A fermented product of Stemona tuberosa for treating H1N1 virus infection, and its preparation method and application

Abstract

The invention discloses a stemona fermented product for treating H1N1 virus infection and its preparation method and application, which belongs to the field of medical technology, wherein the preparation method of the stemona fermented product comprises the following steps: adding stemona, a mixed bacterial liquid of probiotics and a glucose solution to a culture medium to obtain a mixed liquid to be fermented; placing the mixed liquid to be fermented at a fermentation temperature of 35-39°C for fermentation treatment to obtain a fermentation product; subjecting the fermentation product to material-liquid separation to obtain a stemona residue and a fermentation liquid; extracting half of the stemona residue with ethanol to obtain an alcohol extract; extracting the remaining stemona residue with water to obtain an aqueous extract; mixing the fermentation liquid, the alcohol extract and the aqueous extract, and concentrating the mixture to obtain the stemona fermented product. The present invention can further promote the conversion and release of its active ingredients, improve drug efficacy, and produce beneficial compounds by introducing a probiotic compound of Lactobacillus rhamnosus and Lactobacillus plantarum into the fermentation of the stemona.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine, and in particular to a stemonae fermentation product for treating H1N1 virus infection, and a preparation method and application thereof. Background Art

[0002] Influenza viruses such as H1N1 are highly contagious and can cause severe complications, making them a potential trigger for major public health emergencies worldwide. After the H1N1 virus infects a host, the body's innate immune response is first activated. Overactivation of innate immune cells leads to excessive release of cytokines, triggering an inflammatory cytokine storm, causing lung damage and even more severe acute respiratory distress syndrome (ARDS). This H1N1-induced lung damage has been confirmed in numerous studies to be a major cause of severe viral infection and mortality. Oseltamivir, an inhibitor of the influenza virus neuraminidase, is often used as the first-line treatment for influenza because it blocks the release of viral progeny particles and controls viral spread. However, the antiviral efficacy of oseltamivir is challenged by factors such as genetic variation within the virus. Studies have shown that the development of resistance to oseltamivir by influenza viruses limits its clinical effectiveness. Therefore, the development of novel and effective therapeutic strategies is crucial.

[0003] Furthermore, compared to Western medicine, traditional Chinese medicine (TCM) boasts low toxicity, immunomodulatory properties, and a low resistance to drug resistance. The main active ingredients in Stemona radix are alkaloids, which are unique to this plant family and contain pyrrole- or pyridine-nitrogen heterocycles. They exhibit anthelmintic, insecticidal, antitussive, antiasthmatic, antitumor, and antibacterial properties. Chinese herbal medicines primarily combat pathogens by mobilizing the body's own antimicrobial factors, making them less susceptible to drug resistance. However, the active ingredients of Chinese medicinal materials like Stemona radix are primarily located in the cytoplasm of cells in various locations, such as roots, stems, and leaves, protected by the plant cell wall. Traditional processing methods are unable to effectively and quickly penetrate the protective cell wall protecting the active ingredients, preventing the release of their medicinal properties and resulting in low utilization of the medicinal materials. Summary of the Invention

[0004] The object of the present invention is to provide a method for preparing a fermented product of Stemona japonica for treating H1N1 virus infection, so as to solve the problems raised in the above background technology.

[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] A method for preparing a fermented product of Stemona tuberosa for treating H1N1 virus infection, comprising the following steps:

[0007] Adding the stemona root, a probiotic mixed bacterial solution, and a glucose solution to a culture medium to obtain a fermented mixed solution; the probiotic mixed bacterial solution is composed of a bacterial solution of Lactobacillus rhamnosus and Lactobacillus plantarum; the amount of the probiotic mixed bacterial solution added is 20%-40% of the volume of the fermented mixed solution; the material-liquid ratio of the stemona root to the fermented mixed solution is 1 g:(10-30) mL;

[0008] placing the mixed liquid to be fermented at a fermentation temperature of 35-39° C. for fermentation to obtain a fermentation product;

[0009] Separating the fermentation product into a material-liquid mixture to obtain a Stemona slag and a fermentation liquid;

[0010] Extract half of the stemona radix residue with ethanol to obtain an ethanol extract;

[0011] Extracting the remaining Stemona radix residue with water to obtain a water extract;

[0012] The fermentation liquid, the alcohol extract and the water extract are mixed and concentrated to obtain the Stemona tuberosa fermentation product.

[0013] Preferably, the Lactobacillus rhamnosus is Lactobacillus rhamnosus GG strain, and its preservation number is CGMCC 1.3724.

[0014] Preferably, the deposit number of the Lactobacillus plantarum is BNCC 194165.

[0015] Preferably, the absorbance of the bacterial solution of Lactobacillus rhamnosus is 2-4, and the absorbance of the bacterial solution of Lactobacillus plantarum is 2-4; the volume ratio of the bacterial solution of Lactobacillus rhamnosus to the bacterial solution of Lactobacillus plantarum is 1:(0.5-2).

[0016] Preferably, the fermentation treatment time is 36-60 hours.

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

[0018] More preferably, the amount of the probiotic mixed bacterial liquid added is 30% of the volume of the mixed liquid to be fermented; the material-liquid ratio of the Stemona tuber to the mixed liquid to be fermented is 1 g:25 mL; the absorbance of the Lactobacillus rhamnosus bacterial liquid is 3, and the absorbance of the Lactobacillus plantarum bacterial liquid is 3; the volume ratio of the Lactobacillus rhamnosus bacterial liquid to the Lactobacillus plantarum bacterial liquid is 1:1; the concentration of the glucose solution is 1 g / mL; the amount of the glucose solution added is 30% of the volume of the mixed liquid to be fermented; the fermentation temperature is 37°C; and the fermentation treatment time is 48 h.

[0019] Preferably, the step of extracting half of the Stemona radix residue with ethanol to obtain an ethanol extract specifically comprises:

[0020] Mix half of the Stemona radix residue with an ethanol solution having a volume concentration of 50%-70%, heat and reflux, and then filter. Repeat this step for 1-3 times, and combine the filtered liquids to obtain a first filtrate.

[0021] The first filtrate is prepared with anhydrous ethanol to prepare an ethanol solution with a volume concentration of 80%-90% for alcohol precipitation. After the alcohol precipitation is completed, centrifugation is performed, the precipitate is discarded, and after suction filtration, the alcohol is removed by rotary evaporation to obtain an alcohol extract.

[0022] Preferably, the step of extracting the remaining Stemona radix residue with water to obtain a water extract specifically comprises:

[0023] Mix the remaining Stemona radix residue with distilled water, heat and reflux, and then filter. Repeat this step for 1-3 times, and combine the filtered liquids to obtain a second filtrate.

[0024] The second filtrate is centrifuged, the precipitate is discarded, and the water extract is obtained after suction filtration.

[0025] Another object of the present invention is to provide a fermented product of Stemona japonica prepared by the above preparation method.

[0026] Another object of the present invention is to provide a use of the above-mentioned Stemona tuberosa fermentation product in the preparation of a medicament for treating or preventing H1N1 virus infection.

[0027] The present invention can further promote the conversion and release of active ingredients in Stemona by introducing a compound of Lactobacillus rhamnosus and Lactobacillus plantarum into the fermentation of Stemona, thereby improving the efficacy of the drug and producing beneficial compounds. This provides a theoretical basis for the application of Stemona fermentation products in the field of anti-bacterial infection and provides technical support for improving the bioavailability of Stemona. Compared with traditional methods, probiotic fermentation helps to increase the release level of active ingredients in Stemona and then strengthen its pharmacological effects, achieving synergistic results. Specifically, during the fermentation process, microorganisms such as Lactobacillus rhamnosus and Lactobacillus plantarum induce a variety of lignin-degrading enzymes and hydrolases through metabolic activities. These enzymes can decompose the fiber structure of Stemona and then cause structural damage to the cell wall, thereby releasing active substances. After the Stemona is acted upon by the enzyme system of the microorganisms, the fiber and wood part of the Stemona become loose, the active ingredients are effectively released, or new active substances are produced, which greatly improves and enhances the efficacy of the drug. The prebiotics produced by Stemona after fermentation can promote the reproduction of Lactobacillus rhamnosus and Lactobacillus plantarum. Lactobacillus rhamnosus and Lactobacillus plantarum can also promote the absorption and utilization of the active ingredients of Stemona. The two complement each other and enhance synergistically.

[0028] Furthermore, the Stemona tuber fermented product produced by the present invention demonstrated significant therapeutic effects on mice infected with influenza A (H1N1) virus by modulating both innate and adaptive immune responses. Specifically, by reducing lung tissue macrophage and neutrophil infiltration, enhancing the expression of Th2 cytokines and virus-specific antibodies, and alleviating lung inflammation, the product effectively improved the survival rate of the mice. This provides important scientific evidence for the use of Stemona tuber fermented product as an immunotherapy strategy for treating influenza virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a comparison chart of the total flavonoids and total alkaloids in the fermented materials of Stemona tuberosa prepared at different fermentation times.

[0030] Figure 2 This is a comparison chart of the total flavonoids and total alkaloids in the fermented materials of Stemona tuberosa prepared at different fermentation temperatures.

[0031] Figure 3 This is a comparison chart of the total flavonoids and total alkaloids in the fermented products of Stemona tuber prepared with different addition amounts of probiotic mixed bacteria.

[0032] Figure 4 This is a comparison chart of the total flavonoids and total alkaloids in the fermented products of Stemona tuberosa prepared with different addition amounts of glucose solution.

[0033] Figure 5 This is a comparison chart of the total flavonoids and total alkaloids in the fermented products of Stemona tuberosa prepared with different material-liquid ratios.

[0034] Figure 6 The following are the results of HE staining observation of mice in different groups. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Example 1: This example provides a method for preparing a fermented product of Stemona japonica for treating H1N1 virus infection, comprising the following steps: S1, adding 4 g of Stemona japonica, 30 mL of a probiotic mixed bacterial solution, and 30 mL of a glucose solution with a concentration of 1 g / mL to an MRS culture medium, and replenishing the MRS culture medium to 100 mL to obtain a mixed solution to be fermented; wherein the probiotic mixed bacterial solution consists of 15 mL of a Lactobacillus rhamnosus bacterial solution and 15 mL of a Lactobacillus plantarum bacterial solution; the amount of glucose solution added is 30% of the volume of the mixed solution to be fermented, the amount of probiotic mixed bacterial solution added is 30% of the volume of the mixed solution to be fermented, and the material-liquid ratio of Stemona japonica to the mixed solution to be fermented is 1 g:25 mL; Stemona japonica is an existing commercially available product purchased from Kangmei Pharmaceutical Co., Ltd.

[0037] 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 heated distilled water while stirring 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.

[0038] The preparation method of the Lactobacillus plantarum (LP) bacterial liquid is as follows: Lactobacillus plantarum with a known preservation number of BNCC 194165 is inoculated at a 1% inoculation rate into a sterilized MRS culture medium, 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 at a 1% inoculation rate into the MRS culture medium 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 3 to obtain the Lactobacillus plantarum bacterial liquid.

[0039] The preparation method of the bacterial liquid of Lactobacillus rhamnosus (LGG) is as follows: Lactobacillus rhamnosus GG strain with a known preservation number of 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 3 to obtain the bacterial liquid of Lactobacillus rhamnosus.

[0040] The preparation method of glucose solution (GLU) is as follows: weigh 30 g of glucose, add distilled water, and dilute the volume to 30 mL with distilled water. Stir while adding water until the glucose is completely dissolved, and then place the dissolved solution into an autoclave for high-pressure sterilization (121°C, 15 min) to obtain glucose solution for later use.

[0041] S2, placing the fermented mixed liquid in a constant temperature incubator at 37° C. for fermentation for 48 hours to obtain a fermentation product;

[0042] S3, separating the fermentation product into a material-liquid separation to obtain a Stemona slag and a fermentation liquid;

[0043] S4. Divide the above-mentioned Stemona slag into two parts, place one of the Stemona slag in a round-bottom flask, add 36mL of 70% ethanol solution by volume, heat and reflux for 2h, filter with filter paper, extract twice more according to the same method, collect the filtered liquid three times, and obtain a first filtrate; modulate the first filtrate with anhydrous ethanol to an ethanol solution with a volume concentration of 85%, and precipitate it with alcohol overnight. After the alcohol precipitation is completed, centrifuge at a rate of 4000r / min for 15min, discard the precipitate, connect a vacuum pump to a Buchner funnel, filter the filtrate, and carry out dealcoholization by rotary evaporation to obtain an alcohol-free alcohol extract;

[0044] S5. Place another portion of the Stemona slag in a round-bottom flask, add 36 mL of distilled water, heat and reflux for 2 h, filter with filter paper, extract twice more using the same method, collect the filtered liquid three times, and obtain a second filtrate; centrifuge the second filtrate at 4000 r / min for 15 min, discard the precipitate, connect a Buchner funnel to a vacuum pump, and filter the filtrate to obtain an aqueous extract;

[0045] S6. The fermentation liquid, alcohol extract and water extract are mixed and concentrated to 34 mL to obtain the Stemona tuberosa fermentation product; the Stemona tuberosa fermentation product is filtered through a sterile 0.22 μm filter membrane and set aside.

[0046] Example 2: This example provides a method for preparing a fermented product of Stemona radix for treating H1N1 virus infection, comprising the following steps: S1, adding 4 g of Stemona radix, 30 mL of a mixed probiotic solution, and 30 mL of a glucose solution with a concentration of 0.8 g / mL to an MRS culture medium, and replenishing the MRS culture medium to 100 mL to obtain a mixed solution to be fermented; wherein the mixed probiotic solution consists of 20 mL of a Lactobacillus rhamnosus solution and 10 mL of a Lactobacillus plantarum solution; Stemona radix is ​​an existing commercially available product purchased from Kangmei Pharmaceutical Co., Ltd.

[0047] 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 heated distilled water while stirring 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.

[0048] The preparation method of the bacterial liquid of Lactobacillus plantarum (LP) is as follows: Lactobacillus plantarum with a known preservation number of BNCC 194165 is inoculated at a 1% inoculation rate into a sterilized MRS culture medium, 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 at a 1% inoculation rate into the MRS culture medium 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 2 to obtain the bacterial liquid of Lactobacillus plantarum.

[0049] The preparation method of the bacterial liquid of Lactobacillus rhamnosus (LGG) is as follows: Lactobacillus rhamnosus GG strain with a known preservation number of 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 2 to obtain the Lactobacillus rhamnosus bacterial liquid.

[0050] The preparation method of glucose solution (GLU) is as follows: weigh 30 g of glucose, add distilled water, and dilute the volume to 30 mL with distilled water. Stir while adding water until the glucose is completely dissolved, and then place the dissolved solution into an autoclave for high-pressure sterilization (121°C, 15 min) to obtain glucose solution for later use.

[0051] S2, placing the fermented mixed liquid in a constant temperature incubator at 35° C. for fermentation for 36 hours to obtain a fermentation product;

[0052] S3, separating the fermentation product into a material-liquid separation to obtain a Stemona slag and a fermentation liquid;

[0053] S4. Divide the above-mentioned Stemona slag into two parts, place one of the Stemona slag in a round-bottom flask, add 36mL of 60% ethanol solution by volume, heat and reflux for 2h, filter with filter paper, extract twice more according to the same method, collect the filtered liquid three times, and obtain a first filtrate; modulate the first filtrate with anhydrous ethanol to an ethanol solution with a volume concentration of 80%, and precipitate it with alcohol overnight. After the alcohol precipitation is completed, centrifuge at a rate of 4000r / min for 15min, discard the precipitate, connect a vacuum pump to a Buchner funnel, filter the filtrate, and carry out dealcoholization by rotary evaporation to obtain an alcohol-free alcohol extract;

[0054] S5. Place another portion of the Stemona slag in a round-bottom flask, add 36 mL of distilled water, heat and reflux for 2 h, filter with filter paper, extract twice more using the same method, collect the filtered liquid three times, and obtain a second filtrate; centrifuge the second filtrate at 4000 r / min for 15 min, discard the precipitate, connect a Buchner funnel to a vacuum pump, and filter the filtrate to obtain an aqueous extract;

[0055] S6. The fermentation liquid, alcohol extract and water extract are mixed and concentrated to obtain the Stemona tuber fermentation product; the Stemona tuber fermentation product is filtered through a sterile 0.22 μm filter membrane and set aside.

[0056] Example 3: This example provides a method for preparing a fermented product of Stemona radix for treating H1N1 virus infection, which comprises the following steps: S1, adding 4 g of Stemona radix, 30 mL of a mixed probiotic solution, and 30 mL of a glucose solution with a concentration of 1.2 g / mL to an MRS culture medium, and replenishing the MRS culture medium to 100 mL to obtain a mixed solution to be fermented; wherein the mixed probiotic solution consists of 10 mL of a Lactobacillus rhamnosus solution and 20 mL of a Lactobacillus plantarum solution; Stemona radix is ​​an existing commercially available product purchased from Kangmei Pharmaceutical Co., Ltd.

[0057] MRS culture 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 heated distilled water while stirring until the MRS was completely dissolved. The medium was then placed in an autoclave for high-pressure sterilization (121°C, 20 min) for later use.

[0058] The preparation method of the Lactobacillus plantarum (LP) bacterial liquid is as follows: Lactobacillus plantarum with a known preservation number of BNCC 194165 is inoculated at a 1% inoculation rate into a sterilized MRS culture medium, 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 at a 1% inoculation rate into the MRS culture medium 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 Lactobacillus plantarum bacterial liquid.

[0059] The preparation method of the bacterial liquid of Lactobacillus rhamnosus (LGG) is as follows: Lactobacillus rhamnosus GG strain with a known preservation number of 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.

[0060] The preparation method of glucose solution (GLU) is as follows: weigh 30 g of glucose, add distilled water, and dilute the volume to 30 mL with distilled water. Stir while adding water until the glucose is completely dissolved, and then place the dissolved solution into an autoclave for high-pressure sterilization (121°C, 15 min) to obtain glucose solution for later use.

[0061] S2, placing the mixed solution to be fermented in a constant temperature incubator at 39° C. for fermentation for 60 hours to obtain a fermentation product;

[0062] S3, separating the fermentation product into a material-liquid separation to obtain a Stemona slag and a fermentation liquid;

[0063] S4. Divide the above-mentioned Stemona slag into two parts, place one of the Stemona slag in a round-bottom flask, add 36mL of 50% ethanol solution by volume, heat and reflux for 2h, filter with filter paper, extract twice more according to the same method, collect the filtered liquid three times, and obtain a first filtrate; modulate the first filtrate with anhydrous ethanol to an ethanol solution with a volume concentration of 90%, and precipitate it with alcohol overnight. After the alcohol precipitation is completed, centrifuge at a rate of 4000r / min for 15min, discard the precipitate, connect a vacuum pump to a Buchner funnel, filter the filtrate, and carry out dealcoholization by rotary evaporation to obtain an alcohol-free alcohol extract;

[0064] S5. Place another portion of the Stemona slag in a round-bottom flask, add 36 mL of distilled water, heat and reflux for 2 h, filter with filter paper, extract twice more using the same method, collect the filtered liquid three times, and obtain a second filtrate; centrifuge the second filtrate at 4000 r / min for 15 min, discard the precipitate, connect a Buchner funnel to a vacuum pump, and filter the filtrate to obtain an aqueous extract;

[0065] S6. The fermentation liquid, alcohol extract and water extract are mixed and concentrated to obtain the Stemona tuber fermentation product; the Stemona tuber fermentation product is filtered through a sterile 0.22 μm filter membrane and set aside.

[0066] Experimental Example 1: According to the preparation method provided in Example 1, several factors that may affect the fermentation effect were selected, including fermentation temperature (35°C, 36°C, 37°C, 38°C, 39°C), fermentation time (24h, 36h, 48h, 60h, 72h), amount of glucose solution added (0, 10%, 20%, 30%, 40%), amount of probiotic mixed bacterial solution added (5%, 10%, 20%, 30%, 40%), and solid-liquid ratio (1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL, 1g:30mL). The total flavonoids and total alkaloids extraction rates were used as response values ​​for a single-factor experiment.

[0067] Among them, the total flavonoids detection method is as follows: accurately measure 200 μL of rutin standard solution (0.2 g / mL) and the sample solution to be tested (fermented product of Stemona tuber), add 400 μL of 70% ethanol and 40 μL of 5% sodium nitrite solution into the tube in sequence, mix thoroughly, and let stand for 6 minutes; continue to add 40 μL of 10% aluminum nitrate, mix again, and let stand for 6 minutes; 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 total flavonoids content according to the absorbance. The specific calculation method can be referred to the existing technology and will not be repeated here.

[0068] The detection method of total alkaloids is as follows: adjust the pH of the fermented material of Stemona to 2-3 with hydrochloric acid, make the volume to 2.5mL, and add 1mL of freshly prepared 2% saturated solution of Reindet's ammonium salt. After standing in ice water for 1h, centrifuge at 3000r / min and 4℃ for 10min. Discard the supernatant, add ice water mixture to blow and wash, centrifuge, and repeat the above steps until the alkaloid Reindet's salt precipitate is colorless. All the above operations are performed on ice. Acetone can dissolve the filtered and washed alkaloid Reindet's salt precipitate. Finally, measure the absorbance of the sample at the maximum absorption wavelength of 523nm. Use acetone as a blank control to calculate the total alkaloid content of Stemona, and use stemone (C 22 H 33 The specific calculation method can refer to the existing technology and will not be described in detail here.

[0069] The results of the above experiments are as follows: The effect of fermentation time on the content of total flavonoids and total alkaloids in the fermented product of Stemona tuberosa is as follows: Figure 1 As shown, from Figure 1 It can be seen that when the fermentation time is 24-48h, the total alkaloids and total flavonoids increase with the increase of fermentation time; when the fermentation time is more than 48h, the content of total alkaloids and total flavonoids shows a decreasing trend with the extension of fermentation time. When the fermentation time reaches 48h, the content of total alkaloids and total flavonoids reaches the highest value, so the most suitable fermentation time is 36-60h.

[0070] Effects of fermentation temperature on the contents of total flavonoids and total alkaloids in the fermented product of Stemona tuberosa Figure 2 As shown, from Figure 2 It can be seen that when the fermentation temperature is between 35°C and 37°C, the total alkaloid and flavonoid contents increase as the fermentation temperature rises; however, when the fermentation temperature exceeds 37°C, the total alkaloid and flavonoid contents decrease as the fermentation temperature rises. The total alkaloid and flavonoid contents reach their highest values ​​at 37°C. Therefore, the optimal fermentation temperature range is 36°C to 38°C.

[0071] Effects of the amount of probiotics mixed culture added on the contents of total flavonoids and total alkaloids in the fermented product of Stemona tuberosa Figure 3 As shown, from Figure 3 It can be seen that when the probiotic mixture addition level is between 5% and 30%, the total alkaloid and total flavonoid contents increase with the increase in the probiotic mixture addition level. When the probiotic mixture addition level is between 30% and 40%, the total alkaloid and total flavonoid contents decrease with the increase in the probiotic mixture addition level. When the probiotic mixture addition level is 30%, the total alkaloid and total flavonoid contents reach their highest values. Therefore, it can be determined that the optimal probiotic mixture addition level is between 20% and 40%.

[0072] Effects of glucose solution addition on the contents of total flavonoids and total alkaloids in the fermented product of Stemona tuberosa Figure 4 As shown, from Figure 4 It can be seen that when the addition amount of glucose solution is between 10% and 30%, the total alkaloids and total flavonoids increase with the increase of glucose solution addition amount. When the addition amount of glucose solution is between 30% and 40%, the total alkaloids and total flavonoids decrease with the increase of glucose solution addition amount. When the addition ratio is 30%, the total alkaloids and total flavonoids reach the peak. Therefore, the optimal addition amount of glucose solution is 20% to 40%.

[0073] Effect of material-liquid ratio on the content of total flavonoids and total alkaloids in the fermentation product of Stemona tuberosa Figure 5 As shown, from Figure 5It can be seen that when the material-to-liquid ratio is between 1:15 and 1:25, the total alkaloid and total flavonoid contents increase with the increase in the material-to-liquid ratio. When the material-to-liquid ratio is between 1:25 and 1:30, the total alkaloid and total flavonoid contents decrease slightly with the increase in the material-to-liquid ratio. The total alkaloid and total flavonoid contents reach their highest values ​​at a material-to-liquid ratio of 1:25. Therefore, the optimal material-to-liquid ratio for Stemona tuberosa to the fermentation system is 1:20-1:30.

[0074] Test Example 2: Detection of the Probiotic Effect of Stemona tuberosa Fermentation

[0075] 96 microplate culture was used, and 250 μL of the Stemona extract prepared in Example 1, 1750 μL of MRS culture medium, and 10 μL of Lactobacillus plantarum (LP) bacterial solution (OD 600 =3), 10 μL of Lactobacillus rhamnosus (LGG) bacterial solution (OD 600 =3) was added to one well, and the reaction well was sealed with a sterile sealing film. The real-time OD value was measured at 600 nm using a multifunctional microplate reader. After shaking for 10 seconds, the static measurement was performed. The data was automatically recorded every 30 minutes. The measurement was continued for 24 hours to obtain a complete growth curve. The maximum biomass of the Stemona tuber-probiotic fermentation model was determined to be 0.03125 g / mL. The LP addition amount was 1% (OD 600 =3), LGG addition amount 1% (OD 600 =3), in order to establish the optimal concentration of Stemona tuberosa-probiotic co-fermentation system.

[0076] The results are as follows: The concentration of exopolysaccharides increased by 20.40% in the co-culture of Stemona, LP, and LGG (0.1092 mg / mL) compared to the LP-LGG culture (0.0907 mg / mL). The concentration of L-lactic acid increased by 116.56% in the co-culture of Stemona, LP, and LGG (636.74 ng / mL) compared to the LP-LGG culture (294.02 ng / mL). The concentration of folic acid increased by 121.93% in the co-culture of Stemona, LP, and LGG (940.16 ng / mL) compared to the LP-LGG culture (423.61 ng / mL). Furthermore, the concentrations of 6-hydroxyhexanoic acid, L-phenylalanine, N-benzylformamide, and salicylic acid increased by 773%, 643%, 307%, and 161%. After fermentation, new antibacterial and anti-inflammatory ingredients 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-octadecanetriol, D-raffinose, calcitriol, yamase III, and 16-hydroxypalmitic acid are added.

[0077] Experimental Example 3: Immunomodulatory Effects of Stemona tuberosa Fermentation on Pneumonia in Mice Infected with H1N1 Virus

[0078] BALB / c mice were infected with the H1N1 virus and served as a model group; mice not infected with the H1N1 virus served as a blank control group. The experimental groups were treated with the Stemona radix fermented product prepared in Example 1 and with unfermented extracts (alcoholic and aqueous extracts of Stemona radix, extracted using the same method as in Example 1); and the control group was treated with PBS. From day 1 to day 7 after H1N1 infection, mice were given 100 μL of either the Stemona radix fermented product or the unfermented extract (0.78 g / kg) by gavage, while the control group received 200 μL of PBS. The results showed that the Stemona radix fermented product significantly prolonged the survival of H1N1-infected mice (P < 0.05), while all mice in the PBS group died within 14 days of infection. These results demonstrate that the Stemona radix fermented product prepared in this example can alleviate H1N1-induced acute lung injury.

[0079] On the seventh day after treatment with the fermented stemonae, HE observations showed that the alveolar wall thickening, interstitial inflammatory cell infiltration, lymphocyte increase, and capillary congestion in mice were significantly improved. Figure 6 As shown, Figure 6 Figure a shows interstitial inflammatory cell infiltration, b shows alveolar hemorrhage, and c shows alveolar wall thickening.

[0080] Flow cytometry was used to detect the proportion of immune cells in the bronchoalveolar lavage fluid of mice in each group. Compared with the model group, in the bronchoalveolar lavage fluid of mice in the Stemona tuberosa fermentation product group, the proportion of natural killer cells increased by 75% (increased by 120% in the non-fermented extract group), the proportion of cytotoxic T cells increased by 55% (increased by 56% in the non-fermented extract group), the proportion of macrophages increased by 86% (increased by 111% in the non-fermented extract group), the proportion of neutrophils increased by 126% (increased by 120% in the non-fermented extract group), and the proportion of B cells decreased by 51% (increased by 16% in the non-fermented extract group).

[0081] ELISA was used to measure inflammatory cytokine levels in lung tissue and blood. In the Stemona tuberosa fermentation product group, INF-γ levels in the lung tissue and blood decreased by 23.29% and 37.30%, respectively (compared to 18.08% and 30.82%, respectively, in the unfermented extract group), while IL-10 levels in the lung tissue and blood increased by 25.56% and 30.84%, respectively (compared to 21.76% and 23.42%, respectively, in the unfermented extract group).

[0082] Based on the above-mentioned ideal embodiment of the present invention, and through the above description, relevant personnel can make various changes and modifications without departing from the scope of the technical concept of the present invention. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. A method for preparing a fermented product of Stemona tuberosa for treating H1N1 virus infection, characterized in that: The following steps are involved: Adding the stemona root, a probiotic mixed bacterial solution, and a glucose solution to a culture medium to obtain a fermented mixed solution; the probiotic mixed bacterial solution is composed of a bacterial solution of Lactobacillus rhamnosus and Lactobacillus plantarum; the amount of the probiotic mixed bacterial solution added is 20%-40% of the volume of the fermented mixed solution; the material-liquid ratio of the stemona root to the fermented mixed solution is 1 g:(10-30) mL; placing the mixed liquid to be fermented at a fermentation temperature of 35-39° C. for fermentation to obtain a fermentation product; Separating the fermentation product into a material-liquid mixture to obtain a Stemona slag and a fermentation liquid; Extract half of the stemona radix residue with ethanol to obtain an ethanol extract; Extracting the remaining Stemona radix residue with water to obtain a water extract; The fermentation liquid, the alcohol extract and the water extract are mixed and concentrated to obtain the Stemona tuber fermentation product; The Lactobacillus rhamnosus is the Lactobacillus rhamnosus GG strain, and its preservation number is CGMCC 1.3724; The deposit number of the Lactobacillus plantarum is BNCC 194165; The absorbance of the bacterial solution of Lactobacillus rhamnosus is 2-4, and the absorbance of the bacterial solution of Lactobacillus plantarum is 2-4; the volume ratio of the bacterial solution of Lactobacillus rhamnosus to the bacterial solution of Lactobacillus plantarum is 1:(0.5-2); The concentration of the glucose solution is 0.8-1.2 g / mL; the amount of the glucose solution added is 20%-40% of the volume of the mixed solution to be fermented.

2. The method for preparing a fermented product of Stemona tuberosa for treating H1N1 virus infection according to claim 1, characterized in that: The fermentation time is 36-60h.

3. The method for preparing a fermented product of Stemona tuberosa for treating H1N1 virus infection according to claim 1, characterized in that: The step of extracting half of the Stemona radix residue with ethanol to obtain an ethanol extract specifically comprises: Mix half of the Stemona radix residue with an ethanol solution having a volume concentration of 50%-70%, heat and reflux, and then filter. Repeat this step for 1-3 times, and combine the filtered liquids to obtain a first filtrate. The first filtrate is prepared with anhydrous ethanol to prepare an ethanol solution with a volume concentration of 80%-90% for alcohol precipitation. After the alcohol precipitation is completed, centrifugation is performed, the precipitate is discarded, and after suction filtration, the alcohol is removed by rotary evaporation to obtain an alcohol extract.

4. The method for preparing a fermented product of Stemona tuberosa for treating H1N1 virus infection according to claim 1, characterized in that: The step of extracting the remaining Stemona radix residue with water to obtain a water extract specifically comprises: Mix the remaining Stemona radix residue with distilled water, heat and reflux, and then filter. Repeat this step for 1-3 times, and combine the filtered liquids to obtain a second filtrate. The second filtrate is centrifuged, the precipitate is discarded, and the water extract is obtained after suction filtration.

5. A fermented product of Stemona tuberosa obtained by the preparation method according to any one of claims 1 to 4.

6. Use of the fermented product of Stemona tuberosa according to claim 5 in the preparation of a medicament for treating or preventing H1N1 virus infection.

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