase A: Glycerol 0.5%; Hyaluronic acid 0.4%; Sodium hyaluronate 0.4%; 1,2-Butanediol 0.8%; Xanthan gum 0.1%; Salicylic acid 1%; Water 47%; Phase B: Niacinamide 1.5%; Arbutin 1%; Phase C: 0.05% of PEG - 40 hydrogenated castor oil; 0.1% of L - ascorbic acid; 0.1% of tea polyphenols; 1% of centella asiatica extract; 3% of the above - mentioned stemona japonica probiotic ferment; 0.1% of ganoderma lucidum extract; 0.1% of calendula officinalis extract; 0.1% of sodium bicarbonate; 0.8% of mandelic acid; the balance is water.

[0046] The preparation method of the above acne - resistant facial mask is as follows: Step 1: Put Phase A into a water - bath pot, heat up to 85°C, stop heating when the colloid - like substances are completely dispersed, then keep warm for 20 minutes and stir until completely dissolved. Step 2: Transfer it to a 60°C water - bath pot, add Phase B, stir evenly, then transfer it to a 45°C water - bath pot, add Phase C, stir evenly, and use a 0.22μm sterile filter membrane to filter and sterilize the facial mask liquid.

[0047] Step 3: Put the paper mask into the facial mask liquid, stir manually to make it evenly moist, after stirring, gently squeeze it to keep it at an appropriate moist level; fold the soaked facial mask into a rectangular shape and package it, then store it in a 4°C refrigerator.

[0048] Example 3: This example provides a preparation method of a stemona japonica probiotic ferment for treating acne, including the following steps: S1. Prepare a suspension of 2g of stemona japonica, add an enzyme preparation, and carry out enzymatic hydrolysis at 65°C under constant - temperature water - bath conditions for 90 min, then place it under high - pressure treatment at 121°C for 15 min and let it cool to obtain an enzymolysis product; among them, the enzyme preparation is cellulase, and its addition amount is 0.3% of the mass of stemona japonica.

[0049] S2. Add the above - mentioned enzymolysis product, 15 mL of probiotic bacterial liquid, and 20 mL of a glucose solution with a concentration of 1.2 g / mL to MRS medium, and make up the MRS medium to 50 mL to obtain a mixed liquid to be fermented; among them, the probiotic bacterial liquid is the bacterial liquid of lactobacillus rhamnosus. The MRS medium is prepared with 50g of distilled water and 2.622g of MRS. Weigh 2.622g of MRS accurately with an electronic balance, and slowly add it to the 50g of distilled water that is being heated, and at the same time stir until the MRS is completely dissolved, then put it into an autoclave for high - pressure sterilization (121°C, 15 min) for standby.

[0050] The preparation method of the bacterial liquid of Lactobacillus rhamnosus (LGG) is as follows: the Lactobacillus rhamnosus GG strain with the existing publicly known preservation number CGMCC 1.3724 is inoculated into a sterilized MRS culture medium at a 1% inoculation rate, mixed thoroughly, and then cultured at 37°C for 24 hours to obtain a first-generation bacterial liquid for use; the first-generation bacterial liquid is further inoculated into the MRS culture medium at a 1% inoculation rate and added to a new sterile test tube, mixed thoroughly, and then cultured for another 24 hours to obtain a second-generation bacterial liquid for use; similar to the preparation of the second-generation bacterial liquid, the second-generation bacterial liquid is aspirated and mixed with the MRS culture medium at a 1% inoculation rate, cultured for 20 hours, and the absorbance of the bacterial liquid is adjusted to 4 to obtain the bacterial liquid of Lactobacillus rhamnosus.

[0051] The preparation method of glucose solution (GLU) is as follows: weigh 30 g of glucose, add distilled water, and dilute to 30 mL with distilled water. Stir while adding water until the glucose is completely dissolved, then place the dissolved solution into an autoclave for high-pressure sterilization (121°C, 15 min) to obtain glucose solution for later use.

[0052] S3. The mixed solution to be fermented is placed in a constant temperature incubator at 39° C. for fermentation for 4 days to obtain a fermentation product.

[0053] S4. Separate the fermentation product into a material-liquid separation to obtain a Stemona slag and a fermentation liquid, and place the fermentation liquid in a 1000 mL volumetric flask for later use.

[0054] S5. Place the above-mentioned Stemona residue in a round-bottom flask, and add 36 mL of 70% ethanol solution by volume. After heating and refluxing for 2 h, filter with filter paper, extract twice more using the same method, collect the liquid obtained by filtration three times, and obtain the alcohol extract.

[0055] S6. The fermentation broth and the alcohol extract were mixed, and anhydrous ethanol was used to prepare an ethanol solution with a volume concentration of 90%, and the mixture was precipitated overnight. After the precipitation, the mixture was centrifuged at a rate of 4000 r / min for 15 min, and the precipitate was discarded. A vacuum pump was connected to a Buchner funnel, and the filtrate was filtered. The clarified portion was concentrated by rotary evaporation to 2 mL to obtain a fermentation product of Stemona probiotics.

[0056] In addition, this embodiment also provides an anti-acne facial mask, comprising the following components by mass fraction: 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%; Phase B: Niacinamide 2%; Arbutin 1.5%; Phase C: 0.15% of PEG - 40 hydrogenated castor oil; 0.5% of L - ascorbic acid; 0.5% of tea polyphenols; 3% of centella asiatica extract; 1% of the above - mentioned stemona japonica probiotic ferment; 1% of ganoderma lucidum extract; 1% of calendula officinalis extract; 0.3% of sodium bicarbonate; 1.2% of mandelic acid; the balance is water.

[0057] The preparation method of the above acne - fighting facial mask is as follows: Step 1: Put Phase A into a water - bath kettle, heat up to 85°C, stop heating when the colloid - like substances are completely dispersed, then keep warm for 20 minutes and stir until completely dissolved. Step 2: Transfer it to a 60°C water - bath kettle, add Phase B, stir evenly, then transfer it to a 45°C water - bath kettle, add Phase C, stir evenly, and then use a 0.22μm sterile filter membrane to filter and sterilize the facial mask liquid.

[0058] Step 3: Put the paper mask into the facial mask liquid, stir manually to make it evenly moist, after stirring, gently squeeze it to keep it at an appropriate moist level; fold the soaked facial mask into a rectangle and package it, then store it in a 4°C refrigerator.

[0059] Experimental Example 1: Referring to the preparation method provided in Example 1, select several factors that may affect the fermentation effect, such as fermentation time (2d, 3d, 4d, 5d, 6d), addition amount of glucose solution (0, 10%, 20%, 30%, 40%), addition amount of probiotic bacteria liquid (5%, 10%, 20%, 30%, 40%), solid - liquid ratio (1g:2.5mL, 1g:5mL, 1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL), enzyme preparations (including pectinase, cellulase, glucosidase, papain, a mixture of pectinase and cellulase), addition amount of enzyme preparations (0.1%, 0.2%, 0.3%, 0.4%, 0.5%), and enzymatic hydrolysis temperature (50°C, 55°C, 60°C, 65°C, 70°C). Use the extraction rates of total flavonoids and total alkaloids as response values to conduct single - factor experiments.

[0060] Among them, the detection method of total flavonoids is as follows: Precisely measure 200μL of rutin standard solution (0.2g / mL) and the sample solution to be measured (stemona japonica probiotic ferment), sequentially add 400μL of 70% ethanol, 40μL of 5% sodium nitrite solution into the tube, mix well, and let it stand for 6 minutes; then continue to add 40μL of 10% aluminum nitrate, mix well again, and let it stand for 6 minutes; finally, add 80μL of 4mol / L sodium hydroxide solution and 240μL of 70% ethanol, shake well, place it for 15 minutes, and measure the absorbance at a wavelength of 510nm; calculate the content of total flavonoids according to the absorbance. The specific calculation method can refer to the existing technology and will not be elaborated here.

[0061] The detection method of total alkaloids is as follows: The Stemona probiotic fermentate is adjusted to pH 2 - 3 with hydrochloric acid, fixed volume to 2.5 mL, and 1 mL of freshly prepared saturated solution of 2% ammonium reineckate is added. After standing in ice water for 1 h, centrifuge at 3000 r / min and 4 °C for 10 min. Discard the supernatant, add ice water mixture to blow, wash and centrifuge, repeat the above steps until the precipitate of alkaloid ammonium reineckate is colorless. All the above operations are carried out on ice. Acetone can dissolve the filtered and washed precipitate of alkaloid ammonium reineckate. Finally, measure the absorbance of the sample at the maximum absorption wavelength of 523 nm, use acetone as the blank control, calculate the content of total alkaloids in Stemona, and calculate it based on tuberostemonine (C 22 H 33 O4N). For the specific calculation method, refer to the existing technology, and it will not be elaborated here.

[0062] The above test results are as follows: Under the conditions of a material - liquid ratio of 1:20, a glucose solution addition amount of 30%, and a probiotic bacteria solution addition amount of 30%, the effects of fermentation time on the contents of total flavonoids and total alkaloids in the Stemona probiotic fermentate were investigated. The results are as Figure 1 shown. As the fermentation time extends, the yields of total flavonoids and total alkaloids first increase and then decrease, and the yields are relatively high when the fermentation time is 3 d.

[0063] Under the conditions of a material - liquid ratio of 1:20, a fermentation time of 3 d, and a probiotic bacteria solution addition amount of 30%, the effects of the glucose solution addition amount on the contents of total flavonoids and total alkaloids in the Stemona probiotic fermentate were investigated. The results are as Figure 2 shown. When the glucose solution addition amount is 20%, the yield of total flavonoids is relatively high. When the glucose solution addition amount is 30%, the yield of total alkaloids is relatively high.

[0064] Under the conditions of a material - liquid ratio of 1:20, a fermentation time of 3 d, and a glucose solution addition amount of 30%, the effects of the probiotic bacteria solution addition amount on the contents of total flavonoids and total alkaloids in the Stemona probiotic fermentate were investigated. The results are as Figure 3 shown. When the probiotic bacteria solution addition amount is 10%, the yield of total flavonoids is relatively high. When the probiotic bacteria solution addition amount is 30%, the yield of total alkaloids is relatively high.

[0065] Under the conditions of a fermentation time of 3 d, a probiotic bacteria solution addition amount of 30%, and a glucose solution addition amount of 30%, the effects of the material - liquid ratio on the contents of total flavonoids and total alkaloids in the Stemona probiotic fermentate were investigated. The results are as Figure 4 shown. As the material - liquid ratio increases, the yield of total flavonoids first increases and then decreases, and the yield is the highest at 1:10. The alkaloids increase with the increase of the material - liquid ratio, and the yield is relatively high when the material - liquid ratio is about 1:25.

[0066] Under the condition that other conditions remain unchanged, without adding enzyme preparations and adding different enzyme preparations, the contents of active ingredients were extracted after fermentation. The results are as Figure 5As shown. It can be seen from the figure that the contents of total flavonoids and total alkaloids in the Stemona sessilifolia probiotic fermentate are the highest when the enzyme preparation added is a mixture of pectinase and cellulase.

[0067] Under the condition that other conditions remain unchanged, different amounts of the mixture of pectinase and cellulase were added, and the contents of active ingredients were extracted after fermentation. The results are as Figure 6 shown. It can be seen from the figure that when the addition amounts of pectinase and cellulase in the Stemona sessilifolia probiotic fermentate exceed 0.2%, the content of total alkaloids begins to decrease and precipitate. When the addition amount of the mixed enzyme exceeds 0.3%, the total flavonoids decrease somewhat. This shows that the greater the addition amount of the mixed enzyme, the more obvious the inhibition of the enzymolysis effect.

[0068] Under the condition that other conditions remain unchanged, the same amount of the mixture of pectinase and cellulase was added, and at different enzymolysis temperatures, the contents of active ingredients were extracted after fermentation. The results are as Figure 7 shown. It can be seen from the figure that the contents of active ingredients in the Stemona sessilifolia probiotic fermentate all show a trend of first increasing and then decreasing with the increase of the enzymolysis temperature. And the contents of active ingredients in the Stemona sessilifolia probiotic fermentate decrease rapidly and tend to be flat after 60 °C, indicating that the temperature after 60 °C has an inhibitory effect on the precipitation of the Stemona sessilifolia enzymolysis product.

[0069] Experimental Example 2: Detection of the promoting effect of Stemona sessilifolia probiotic fermentate on probiotics: Using 96-well plate culture, 250 μL of the alcohol extract of Stemona sessilifolia prepared according to the method provided in Example 1, 1750 μL of MRS medium, and 10 μL of Lactobacillus rhamnosus (LGG) bacterial solution (OD 600 = 3) were added to one well. The reaction wells were sealed with a sterile sealing film. Real-time OD value detection was carried out at 600 nm using a multifunctional microplate reader. After shaking for 10 s, static measurement was performed, and data was automatically recorded every 30 min, and continuous detection was carried out for 24 hours to obtain a complete growth curve. The concentration value of Stemona sessilifolia corresponding to the maximum biomass in the Stemona sessilifolia-LGG co-culture fermentation model was determined to be 0.03125 g / mL, and the addition amount of LGG was 1% (OD 600 = 3). Thus, an optimal concentration Stemona sessilifolia-LGG co-culture fermentation system was established. At this concentration, the OD 600 of LGG reached 2.6, while that of the blank control group was 0.9, indicating that Stemona sessilifolia can promote the growth of LGG.

[0070] The polysaccharide concentration was determined using the plant soluble polysaccharide detection kit Solarbio BC0030 of the anthrone colorimetric method. Compared with the pure LGG culture (0.0839 mg / mL), the extracellular polysaccharide concentration in the Stemona sessilifolia-LGG co-culture (0.1092 mg / mL) increased by 30.2%.

[0071] The double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine L-lactic acid. Compared with the pure LGG culture (189.2514 ng / ml), the concentration of L-lactic acid in the Stemona-LGG co-culture (531.9712 ng / mL) increased by 181.1%.

[0072] The double-antibody one-step sandwich enzyme-linked immunosorbent assay was used to determine the folic acid content. Compared with the pure LGG culture (237.7178 ng / mL), the concentration of folic acid in the Stemona-LGG co-culture (754.2683 ng / mL) increased by 217.3%.

[0073] Test Example 3: The anti-acne facial mask prepared in Example 1 above was subjected to finished product inspection, and the results are as follows: (1) The color of the facial mask is light brown, with a smooth texture and a slight luster. The smell is an elegant medicinal fragrance, a single linear incense, without an irritating smell.

[0074] (2) The results of the physical and chemical experiments showed that the pH value of the facial mask liquid was 5 - 6, and the results of the heat resistance, cold resistance, and centrifugal stability experiments were all without stratification.

[0075] (3) The results of the microbial detection showed that the total number of colonies in the facial mask liquid was 640 CFU / mL, no Staphylococcus aureus was detected, and no thermotolerant coliforms were detected.

[0076] (4) The total flavonoid concentration of the facial mask was 1.009 mg / mL, not lower than the total flavonoid concentration of the Stemona extract used as the raw material, and the active ingredients were effectively retained. The total alkaloid concentration of the facial mask was 1.620 mg / mL, not lower than the total alkaloid concentration of the Stemona extract used as the raw material.

[0077] Test Example 4: The product prepared in Example 1 above was subjected to in vitro antibacterial and anti-acne tests. Specifically, 28 SPF-grade healthy female ICR mice aged 6 - 8 weeks and weighing 36 - 45 g were selected. These mice were purchased from Changchun Yisi Biotechnology Co., Ltd. After being freely fed and drinking water at room temperature of 20 - 26 °C for one week, the experiment was carried out. They 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 probiotic fermentation product group. The concentration of Propionibacterium acnes was adjusted to (1×10 8 CFU / 20 μL) suspension bacteria solution using PBS buffer. Each group of mice was anesthetized with sodium pentobarbital anesthetic, and dorsal marking was carried out.

[0078] A mouse acne model was constructed. Except for the normal group, in the other groups, 20 μL of Propionibacterium acnes bacterial solution was injected intradermally into the auricular skin of mice using a 100 μL syringe, and then the mice were raised normally. When there were small bumps on the auricles of the mice accompanied by symptoms of inflammation, redness, and swelling, it indicated that the modeling was successful. After successful modeling, in the normal group, the mice did not receive any special treatment throughout the experiment. The mice in the negative group were smeared with PBS buffer solution at the acne lesion site, 20 μL each time, twice a day. The mice in the positive group were smeared with erythromycin at the acne lesion site, 20 μL each time, twice a day. The mice in the unfermented extract group were smeared with the unfermented Stemona tuberosa alcohol extract (prepared according to the method provided in Example 1) at the acne lesion site, 20 μL each time, twice a day. The mice in the Stemona tuberosa probiotic fermentation product group were smeared with the Stemona tuberosa probiotic fermentation product prepared in Example 1 at the acne lesion site, 20 μL each time, twice a day. This treatment lasted for 7 days to simulate the treatment process. At the end of the experiment, the mice were intraperitoneally anesthetized with pentobarbital, and then their ear skin lesion tissues and sera were taken for research. These ear tissues were subsequently fixed with formaldehyde, and finally the mice were sacrificed by cervical dislocation to ensure the smooth progress of the experiment.

[0079] The test results were as follows: The minimum inhibitory concentration (MIC) of the Stemona tuberosa probiotic fermentation product against Propionibacterium acnes was 31.25 mg / mL, the minimum bactericidal concentration (MBC) was 0.5 g / mL, the MBC / MIC ratio ≤ 4, the MIC values of the Stemona tuberosa probiotic fermentation product against Escherichia coli and Staphylococcus aureus in vitro were 0.0625 g / mL, and the MIC value against Propionibacterium acnes was 0.0625 g / mL.

[0080] The swelling of the auricles of the mice in the Stemona tuberosa probiotic fermentation product group gradually subsided, and the thickness of the auricles of the mice decreased by 0.71 ± 0.03 mm (the unfermented extract group decreased by 0.633 ± 0.05 mm). HE staining showed that the inflammatory cells were significantly reduced, the vasodilation effect of the mice weakened, and the degree of edema improved, as shown in Figure 8 shown.

[0081] The contents of TNF-α, IL-8, and IL-6 in the auricular skin lesion tissues of the mice in the Stemona tuberosa probiotic fermentation product group decreased by 36.71%, 17.64%, and 26.83% respectively (the unfermented extract group decreased by 25.45%, 15.08%, and 23.62% respectively), the contents of TNF-α, IL-8, and IL-6 in the blood decreased by 35.56%, 40.84%, and 33.76% respectively (the unfermented extract group decreased by 23.42%, 38.30%, and 30.93% respectively), and the numbers of immune cells producing TNF-α and IL-6 in the spleen decreased by 59% and 65% respectively (the unfermented extract group decreased by 46% and 60% respectively).

[0082] Inspired by the above-described ideal embodiments of the present invention, through the above description, relevant staff can make various changes and modifications completely within the scope without departing from the technical idea of this invention. The technical scope of this invention is not limited to the content in the specification.

Claims

1. A preparation method of Stemona tuberosa probiotic ferment for treating acne, characterized in that, It includes the following steps: Prepare Stemona sessilifolia Miq. into a suspension, add an enzyme preparation for enzymatic hydrolysis to obtain a hydrolysate; the enzyme preparation is pectinase and / or cellulase, and its addition amount is 0.1%-0.3% of the mass of Stemona sessilifolia Miq.; the temperature of enzymatic hydrolysis is 50-70°C; Add the hydrolysate, probiotic bacterial liquid, and glucose solution to a culture medium to obtain a mixed liquid to be fermented; the probiotic bacterial liquid is the bacterial liquid of Lactobacillus rhamnosus; Perform fermentation treatment on the mixed liquid to be fermented to obtain a fermentation product; Perform liquid-solid separation on the fermentation product to obtain Stemona sessilifolia Miq. residue and fermentation broth; Extract the Stemona sessilifolia Miq. residue with ethanol to obtain an ethanol extract; Mix the fermentation broth and the ethanol extract, perform ethanol precipitation, after the ethanol precipitation is completed, perform centrifugation, discard the precipitate, and then perform suction filtration and concentration treatment to obtain the Stemona sessilifolia Miq. probiotic ferment.

2. The preparation method of the stemona probiotic ferment for treating acne according to claim 1, wherein The Lactobacillus rhamnosus is Lactobacillus rhamnosus GG strain, and its preservation number is CGMCC 1.3724.

3. The preparation method of the stemona probiotic ferment for treating acne according to claim 1 or 2, characterized in that, The absorbance of the probiotic bacterial liquid is 2-4, and its addition amount is 10%-40% of the volume of the mixed liquid to be fermented.

4. The preparation method of the stemona probiotic ferment for treating acne according to claim 1, wherein The temperature of fermentation treatment is 35-39°C, and the time is 2-4 d.

5. The preparation method of the stemona-probiotic ferment for treating acne according to claim 1, wherein, The concentration of the glucose solution is 0.8-1.2 g / mL; the addition amount of the glucose solution is 20%-40% of the volume of the mixed liquid to be fermented.

6. The preparation method of the stemona probiotic ferment for treating acne according to claim 1, characterized in that, The liquid-solid ratio of Stemona sessilifolia Miq. to the mixed liquid to be fermented is 1 g:(10-25) mL.

7. The preparation method of the stemona probiotic ferment for treating acne according to claim 1, characterized in that, The step of extracting the Stemona sessilifolia Miq. residue with ethanol to obtain an ethanol extract specifically includes: Mix a part of the Stemona sessilifolia Miq. residue with an ethanol solution with a volume concentration of 50%-70%, perform heating under reflux, and then perform filtration. Repeat this step for extraction several times, and combine the filtered liquids to obtain an ethanol extract.

8. A Stemona sessilifolia Miq. probiotic ferment prepared by the preparation method according to any one of claims 1-7.

9. Use of the Stemona sessilifolia Miq. probiotic ferment according to claim 8 in the preparation of anti-acne drugs or skin care products.

10. An anti-acne facial mask, characterized in that, It includes the following components by mass fraction: Phase A: glycerol 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: niacinamide 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%; the Stemona sessilifolia Miq. probiotic ferment according to 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%; the balance is water.

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