Stemona japonica leavening for treating H1N1 virus infection as well as preparation method and application of stemona japonica leavening

Abstract

The invention discloses a radix stemonae fermentation product for treating H1N1 virus infection and a preparation method and application thereof, and belongs to the technical field of medicines.The preparation method of the radix stemonae fermentation product comprises the following steps that radix stemonae, probiotic mixed bacterium liquid and a glucose solution are added into a culture medium, and mixed liquid to be fermented is obtained; placing the to-be-fermented mixed solution at a fermentation temperature of 35-39 DEG C for fermentation treatment to obtain a fermentation product; performing material-liquid separation on the fermentation product to obtain radix stemonae material residues and fermentation liquid; extracting half of the radix stemonae residues with ethanol to obtain an alcohol extract; extracting the residual stemona root residues with water to obtain a water extract; and mixing the fermentation liquor, the alcohol extract and the water extract, and performing concentration treatment to obtain the stemona japonica fermented product. Probiotics compounded by lactobacillus rhamnosus and lactobacillus plantarum are introduced in the radix stemonae fermentation process, conversion and release of active ingredients of the radix stemonae can be further promoted, the medicine effect is improved, and beneficial compounds are generated.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a Stemona ferment for treating H1N1 virus infection, its preparation method and application. Background Art

[0002] Influenza caused by influenza viruses such as H1N1 is characterized by strong infectivity and severe complications, and is one of the potential factors leading to major public health events worldwide. After the H1N1 virus infects a host, the innate immune response of the body is first activated. Over-activation of innate immune cells will lead to the excessive release of cytokines, causing a cytokine storm, resulting in lung injury and even more severe acute respiratory distress syndrome. This kind of lung injury caused by the H1N1 virus has been confirmed by many studies and is the main cause of severe viral infection and death. Oseltamivir is an inhibitor of influenza virus neuraminidase. Because it can prevent the release of virus progeny particles and control the spread of the virus, it is often used as the first choice drug for treating influenza. However, the antiviral effect of oseltamivir has been challenged due to factors such as gene mutations of virus strains. Research shows that the emergence of drug resistance of influenza viruses to oseltamivir limits its antiviral effectiveness in clinical applications. Therefore, it is particularly important to explore new and effective treatment strategies.

[0003] In addition, compared with Western medicine, traditional Chinese medicine has the characteristics of low toxicity, immunomodulation and low drug resistance. Among them, the main active ingredient of Stemona is Stemona alkaloids, which are alkaloids with a pyrrole heterocyclic or pyridine heterocyclic mother nucleus structure unique to this family of plants, and have functions such as expelling parasites, killing insects, relieving cough and asthma, anti-tumor and antibacterial. Chinese herbal medicines mainly use the body's own antibacterial factors to fight against pathogenic bacteria and are not prone to drug resistance. However, the active ingredients of Chinese herbal medicines such as Stemona are mostly located in the cytoplasm of cells in different positions such as roots, rhizomes and leaves, and are protected by plant cell walls; traditional processing methods cannot effectively and quickly break through the protection of cell walls on active ingredients, and the medicinal properties cannot be extracted, resulting in low utilization rate of Chinese herbal medicines. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of a Stemona ferment for treating H1N1 virus infection, so as to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the embodiments of the present invention provide the following technical solutions: A preparation method of a Stemona ferment for treating H1N1 virus infection, which comprises the following steps: Add Stemona sessilifolia, probiotic mixed bacterial liquid, and glucose solution to the culture medium to obtain the mixed liquid to be fermented; the probiotic mixed bacterial liquid is composed of the bacterial liquids of Lactobacillus rhamnosus and Lactobacillus plantarum; the addition amount of the probiotic mixed bacterial liquid is 20%-40% of the volume of the mixed liquid to be fermented; the material-liquid ratio of Stemona sessilifolia to the mixed liquid to be fermented is 1 g:(10-30) mL; Place the mixed liquid to be fermented at a fermentation temperature of 35-39 °C for fermentation treatment to obtain a fermentation product; Separate the material and liquid of the fermentation product to obtain Stemona sessilifolia dregs and fermentation broth; Extract half of the Stemona sessilifolia dregs with ethanol to obtain an ethanol extract; Extract the remaining Stemona sessilifolia dregs with water to obtain a water extract; Mix the fermentation broth, ethanol extract, and water extract, and perform concentration treatment to obtain the Stemona sessilifolia ferment.

[0006] Preferably, the Lactobacillus rhamnosus is Lactobacillus rhamnosus GG strain, and its preservation number is CGMCC 1.3724.

[0007] Preferably, the preservation number of the Lactobacillus plantarum is BNCC 194165.

[0008] Preferably, the absorbance of the bacterial liquid of Lactobacillus rhamnosus is 2-4, and the absorbance of the bacterial liquid of Lactobacillus plantarum is 2-4; the volume ratio of the bacterial liquid of Lactobacillus rhamnosus to the bacterial liquid of Lactobacillus plantarum is 1:(0.5-2).

[0009] Preferably, the fermentation treatment time is 36-60 h.

[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 liquid to be fermented.

[0011] More preferably, the addition amount of the probiotic mixed bacterial liquid is 30% of the volume of the mixed liquid to be fermented; the material-liquid ratio of Stemona sessilifolia to the mixed liquid to be fermented is 1 g:25 mL; the absorbance of the bacterial liquid of Lactobacillus rhamnosus is 3, and the absorbance of the bacterial liquid of Lactobacillus plantarum is 3; the volume ratio of the bacterial liquid of Lactobacillus rhamnosus to the bacterial liquid of Lactobacillus plantarum is 1:1; the concentration of the glucose solution is 1 g / mL; the addition amount of the glucose solution is 30% of the volume of the mixed liquid to be fermented; the fermentation temperature is 37 °C; the fermentation treatment time is 48 h.

[0012] Preferably, the step of extracting half of the Stemona sessilifolia dregs with ethanol to obtain an ethanol extract specifically includes: Mix half of the Stemona sessilifolia residue with an ethanol solution having a volume concentration of 50%-70%, perform heating under reflux, then filter, repeat this step for extraction 1-3 times, combine the filtered liquids to obtain a first filtrate; Adjust the first filtrate with absolute ethanol to an ethanol solution having a volume concentration of 80%-90% for ethanol precipitation. After the ethanol precipitation is completed, perform centrifugation, discard the precipitate, after suction filtration, then perform rotary evaporation to remove ethanol to obtain an ethanol extract.

[0013] Preferably, the step of extracting the remaining Stemona sessilifolia residue with water to obtain a water extract specifically includes: Mix the remaining Stemona sessilifolia residue with distilled water, perform heating under reflux, then filter, repeat this step for extraction 1-3 times, combine the filtered liquids to obtain a second filtrate; Perform centrifugation on the second filtrate, discard the precipitate, after suction filtration, obtain a water extract.

[0014] Another object of the present invention is to provide a Stemona sessilifolia ferment prepared by the above preparation method.

[0015] Another object of the present invention is to provide an application of the above Stemona sessilifolia ferment in the preparation of a drug for treating or preventing H1N1 virus infection.

[0016] In the present invention, by introducing probiotics compounded with Lactobacillus rhamnosus and Lactobacillus plantarum in the fermentation of Stemona sessilifolia, the transformation and release of its active ingredients can be further promoted, the drug efficacy can be improved, and beneficial compounds can be produced, providing a theoretical basis for the application of Stemona sessilifolia ferment in the field of anti-bacterial infection and providing technical support for improving the bioavailability of Stemona sessilifolia. Compared with the traditional method, probiotic fermentation helps to increase the release level of active ingredients in Stemona sessilifolia and then strengthen its pharmacological effect, achieving a synergistic effect. Specifically, during the fermentation process, microorganisms such as Lactobacillus rhamnosus and Lactobacillus plantarum trigger various lignin-degrading enzymes and hydrolases through metabolic activities. These enzymes can decompose the fiber structure of Stemona sessilifolia and then cause structural damage to the cell wall, thereby releasing active substances. After Stemona sessilifolia is acted on by the enzyme system of microorganisms, the fiber and lignin parts become loose, the active ingredients are effectively released, or new active substances are produced, greatly improving and enhancing the drug efficacy. The prebiotics produced by Stemona sessilifolia after fermentation can promote the reproduction of Lactobacillus rhamnosus and Lactobacillus plantarum, and Lactobacillus rhamnosus and Lactobacillus plantarum can also promote the absorption and utilization of the active ingredients of Stemona sessilifolia. The two complement each other and work synergistically.

[0017] In addition, the Stemona sessilifolia ferment produced by the present invention has an obvious therapeutic effect on mice infected with the H1N1 influenza virus through its regulatory effects on innate immunity and adaptive immune responses. That is, by reducing the infiltration of macrophages and neutrophils in lung tissues, enhancing the expression levels of Th2 cytokines and virus-specific antibodies, and alleviating lung inflammation, the survival rate of mice is effectively improved, which provides an important scientific basis for the immunotherapy strategy of Stemona sessilifolia ferment in the treatment of influenza virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a comparison chart of the total flavonoid and total alkaloid contents in the Stemona sessilifolia ferment prepared at different fermentation times.

[0019] Figure 2 It is a comparison chart of the total flavonoid and total alkaloid contents in the Stemona sessilifolia ferment prepared at different fermentation temperatures.

[0020] Figure 3 It is a comparison chart of the total flavonoid and total alkaloid contents in the Stemona sessilifolia ferment prepared with different addition amounts of probiotic mixed bacterial solutions.

[0021] Figure 4 It is a comparison chart of the total flavonoid and total alkaloid contents in the Stemona sessilifolia ferment prepared with different addition amounts of glucose solutions.

[0022] Figure 5 It is a comparison chart of the total flavonoid and total alkaloid contents in the Stemona sessilifolia ferment prepared with different solid-to-liquid ratios.

[0023] Figure 6 It is an observation result chart of HE staining of mice in different groups. DETAILED DESCRIPTION OF THE INVENTION

[0024] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0025] Example 1: This example provides a method for preparing Stemona fermentum for treating H1N1 virus infection, which includes the following steps: S1. Add 4 g of Stemona, 30 mL of probiotic mixed bacterial liquid, and 30 mL of glucose solution with a concentration of 1 g / mL into MRS medium, and make up the MRS medium to 100 mL to obtain a mixed liquid to be fermented. Among them, the probiotic mixed bacterial liquid is composed of 15 mL of Lactobacillus rhamnosus bacterial liquid and 15 mL of Lactobacillus plantarum bacterial liquid. The addition amount of the glucose solution is 30% of the volume of the mixed liquid to be fermented, the addition amount of the probiotic mixed bacterial liquid is 30% of the volume of the mixed liquid to be fermented, and the material-liquid ratio of Stemona to the mixed liquid to be fermented is 1 g:25 mL. Stemona is an existing commercially available product purchased from Kangmei Pharmaceutical Co., Ltd.

[0026] The MRS medium is prepared by mixing 50 g of distilled water and 2.622 g of MRS. Accurately weigh 2.622 g of MRS with an electronic balance and slowly add it to 50 g of distilled water that is being heated. At the same time, stir until the MRS is completely dissolved, and then put it into an autoclave for autoclaving (121 °C, 15 min) for standby.

[0027] The preparation method of the bacterial liquid of Lactobacillus plantarum (LP) is as follows: Inoculate the existing publicly known Lactobacillus plantarum with the preservation number of BNCC 194165 into the sterilized MRS medium at an inoculation amount of 1%, fully mix it, and then statically culture it at 37 °C for 24 h to obtain a first-generation bacterial liquid for standby; continue to inoculate the first-generation bacterial liquid into the MRS medium in a new sterile test tube at an inoculation ratio of 1%, fully mix it, and then continue to statically culture it for 24 h to obtain a second-generation bacterial liquid for standby; similar to the preparation of the second-generation bacterial liquid, suck the second-generation bacterial liquid and mix it with the MRS medium at an inoculation ratio of 1%, culture it for 20 h, and adjust the absorbance of the bacterial liquid to 3 to obtain the bacterial liquid of Lactobacillus plantarum.

[0028] The preparation method of the bacterial liquid of Lactobacillus rhamnosus (LGG) is as follows: Inoculate the existing publicly known Lactobacillus rhamnosus GG strain with the preservation number of CGMCC 1.3724 into the sterilized MRS medium at an inoculation amount of 1%, fully mix it, and then statically culture it at 37 °C for 24 h to obtain a first-generation bacterial liquid for standby; continue to inoculate the first-generation bacterial liquid into the MRS medium in a new sterile test tube at an inoculation ratio of 1%, fully mix it, and then continue to statically culture it for 24 h to obtain a second-generation bacterial liquid for standby; similar to the preparation of the second-generation bacterial liquid, suck the second-generation bacterial liquid and mix it with the MRS medium at an inoculation ratio of 1%, culture it for 20 h, and adjust the absorbance of the bacterial liquid 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, make up the volume to 30 mL with distilled water, stir while adding water until the glucose is completely dissolved, then put the dissolved solution into an autoclave for autoclaving (121 °C, 15 min) to obtain the glucose solution for standby.

[0030] S2. Place the above-mentioned fermentation mixture to be fermented in a constant temperature incubator at 37 °C for fermentation treatment for 48 h to obtain a fermentation product. S3. Separate the liquid and solid of the above-mentioned fermentation product to obtain Stemona sessilifolia residue and fermentation broth. S4. Divide the above-mentioned Stemona sessilifolia residue into two equal parts on average. Place one part of the Stemona sessilifolia residue in a round-bottom flask, add 36 mL of ethanol solution with a volume concentration of 70%, heat under reflux for 2 h, filter with filter paper, extract twice more according to the same method, collect the liquids obtained from the three filtrations to obtain the first filtrate; Adjust the first filtrate to an ethanol solution with a volume concentration of 85% with absolute ethanol and perform 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 perform rotary evaporation to remove ethanol on the clarified part to obtain an ethanol-free ethanol extract. S5. Place the other part of the Stemona sessilifolia residue in a round-bottom flask, add 36 mL of distilled water, heat under reflux for 2 h, filter with filter paper, extract twice more according to the same method, collect the liquids obtained from the three filtrations to obtain the second filtrate; Centrifuge the second filtrate at a rate of 4000 r / min for 15 min, discard the precipitate, connect a Buchner funnel to a vacuum pump, filter the filtrate to obtain a water extract. S6. Mix the above-mentioned fermentation broth, ethanol extract and water extract, and perform concentration treatment to 34 mL to obtain Stemona sessilifolia fermented product; The Stemona sessilifolia fermented product is filtered through a sterile 0.22 μm filter membrane for standby.

[0031] Example 2: This example provides a preparation method of a Stemona sessilifolia fermented product for treating H1N1 virus infection, which includes the following steps: S1. Add 4 g of Stemona sessilifolia, 30 mL of probiotic mixed bacteria solution, and 30 mL of glucose solution with a concentration of 0.8 g / mL to MRS medium, and make up the MRS medium to 100 mL to obtain a fermentation mixture to be fermented; Among them, the probiotic mixed bacteria solution is composed of 20 mL of Lactobacillus rhamnosus bacteria solution and 10 mL of Lactobacillus plantarum bacteria solution; Stemona sessilifolia is an existing commercially available product, purchased from Kangmei Pharmaceutical Co., Ltd.

[0032] 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 50 g of distilled water that was being heated. At the same time, the medium was stirred until the MRS was completely dissolved. The medium was then placed in an autoclave for high-pressure sterilization (121°C, 15 min) for later use.

[0033] The preparation method of the bacterial liquid of Lactobacillus plantarum (LP) is as follows: Lactobacillus plantarum with an existing known preservation number of BNCC 194165 is inoculated at a 1% inoculation rate into a sterilized MRS culture medium, and the mixture is cultured at 37°C for 24 hours after being fully mixed to obtain a first-generation bacterial liquid for use; the first-generation bacterial liquid is then inoculated at a 1% inoculation rate into the MRS culture medium and added to a new sterile test tube, and the mixture is cultured for 24 hours after being fully mixed 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, and the mixture is cultured for 20 hours, and the absorbance of the bacterial liquid is modulated to 2 to obtain the bacterial liquid of Lactobacillus plantarum.

[0034] The preparation method of the bacterial solution of Lactobacillus rhamnosus (LGG) is as follows: the Lactobacillus rhamnosus GG strain with the existing known preservation number of CGMCC 1.3724 is inoculated with a 1% inoculation amount into a sterilized MRS medium, and the mixture is fully mixed and then statically cultured at 37°C for 24 hours to obtain a first-generation bacterial solution for use; the first-generation bacterial solution is continuously inoculated with an inoculation ratio of 1% into the MRS medium and added to a new sterile test tube, and the mixture is fully mixed and then statically cultured for 24 hours to obtain a second-generation bacterial solution for use; similar to the preparation of the second-generation bacterial solution, the second-generation bacterial solution is aspirated and mixed with the MRS medium at an inoculation ratio of 1%, and the mixture is cultured for 20 hours, and the absorbance of the bacterial solution is modulated to 2 to obtain the bacterial solution of Lactobacillus rhamnosus.

[0035] The preparation method of glucose solution (GLU) is as follows: weigh 30 g of glucose, add distilled water, and make 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 high-pressure sterilization (121°C, 15min) to obtain a glucose solution for use.

[0036] S2, placing the mixed solution to be fermented in a constant temperature incubator at 35° C. for fermentation for 36 hours to obtain a fermentation product; S3, separating the fermentation product into a material-liquid separation to obtain a Stemona slag and a fermentation liquid; S4. Divide the above-mentioned Stemona residue into two equal parts. Place one part of the Stemona 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 two more times in the same way, collect the liquid obtained from the three filtrations to get the first filtrate; adjust the first filtrate to an ethanol solution with a volume concentration of 80% with absolute ethanol and perform 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 subject the clarified part to rotary evaporation to remove alcohol to obtain an alcohol-free alcohol extract; S5. Place the other part of the Stemona residue in a round-bottom flask, add 36 mL of distilled water, heat under reflux for 2 h, filter with filter paper, extract two more times in the same way, collect the liquid obtained from the three filtrations to get the second filtrate; centrifuge the second filtrate at a rate of 4000 r / min for 15 min, discard the precipitate, connect a Buchner funnel to a vacuum pump, filter the filtrate to obtain a water extract; S6. Mix the above-mentioned fermentation broth, alcohol extract and water extract, and perform concentration treatment to obtain a Stemona ferment; filter the Stemona ferment through a sterile 0.22 μm filter membrane for standby.

[0037] Example 3: This example provides a method for preparing a Stemona ferment for treating H1N1 virus infection, which includes the following steps: S1. Add 4 g of Stemona, 30 mL of probiotic mixed bacteria solution, and 30 mL of glucose solution with a concentration of 1.2 g / mL to MRS medium, and make up the MRS medium to 100 mL to obtain a mixed solution to be fermented; among them, the probiotic mixed bacteria solution is composed of 10 mL of Lactobacillus rhamnosus bacteria solution and 20 mL of Lactobacillus plantarum bacteria solution; the Stemona is a commercially available product, purchased from Kangmei Pharmaceutical Co., Ltd.

[0038] The MRS medium is prepared with 50 g of distilled water and 2.622 g of MRS. Weigh 2.622 g of MRS accurately with an electronic balance, and slowly add it to the 50 g of distilled water that is being heated. At the same time, stir until the MRS is completely dissolved, and place it in an autoclave for autoclaving (121 °C, 20 min) for standby.

[0039] The preparation method of the bacterial liquid of Lactobacillus plantarum (LP) is as follows: Inoculate Lactobacillus plantarum with the existing publicly known preservation number of BNCC 194165 into the sterilized MRS medium at an inoculation amount of 1%, mix well, and then statically culture at 37°C for 24 h to obtain the first-generation bacterial liquid for standby; continue to inoculate the first-generation bacterial liquid into the MRS medium in a new sterile test tube at an inoculation ratio of 1%, mix well, and then continue to statically culture for 24 h to obtain the second-generation bacterial liquid for standby; similar to the preparation of the second-generation bacterial liquid, draw the second-generation bacterial liquid and mix it with the MRS medium at an inoculation ratio of 1%, culture for 20 h, and adjust the absorbance of the bacterial liquid to 4 to obtain the bacterial liquid of Lactobacillus plantarum.

[0040] The preparation method of the bacterial liquid of Lactobacillus rhamnosus (LGG) is as follows: Inoculate the LGG strain of Lactobacillus rhamnosus with the existing publicly known preservation number of CGMCC 1.3724 into the sterilized MRS medium at an inoculation amount of 1%, mix well, and then statically culture at 37°C for 24 h to obtain the first-generation bacterial liquid for standby; continue to inoculate the first-generation bacterial liquid into the MRS medium in a new sterile test tube at an inoculation ratio of 1%, mix well, and then continue to statically culture for 24 h to obtain the second-generation bacterial liquid for standby; similar to the preparation of the second-generation bacterial liquid, draw the second-generation bacterial liquid and mix it with the MRS medium at an inoculation ratio of 1%, culture for 20 h, and adjust the absorbance of the bacterial liquid to 4 to obtain the bacterial liquid of Lactobacillus rhamnosus.

[0041] 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.

[0042] S2. Place the above-mentioned mixed liquid to be fermented in a constant temperature incubator at 39°C for fermentation treatment for 60 h to obtain a fermentation product; S3. Separate the liquid and solid of the above-mentioned fermentation product to obtain Stemona residue and fermentation broth; S4. Divide the above-mentioned Stemona residue into two equal parts, place one part of the Stemona residue in a round-bottom flask, add 36 mL of ethanol solution with a volume concentration of 50%, heat under reflux for 2 h, filter with filter paper, extract twice more in the same way, collect the liquids obtained from the three filtrations to obtain the first filtrate; adjust the first filtrate to an ethanol solution with a volume concentration of 90% with absolute ethanol and perform 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 vacuum pump with a Buchner funnel, filter the filtrate, and perform rotary evaporation to remove alcohol on the clarified part to obtain an alcohol-free alcohol extract; S5. Place another portion of Stemona tuberosa residue in a round-bottom flask, add 36 mL of distilled water, heat under reflux for 2 h, filter with filter paper, and extract two more times in the same way. Collect the liquid obtained from the three filtrations to get the second filtrate. Centrifuge the second filtrate 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 to obtain the water extract. S6. Mix the above fermentation broth, ethanol extract, and water extract, and perform concentration treatment to obtain the Stemona tuberosa fermented product. Filter the Stemona tuberosa fermented product through a sterile 0.22 μm filter membrane and set aside for use.

[0043] Experimental Example 1: According to the preparation method provided in Example 1, select several factors that may affect the fermentation effect, such as fermentation temperature (35°C, 36°C, 37°C, 38°C, 39°C), fermentation time (24 h, 36 h, 48 h, 60 h, 72 h), addition amount of glucose solution (0, 10%, 20%, 30%, 40%), addition amount of probiotic mixed bacterial solution (5%, 10%, 20%, 30%, 40%), and solid-liquid ratio (1 g:10 mL, 1 g:15 mL, 1 g:20 mL, 1 g:25 mL, 1 g:30 mL). Use the extraction rates of total flavonoids and total alkaloids as response values to conduct single-factor experiments.

[0044] Among them, the detection method for total flavonoids is as follows: Precisely measure 200 μL of rutin standard solution (0.2 g / mL) and the test sample solution (Stemona tuberosa fermented product), and successively add 400 μL of 70% ethanol and 40 μL of 5% sodium nitrite solution to the tube, mix well, and let stand for 6 min; continue to 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, let stand for 15 minutes, and measure the absorbance at a wavelength of 510 nm; calculate the content of total flavonoids based on the absorbance. The specific calculation method can refer to the existing technology and will not be elaborated here.

[0045] The detection method for total alkaloids is as follows: Adjust the pH of the Stemona tuberosa fermented product to 2 - 3 with hydrochloric acid, make the volume up to 2.5 mL, and add 1 mL of freshly prepared saturated solution of 2% ammonium reineckate. Let stand in ice water for 1 h, then centrifuge at 3000 r / min and 4°C for 10 min. Discard the supernatant, add ice water mixture to blow, wash, and centrifuge, and 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 a blank control, and calculate the content of total alkaloids in Stemona tuberosa, calculated based on tuberostemonine (C 22 H 33 O4N). The specific calculation method can refer to the existing technology and will not be elaborated here.

[0046] The above test results are as follows: The effects of fermentation time on the contents of total flavonoids and total alkaloids in Stemonae Radix fermented products are as Figure 1 shown. From Figure 1 it can be seen that when the fermentation time is between 24 - 48 h, the contents of total alkaloids and total flavonoids increase with the increase of fermentation time; when the fermentation time is more than 48 h, with the extension of fermentation time, the contents of total alkaloids and total flavonoids show a decreasing trend. When the fermentation time reaches 48 h, the contents of total alkaloids and total flavonoids reach the highest value. Therefore, the most suitable fermentation time is 36 - 60 h.

[0047] The effects of fermentation temperature on the contents of total flavonoids and total alkaloids in Stemonae Radix fermented products are as Figure 2 shown. From Figure 2 it can be seen that when the fermentation temperature is in the range of 35 - 37 °C, the contents of total alkaloids and total flavonoids increase with the increase of fermentation temperature; when the fermentation temperature exceeds 37 °C, the contents of total alkaloids and total flavonoids decrease with the increase of fermentation temperature. When the fermentation temperature reaches 37 °C, the contents of total alkaloids and total flavonoids reach the highest value. Therefore, the most suitable fermentation temperature range is 36 °C - 38 °C.

[0048] The effects of the addition amount of probiotic mixed bacterial liquid on the contents of total flavonoids and total alkaloids in Stemonae Radix fermented products are as Figure 3 shown. From Figure 3 it can be seen that when the addition amount of probiotic mixed bacterial liquid is 5% - 30%, the contents of total alkaloids and total flavonoids show an increasing trend with the increase of the addition amount of probiotic mixed bacterial liquid; when the addition amount of probiotic mixed bacterial liquid is in the range of 30% - 40%, the contents of total alkaloids and total flavonoids decrease with the increase of the addition amount of probiotic mixed bacterial liquid. When the addition amount of probiotic mixed bacterial liquid is 30%, the contents of total alkaloids and total flavonoids reach the highest value. Therefore, it can be judged that the most suitable addition amount of probiotic mixed bacterial liquid is 20% - 40%.

[0049] The effects of the addition amount of glucose solution on the contents of total flavonoids and total alkaloids in Stemonae Radix fermented products are as Figure 4 shown. From Figure 4 it can be seen that when the addition amount of glucose solution is 10% - 30%, the total alkaloids and total flavonoids increase with the increase of the addition amount of glucose solution. When the addition amount of glucose solution is 30% - 40%, the total alkaloids and total flavonoids decrease with the increase of the addition amount of glucose solution. When the addition ratio is 30%, the total alkaloids and total flavonoids reach the peak value. Therefore, the most suitable addition amount of glucose solution is 20% - 40%.

[0050] The effects of the ratio of material to liquid on the contents of total flavonoids and total alkaloids in Stemonae Radix fermented products are as Figure 5 shown. From Figure 5It can be seen that when the material-liquid ratio is in the range of 1:15 to 1:25, the contents of total alkaloids and total flavonoids will increase with the increase of the material-liquid ratio; when the material-liquid ratio is in the range of 1:25 to 1:30, the contents of total alkaloids and total flavonoids will slightly decrease with the increase of the material-liquid ratio. When the material-liquid ratio is 1:25, the contents of total alkaloids and total flavonoids reach the highest values. Therefore, it can be judged that the optimal material-liquid ratio of Stemona sessilifolia and the fermentation system is 1:20 - 1:30.

[0051] Experimental Example 2: Detection of the promoting effect of the fermented product of Stemona sessilifolia on probiotics: Using 96-well plate culture, 250 μL of the Stemona sessilifolia extract prepared in Example 1, 1750 μL of MRS medium, 10 μL of Lactobacillus plantarum (LP) bacterial solution (OD 600 = 3), and 10 μL of Lactobacillus rhamnosus (LGG) bacterial solution (OD 600 = 3) were added to one well. The reaction well was sealed with a sterile sealing film, and the real-time OD value was detected at 600 nm using a multifunctional microplate reader. After shaking for 10 s, static measurement was carried out, and data was automatically recorded every 30 min, and continuous detection was carried out for 24 hours to obtain a complete growth curve. The Stemona sessilifolia concentration value corresponding to the maximum biomass in the Stemona sessilifolia-probiotic fermentation model was determined to be 0.03125 g / mL, the LP addition amount was 1% (OD 600 = 3), and the LGG addition amount was 1% (OD 600 = 3), and thus a Stemona sessilifolia-probiotic co-fermentation system with the optimal concentration was established.

[0052] The test results are as follows: When the co-culture of Stemona sessilifolia, LP, and LGG (0.1092 mg / mL) was compared with the LP-LGG culture (0.0907 mg / mL), the concentration of exopolysaccharides increased by 20.40%. When the co-culture of Stemona sessilifolia, LP, and LGG (636.74 ng / mL) was compared with the LP-LGG culture (294.02 ng / mL), the concentration of L-lactic acid increased by 116.56%. When the co-culture of Stemona sessilifolia, LP, and LGG (940.16 ng / mL) was compared with the LP-LGG culture (423.61 ng / mL), the concentration of folic acid increased by 121.93%. In addition, 6-hydroxyhexanoic acid increased by 773%, L-phenylalanine increased by 643%, N-benzylformamide increased by 307%, and salicylic acid increased by 161%. After fermentation, new antibacterial and anti-inflammatory components such as citric acid, L-phenylalanine, D-(-)-quinic acid, 6-hydroxyhexanoic acid, N-benzylformamide, DL-arginine, 4-hydroxybenzaldehyde, L-glutamic acid, salicylic acid, porphobilinogen, uridine, propafenone, 2-amino-1,3,4-octadecatriol, D-raffinose, calcitriol, dioscorin III, 16-hydroxypalmitic acid, etc. were added.

[0053] Experimental Example 3: Immunomodulatory effect of the fermented product of Stemona sessilifolia on pneumonia in mice infected with H1N1 virus: BALB / c mice were infected with H1N1 virus to serve as the model group; mice not infected with H1N1 virus served as the blank control group; the experimental groups were the treatment group with the stemona fermented product prepared in Example 1 and the treatment group with the unfermented extract (the alcohol extract and water extract of stemona, with the extraction method the same as that in Example 1); the control group was the PBS treatment group for virus infection. From the 1st day to the 7th day after the mice were infected with H1N1 virus, 100 μL of the stemona fermented product or the unfermented extract (0.78 g / kg) was respectively administered by gavage, and the control group was given 200 μL of PBS. The results showed that the stemona fermented product could significantly prolong the survival period of the mice infected with H1N1 virus (P<0.05), while all the mice in the PBS group died within 14 days after being infected with H1N1 virus. The results indicated that the stemona fermented product prepared in the embodiment of the present invention could alleviate the acute lung injury induced by H1N1 virus.

[0054] On the seventh day after the treatment with the stemona fermented product, obvious improvements were observed in the thickening of the alveolar wall, interstitial inflammatory cell infiltration, lymphocyte increase, capillary congestion, etc. in the mice by HE observation, as Figure 6 shown, Figure 6 in which a was the phenomenon of interstitial inflammatory cell infiltration, b was the phenomenon of alveolar hemorrhage, and c was the phenomenon of thickening of the alveolar wall.

[0055] Flow cytometry was used to detect the proportion of immune cells in the bronchoalveolar lavage fluid of the mice in each group. Compared with the model group, in the bronchoalveolar lavage fluid of the mice in the stemona fermented product group, the proportion of natural killer cells increased by 75% (the unfermented extract group increased by 120%), the proportion of cytotoxic T cells increased by 55% (the unfermented extract group increased by 56%), the proportion of macrophages increased by 86% (the unfermented extract group increased by 111%), the proportion of neutrophils increased by 126% (the unfermented extract group increased by 120%), and the proportion of B cells decreased by 51% (the unfermented extract group increased by 16%).

[0056] The ELISA method was used to detect the levels of inflammatory factors in the lung tissue and blood. In the lung tissue and blood of the mice in the stemona fermented product group, INF-γ decreased by 23.29% and 37.30% respectively (the unfermented extract group decreased by 18.08% and 30.82% respectively), and IL-10 in the lung tissue and blood increased by 25.56% and 30.84% respectively (the unfermented extract group increased by 21.76% and 23.42% respectively).

[0057] Enlightened by the ideal embodiments of the present invention as described above, through the above description, relevant staff can completely make various changes and modifications within the scope not deviating 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 ferment for treating H1N1 virus infection, characterized in that, It includes the following steps: Add Stemona sessilifolia, probiotic mixed bacteria liquid, and glucose solution into a culture medium to obtain a mixed liquid to be fermented; the probiotic mixed bacteria liquid is composed of the bacterial liquids of Lactobacillus rhamnosus and Lactobacillus plantarum; the addition amount of the probiotic mixed bacteria liquid is 20%-40% of the volume of the mixed liquid to be fermented; the material-liquid ratio of Stemona sessilifolia to the mixed liquid to be fermented is 1 g:(10-30) mL; Place the mixed liquid to be fermented at a fermentation temperature of 35-39 °C for fermentation treatment to obtain a fermentation product; Separate the material and liquid of the fermentation product to obtain Stemona sessilifolia residue and fermentation broth; Extract half of the Stemona sessilifolia residue with ethanol to obtain an ethanol extract; Extract the remaining Stemona sessilifolia residue with water to obtain a water extract; Mix the fermentation broth, ethanol extract, and water extract, and perform concentration treatment to obtain the Stemona sessilifolia ferment; 2. The preparation method of the stemona ferment for treating H1N1 virus infection 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 ferment for treating H1N1 virus infection according to claim 1, wherein The preservation number of the Lactobacillus plantarum is BNCC 194165.

4. The preparation method of the stemona ferment for treating H1N1 virus infection according to any one of claims 1-3, characterized in that, The absorbance of the bacterial liquid of Lactobacillus rhamnosus is 2-4, and the absorbance of the bacterial liquid of Lactobacillus plantarum is 2-4; the volume ratio of the bacterial liquid of Lactobacillus rhamnosus to the bacterial liquid of Lactobacillus plantarum is 1:(0.5-2).

5. The preparation method of the stemona ferment for treating H1N1 virus infection according to claim 1, characterized in that, The time of fermentation treatment is 36-60 h.

6. The preparation method of the Stemona ferment for treating H1N1 virus infection 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.

7. The preparation method of the stemona ferment for treating H1N1 virus infection according to claim 1, characterized in that, The step of extracting half of the Stemona sessilifolia residue with ethanol to obtain an ethanol extract specifically includes: Mix half of the Stemona sessilifolia residue with an ethanol solution with a volume concentration of 50%-70%, perform heating and reflux, then filter, repeat this step for extraction 1-3 times, combine the filtered liquids to obtain a first filtrate; Adjust the first filtrate with absolute ethanol to an ethanol solution with a volume concentration of 80%-90% for ethanol precipitation, after the ethanol precipitation ends, perform centrifugation, discard the precipitate, after suction filtration, then perform rotary evaporation to remove ethanol to obtain an ethanol extract.

8. The preparation method of the stemona ferment for treating H1N1 virus infection according to claim 1, wherein The step of extracting the remaining Stemona sessilifolia residue with water to obtain a water extract specifically includes: Mix the remaining Stemona sessilifolia residue with distilled water, perform heating and reflux, then filter, repeat this step for extraction 1-3 times, combine the filtered liquids to obtain a second filtrate; Perform centrifugation on the second filtrate, discard the precipitate, after suction filtration, obtain a water extract.

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

10. Use of the Stemona sessilifolia ferment according to claim 9 in the preparation of a drug for treating or preventing H1N1 virus infection.

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