Composition for inhibiting streptococcus angina and preparation method thereof

Abstract

The invention provides a composition for inhibiting streptococcus angina and a preparation method of the composition, and belongs to the technical field of medicines. The preparation method comprises the following steps: carrying out water extraction on purslane and ginseng to obtain polysaccharide and an anti-inflammatory composition, mixing the anti-inflammatory composition and antibiotics, embedding the mixture in beta-cyclodextrin, forming a compound by the prepared inclusion compound, a magnetic gold nanocage and a probiotic secretion compound, loading the compound on lactobacillus salivarius LS02, uniformly mixing the compound with lactobacillus reuteri FPHC2951, lactobacillus acidophilus LA16 and polysaccharide, and carrying out freeze-drying to obtain the anti-inflammatory compound. The composition for inhibiting the streptococcus angina is prepared. The composition for inhibiting the streptococcus anginiae has good targeted inhibition and killing effects on harmful bacteria streptococcus anginiae, does not have drug resistance, has small side effects, is high in patient compliance, has good anti-oxidation, anti-inflammatory and antibacterial effects, is good in biocompatibility, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a composition for inhibiting Streptococcus anginosus and a preparation method thereof. Background Art

[0002] As a conditional pathogen, Streptococcus anginosus (S. anginosus) is one of the common bacteria causing infections. The clinical manifestations vary, and there are significant differences in treatment methods and disease prognoses. It often causes abscesses in various organs and tissues and suppurative infections in other parts. Previous studies have confirmed that Streptococcus anginosus can produce various exotoxins in vitro to cause invasive suppurative infections. Once it invades the blood, it can spread throughout the body along with the blood circulation, causing severe infectious diseases such as systemic infection, poisoning, and systemic inflammatory response. The condition is often severe. If anti-infective treatment cannot be carried out in a timely manner, the prognosis is poor.

[0003] It can cause a variety of infectious diseases:

[0004] Suppurative infections and abscesses: Streptococcus anginosus can cause suppurative infections and abscess formation in various organs, commonly in the abdomen, skin and soft tissues, and head and neck.

[0005] Gastric cancer: The latest research has found that Streptococcus anginosus can promote the occurrence and development of gastric cancer. It remodels the tumor immune microenvironment through the arginine metabolic pathway, promotes the proliferation and metastasis of cancer cells, and at the same time inhibits the differentiation and infiltration of CD8+ T cells.

[0006] Thoracic infections: The Streptococcus anginosus group can cause thoracic infections, often manifested as empyema, and imaging shows encapsulated pleural effusion.

[0007] Using antibiotics for treatment is likely to cause the problem of bacterial drug resistance. In order to reduce the use of antibacterial drugs and prevent the generation of drug-resistant bacteria, the development of new drugs has important reference value. Summary of the Invention

[0008] The object of the present invention is to provide a composition for inhibiting Streptococcus anginosus and a preparation method thereof, which have good targeted inhibition and killing effects on the harmful bacterium Streptococcus anginosus, have no drug resistance, have small side effects, high patient compliance, have good antioxidant, anti-inflammatory, and antibacterial effects, good biocompatibility, and broad application prospects.

[0009] The technical solution of the present invention is realized as follows:

[0010] The present invention provides a method for preparing a composition for inhibiting Streptococcus anginosus. Purslane and ginseng are subjected to water extraction to obtain polysaccharides and an anti-inflammatory composition. The anti-inflammatory composition is mixed with an antibiotic and embedded in β-cyclodextrin. The obtained inclusion complex forms a complex with a magnetic gold nanocage and a probiotic secretion complex, and is loaded on Lactobacillus salivarius LS02. It is mixed evenly with Lactobacillus reuteri FPHC2951, Lactobacillus acidophilus LA16, and polysaccharides to prepare a composition for inhibiting Streptococcus anginosus.

[0011] As a further improvement of the present invention, it includes the following steps:

[0012] S1. Preparation of the anti-inflammatory composition and polysaccharides: Wash, dry, and pulverize purslane and ginseng, add them to water, heat to boiling for extraction, filter, add ethanol to the filtrate for precipitation, wash the solid, dry it to obtain polysaccharides, recover ethanol from the liquid, dialyze, and dry the undialyzed liquid to obtain the anti-inflammatory composition;

[0013] S2. Preparation of the inclusion complex: Dissolve β-cyclodextrin in an ethanol aqueous solution, add the anti-inflammatory composition and an antibiotic to the solution, stir and mix, and freeze-dry to obtain the inclusion complex;

[0014] S3. Preparation of the magnetic gold nanocage: Disperse the gold nanocage in PBS buffer, add ferric chloride and ferrous chloride, under the protection of an inert gas, dropwise add ammonia water, heat and stir for reaction, centrifuge, wash, and dry to obtain the magnetic gold nanocage;

[0015] S4. Preparation of the probiotic secretion: Inoculate Lactobacillus reuteri FPHC2951 and Lactobacillus acidophilus LA16 in a culture medium, ferment and culture, centrifuge, collect the bacterial sludge, freeze-dry to obtain probiotics, and freeze-dry the supernatant to obtain the probiotic secretion;

[0016] S5. Preparation of the inclusion complex / magnetic gold nanocage / probiotic secretion complex: Add the inclusion complex, magnetic gold nanocage, probiotic secretion, phytic acid, and dipotassium hydrogen phosphate to water, heat and stir, carry out a hydrothermal reaction, adjust the pH value to neutral, centrifuge, wash, and dry to obtain the inclusion complex / magnetic gold nanocage / probiotic secretion complex;

[0017] S6. Preparation of the drug-loaded lactobacillus: Add the inclusion complex / magnetic gold nanocage / probiotic secretion complex to water, add EDC and NHS, stir for activation, add the activated seed solution of Lactobacillus salivarius LS02 strain, stir for reaction, centrifuge, wash, and freeze-dry to obtain the drug-loaded lactobacillus;

[0018] S7. Preparation of the composition for inhibiting Streptococcus anginosus: Mix the drug-loaded lactobacillus, the probiotics in step S4, and the polysaccharides in step S1 evenly to prepare a composition for inhibiting Streptococcus anginosus.

[0019] As a further improvement of the present invention, the mass ratio of purslane and ginseng in step S1 is 3-5:7-10, the extraction time is 1-2 h, the pore size of the dialysis bag used for dialysis is 3-5 kDa, and the dialysis time is 12-24 h.

[0020] As a further improvement of the present invention, the ethanol content in the ethanol aqueous solution in step S2 is 30-40 wt%, the mass ratio of β-cyclodextrin, anti-inflammatory composition and antibiotic is 10-12:4-7:1-2, and the antibiotic is selected from at least one of penicillin, cefixime, cefprozil, cefdinir, erythromycin, azithromycin, vancomycin. Preferably, it is a mixture of penicillin and azithromycin with a mass ratio of 1:1-2, and the stirring and mixing time is 1-2 h.

[0021] As a further improvement of the present invention, the mass ratio of gold nanocages, ferric chloride and ferrous chloride in step S3 is 4-7:3.24:1.26, the pH value of the solution is adjusted to 10-11 by dropping ammonia water, and the temperature of the heating and stirring reaction is 85-95 °C and the time is 3-5 h.

[0022] As a further improvement of the present invention, the medium in step S4 is a liquid Gao's medium, and the fermentation culture conditions are 35-37 °C, 100-200 r / min, and cultured for 24-36 h to obtain a mixture with a bacterial content of 10 10 -10 11 cfu / mL, and the mass ratio of Lactobacillus reuteri FPHC2951 and Lactobacillus acidophilus LA16 is 4-7:2-4.

[0023] As a further improvement of the present invention, the mass ratio of the inclusion complex, magnetic gold nanocages, probiotic secretions, phytic acid, and dipotassium hydrogen phosphate in step S5 is 6-8:3-5:10-12:5-7:1-2, the heating and stirring temperature is 90-100 °C and the time is 1-2 h, and the hydrothermal reaction temperature is 150-160 °C and the time is 8-10 h.

[0024] As a further improvement of the present invention, the mass ratio of the inclusion complex / magnetic gold nanocage / probiotic secretion complex, EDC and NHS in step S6 is 10:2-4:2-4, the inoculation amount of the activated Lactobacillus salivarius LS02 strain seed liquid is 3-5 v / v%, and the bacterial content of the strain seed liquid is 10 8 -10 9 cfu / mL, the stirring and activation temperature is 0-4 °C and the time is 30-40 min, the stirring reaction temperature is 35-37 °C, the CO2 content is 4-6 v / v%, and the time is 10-12 h.

[0025] As a further improvement of the present invention, the mass ratio of the Lactobacillus drug carrier in step S7, the probiotic bacteria in step S4, and the polysaccharide in step S1 is 10:3-5:2-3.

[0026] The present invention further protects a composition for inhibiting Streptococcus anginosus prepared by the above preparation method.

[0027] The present invention has the following beneficial effects:

[0028] A new bioactive antigen SAA (S. anginosus antigen) was isolated and purified from the supernatant of Streptococcus anginosus. It was found that SAA can stimulate macrophages to produce high concentrations of NO (nitric oxide) and inflammatory factors, and the bacteriocin secreted by Lactobacillus salivarius LS02 can bind to this SAA, thereby increasing the concentration of Lactobacillus drug carrier near Streptococcus anginosus. On the one hand, by releasing the loaded antibiotics and anti-inflammatory substances, the production of inflammatory factors is inhibited. At the same time, Lactobacillus can enhance the activity of phagocytes in the body, activate dendritic cells, thereby activating T cells and improving the immune function of the body, playing a role in protecting the body and inhibiting the growth of Streptococcus anginosus. At the same time, probiotic bacteria (Lactobacillus reuteri FPHC2951, Lactobacillus acidophilus LA16) can also reduce the colonization of Streptococcus anginosus in gastric epithelial cells or oral cells by competitive colonization, thereby reducing the harm of Streptococcus anginosus; furthermore, these probiotic bacteria can also release a series of bacteriocins, which have a good inhibitory effect on the harmful bacterium Streptococcus anginosus.

[0029] The present invention also prepared magnetic gold nanocages. Since gold nanomaterials have good coordination with thiol groups, and the formed coordination complexes have good stability, nano-gold can form a gold-DNA complex with the DNA sequence of the harmful bacterium Streptococcus anginosus through thiol groups, so that antibiotic molecules are inserted into the DNA of the harmful bacterium Streptococcus anginosus, effectively avoiding drug resistance. At the same time, due to the good near-infrared light response and magnetic response of magnetic gold nanocages, local temperature will rise under the stimulation of near-infrared light or magnetic field, opening the hydrogen bonds of DNA strands to release drugs into cells, and the local high temperature generated can directly kill harmful bacteria, thus achieving a good effect of inhibiting the growth of harmful bacteria.

[0030] In the present invention, an inclusion complex, magnetic gold nanocages and probiotic secretions are mixed and then linked through phytic acid to form a complex, which is linked to Lactobacillus salivarius LS02 through the condensation reaction of classical EDC and NHS, thereby achieving targeted drug release, reducing the concentration of antibiotic use, reducing side effects and adverse reactions. At the same time, it can also overcome the problem of drug resistance caused by high concentrations of antibiotics, greatly improving the drug efficacy. Since the antibiotic is embedded in cyclodextrin and will not be released under normal circumstances, the activity of the probiotic is also guaranteed. When the carrier moves near the harmful bacterium Streptococcus anginosus, under the action of the surrounding environment, the complex decomposes, and the antibiotic molecules embedded in cyclodextrin are slowly released, thereby playing a role in inhibiting the harmful bacterium Streptococcus anginosus.

[0031] The present invention also adds purslane and ginseng polysaccharide, which have good antibacterial, antioxidant and anti-inflammatory effects, further improving the drug efficacy of the composition. At the same time, the biocompatibility of the composition is improved, avoiding the occurrence of adverse reactions.

[0032] The composition for inhibiting Streptococcus anginosus prepared by the present invention has a good targeted inhibition and killing effect on the harmful bacterium Streptococcus anginosus, has no drug resistance, has small side effects, high patient compliance, has good antioxidant, anti-inflammatory and antibacterial effects, good biocompatibility, and has broad application prospects. Detailed implementation manners

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 shall fall within the protection scope of the present invention.

[0034] EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide.

[0035] Lactobacillus reuteri FPHC2951, Lactobacillus acidophilus LA16, Lactobacillus salivarius LS02, 20 billion cfu / g.

[0036] Lactobacillus salivarius LS02, taxonomically named Lactobacillus salivarius, with the preservation number of CGMCC NO.24667, the preservation date of April 11, 2022, and the preservation unit is the General Microbiology Center of the China National Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode: 100101.

[0037] Lactobacillus acidophilus LA16, classified and named as Lactobacillus acidophilus, with a preservation number of CGMCC NO.22771, a preservation date of June 24, 2021, and a preservation unit of the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code: 100101.

[0038] Limosilactobacillus reuteri FPHC2951, classified and named as Limosilactobacillus reuteri FPHC2951, with a preservation number of GDMCC NO.62946, a preservation date of November 3, 2022, and a preservation unit of the Guangdong Provincial Culture Collection of Biological Species, Address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Zip Code: 510070.

[0039] Preparation of the activated Lactobacillus salivarius LS02 strain seed solution: Inoculate Lactobacillus salivarius LS02 into Gao's medium, and activate and culture it at 37 °C and 100 r / min for 24 h to obtain a strain seed solution with a bacterial content of 10 8 -10 9 cfu / mL.

[0040] Example 1

[0041] This example provides a method for preparing a composition for inhibiting Streptococcus anginosus, including the following steps:

[0042] S1. Preparation of the anti-inflammatory composition and polysaccharide: Wash, dry, and crush 3 g of Portulaca oleracea and 7 g of ginseng, add 200 mL of water, heat to boiling and extract for 1 h, filter, add ethanol to the filtrate until the ethanol content in the system is 90 wt%, precipitate for 1 h, wash the solid, dry to obtain the polysaccharide, recover ethanol from the liquid, dialyze with a dialysis bag with a pore size of 3 kDa for 12 h, and dry the undialyzed liquid to obtain the anti-inflammatory composition;

[0043] S2. Preparation of the inclusion complex: Dissolve 10 g of β-cyclodextrin in 200 mL of 30 wt% ethanol aqueous solution, add 4 g of the anti-inflammatory composition and 1 g of the antibiotic mixture to the above solution, stir and mix for 1 h, and freeze-dry to obtain the inclusion complex;

[0044] The antibiotic is a mixture of penicillin and azithromycin with a mass ratio of 1:1;

[0045] S3. Preparation of magnetic gold nanocages: Disperse 4 g of gold nanocages in 200 mL of PBS buffer with pH = 7, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride. Under nitrogen protection, dropwise add ammonia water to adjust the pH value to 10, heat to 85 °C, stir and react for 3 h, centrifuge, wash, and dry to obtain magnetic gold nanocages;

[0046] S4. Preparation of probiotic secretions: Inoculate 4 g of Lactobacillus reuteri FPHC2951 and 2 g of Lactobacillus acidophilus LA16 into 1 L of liquid Gao's medium, and ferment at 35 °C and 100 r / min for 24 h to obtain a mixed solution with a bacterial content of 10 10 -10 11 cfu / mL. Centrifuge, collect the bacterial sludge, freeze-dry to obtain probiotics, and freeze-dry the supernatant to obtain probiotic secretions;

[0047] S5. Preparation of inclusion complex / magnetic gold nanocage / probiotic secretion complex: Add 6 g of inclusion complex, 3 g of magnetic gold nanocages, 10 g of probiotic secretions, 5 g of phytic acid, and 1 g of dipotassium hydrogen phosphate to 500 mL of water, heat to 90 °C, stir for 1 h, perform hydrothermal reaction at 150 °C for 8 h, adjust the pH value to neutral, centrifuge, wash, and dry to obtain the inclusion complex / magnetic gold nanocage / probiotic secretion complex;

[0048] S6. Preparation of drug-loaded Lactobacillus: Add 10 g of inclusion complex / magnetic gold nanocage / probiotic secretion complex to 200 mL of water, add 2 g of EDC and 2 g of NHS, stir and activate at 0 °C for 30 min, add the activated seed solution of Lactobacillus salivarius LS02 strain, with an inoculation amount of 3 v / v%, react at 35 °C with a CO2 content of 4 v / v%, stir and react for 10 h, centrifuge, wash, and freeze-dry to obtain drug-loaded Lactobacillus;

[0049] S7. Preparation of the composition for inhibiting Streptococcus anginosus: Stir and mix 10 g of drug-loaded Lactobacillus, 3 g of the probiotics in step S4, and 2 g of the polysaccharide in step S1 for 15 min to obtain the composition for inhibiting Streptococcus anginosus.

[0050] Example 2

[0051] This example provides a method for preparing a composition for inhibiting Streptococcus anginosus, including the following steps:

[0052] S1. Preparation of anti-inflammatory composition and polysaccharide: Wash 5 g of purslane and 10 g of ginseng, dry, crush, add to 200 mL of water, heat to boiling for extraction for 2 h, filter, add ethanol to the filtrate until the ethanol content in the system is 90 wt%, precipitate for 1 h, wash the solid, dry to obtain polysaccharide, recover ethanol from the liquid, dialyze with a dialysis bag with a pore size of 5 kDa for 24 h, and dry the non-permeated liquid to obtain the anti-inflammatory composition;

[0053] S2. Preparation of the inclusion complex: Dissolve 12 g of β-cyclodextrin in 200 mL of 40 wt% ethanol aqueous solution, add the mixture of 7 g of the anti-inflammatory composition and 2 g of antibiotics to the above solution, stir and mix for 2 h, and then perform freeze-drying to obtain the inclusion complex;

[0054] The antibiotic is a mixture of penicillin and azithromycin, and the mass ratio is 1:2;

[0055] S3. Preparation of magnetic gold nanocages: Disperse 7 g of gold nanocages in 200 mL of PBS buffer solution with pH = 7, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride, under nitrogen protection, dropwise add ammonia water to adjust the pH value to 11, heat to 95 °C, stir and react for 5 h, centrifuge, wash, and dry to obtain magnetic gold nanocages;

[0056] S4. Preparation of probiotic secretions: Inoculate 7 g of Lactobacillus reuteri FPHC2951 and 4 g of Lactobacillus acidophilus LA16 into 1 L of liquid Czapek's medium, and perform fermentation culture at 37 °C and 200 r / min for 36 h to obtain a mixed solution with a bacterial content of 10 10 -10 11 cfu / mL, centrifuge, collect the bacterial sludge, perform freeze-drying to obtain probiotics, and perform freeze-drying on the supernatant to obtain probiotic secretions;

[0057] S5. Preparation of the inclusion complex / magnetic gold nanocage / probiotic secretion complex: Add 8 g of the inclusion complex, 5 g of magnetic gold nanocages, 12 g of probiotic secretions, 7 g of phytic acid, and 2 g of dipotassium hydrogen phosphate to 500 mL of water, heat to 100 °C, stir for 2 h, perform hydrothermal reaction at 160 °C for 10 h, adjust the pH value to neutral, centrifuge, wash, and dry to obtain the inclusion complex / magnetic gold nanocage / probiotic secretion complex;

[0058] S6. Preparation of drug-loaded lactobacilli: Add 10 g of the inclusion complex / magnetic gold nanocage / probiotic secretion complex to 200 mL of water, add 4 g of EDC and 4 g of NHS, stir and activate at 4 °C for 40 min, add the seed solution of the activated Lactobacillus salivarius LS02 strain, with an inoculation amount of 5 v / v%, at 37 °C and a CO2 content of 6 v / v%, stir and react for 12 h, centrifuge, wash, and perform freeze-drying to obtain drug-loaded lactobacilli;

[0059] S7. Preparation of the composition for inhibiting Streptococcus anginosus: Stir and mix 10 g of drug-loaded lactobacilli, 5 g of the probiotics in step S4, and 3 g of the polysaccharide in step S1 for 15 min to obtain the composition for inhibiting Streptococcus anginosus.

[0060] Example 3

[0061] This embodiment provides a method for preparing a composition for inhibiting Streptococcus anginosus, comprising the following steps:

[0062] S1. Preparation of anti-inflammatory composition and polysaccharide: Wash, dry, and pulverize 4 g of purslane and 8 g of ginseng, add them to 200 mL of water, heat to boiling and extract for 1.5 h, filter, add ethanol to the filtrate until the ethanol content in the system is 90 wt%, precipitate for 1 h, wash the solid, dry to obtain the polysaccharide, recover ethanol from the liquid, dialyze with a dialysis bag with a pore size of 4 kDa for 18 h, and dry the undialyzed liquid to obtain the anti-inflammatory composition;

[0063] S2. Preparation of inclusion complex: Dissolve 11 g of β-cyclodextrin in 200 mL of 35 wt% aqueous ethanol solution, add 5.5 g of anti-inflammatory composition and 1.5 g of antibiotic mixture to the above solution, stir and mix for 1.5 h, and freeze-dry to obtain the inclusion complex;

[0064] The antibiotic is a mixture of penicillin and azithromycin, and the mass ratio is 1:1.5;

[0065] S3. Preparation of magnetic gold nanocages: Disperse 5.5 g of gold nanocages in 200 mL of PBS buffer solution with pH = 7, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride, under nitrogen protection, dropwise add ammonia water to adjust the pH value to 10.5, heat to 90 °C, stir and react for 4 h, centrifuge, wash, and dry to obtain magnetic gold nanocages;

[0066] S4. Preparation of probiotic secretions: Inoculate 5.5 g of Lactobacillus reuteri FPHC2951 and 3 g of Lactobacillus acidophilus LA16 into 1 L of liquid Czapek-Dox medium, ferment and culture at 36 °C and 150 r / min for 30 h to obtain a mixture with a bacterial content of 10 10 -10 11 cfu / mL, centrifuge, collect the bacterial sludge, freeze-dry to obtain probiotics, and freeze-dry the supernatant to obtain probiotic secretions;

[0067] S5. Preparation of inclusion complex / magnetic gold nanocage / probiotic secretion complex: Add 7 g of inclusion complex, 4 g of magnetic gold nanocages, 11 g of probiotic secretions, 6 g of phytic acid, and 1.5 g of dipotassium hydrogen phosphate to 500 mL of water, heat to 95 °C, stir for 1.5 h, carry out a hydrothermal reaction at 155 °C for 9 h, adjust the pH value to neutral, centrifuge, wash, and dry to obtain the inclusion complex / magnetic gold nanocage / probiotic secretion complex;

[0068] S6. Preparation of drug-loaded Lactobacillus: Add 10 g of the inclusion complex / magnetic gold nanocage / probiotic secretion complex to 200 mL of water, add 3 g of EDC and 3 g of NHS, stir and activate at 2°C for 35 min, add the activated seed solution of Lactobacillus salivarius LS02 strain, with an inoculation amount of 4 v / v%, at 36°C and a CO2 content of 5 v / v%, stir and react for 11 h, centrifuge, wash, and freeze-dry to obtain drug-loaded Lactobacillus;

[0069] S7. Preparation of the composition for inhibiting Streptococcus anginosus: Stir and mix 10 g of drug-loaded Lactobacillus, 4 g of the probiotic in step S4, and 2.5 g of the polysaccharide in step S1 for 15 min to obtain the composition for inhibiting Streptococcus anginosus.

[0070] Comparative Example 1

[0071] Compared with Example 3, the difference is that purslane was not added in step S1.

[0072] Specifically as follows:

[0073] S1. Preparation of the anti-inflammatory composition and polysaccharide: Wash, dry, and pulverize 12 g of ginseng, add it to 200 mL of water, heat to boiling and extract for 1.5 h, filter, add ethanol to the filtrate until the ethanol content in the system is 90 wt%, precipitate for 1 h, wash the solid, dry to obtain the polysaccharide, recover ethanol from the liquid, dialyze with a dialysis bag with a pore size of 4 kDa for 18 h, and dry the undialyzed liquid to obtain the anti-inflammatory composition.

[0074] Comparative Example 2

[0075] Compared with Example 3, the difference is that ginseng was not added in step S1.

[0076] Specifically as follows:

[0077] S1. Preparation of the anti-inflammatory composition and polysaccharide: Wash, dry, and pulverize 12 g of purslane, add it to 200 mL of water, heat to boiling and extract for 1.5 h, filter, add ethanol to the filtrate until the ethanol content in the system is 90 wt%, precipitate for 1 h, wash the solid, dry to obtain the polysaccharide, recover ethanol from the liquid, dialyze with a dialysis bag with a pore size of 4 kDa for 18 h, and dry the undialyzed liquid to obtain the anti-inflammatory composition.

[0078] Comparative Example 3

[0079] Compared with Example 3, the difference is that the anti-inflammatory composition was not added in step S2.

[0080] Specifically as follows:

[0081] S2. Preparation of inclusion complex: Dissolve 11 g of β-cyclodextrin in 200 mL of 35 wt% ethanol aqueous solution, add 1.5 g of antibiotic to the above solution, stir and mix for 1.5 h, and then perform freeze-drying to obtain the inclusion complex;

[0082] The antibiotic is a mixture of penicillin and azithromycin, and the mass ratio is 1:1.5.

[0083] Comparative Example 4

[0084] Compared with Example 3, the difference is that step S3 is not carried out, and magnetic gold nanocages are replaced by gold nanocages.

[0085] Specifically as follows:

[0086] S1. Preparation of anti-inflammatory composition and polysaccharide: Wash, dry, and pulverize 4 g of Portulaca oleracea and 8 g of ginseng, add them to 200 mL of water, heat to boiling for extraction for 1.5 h, filter, add ethanol to the filtrate until the ethanol content in the system is 90 wt%, precipitate for 1 h, wash the solid, and dry to obtain the polysaccharide. Recover ethanol from the liquid, dialyze with a dialysis bag with a pore size of 4 kDa for 18 h, and dry the undialyzed liquid to obtain the anti-inflammatory composition;

[0087] S2. Preparation of inclusion complex: Dissolve 11 g of β-cyclodextrin in 200 mL of 35 wt% ethanol aqueous solution, add 5.5 g of anti-inflammatory composition and 1.5 g of antibiotic to the above solution, stir and mix for 1.5 h, and then perform freeze-drying to obtain the inclusion complex;

[0088] The antibiotic is a mixture of penicillin and azithromycin, and the mass ratio is 1:1.5;

[0089] S3. Preparation of probiotic secretion: Inoculate 5.5 g of Lactobacillus reuteri FPHC2951 and 3 g of Lactobacillus acidophilus LA16 into 1 L of liquid Czapek medium, ferment and culture at 36 °C and 150 r / min for 30 h to obtain a mixed solution with a bacterial content of 10 10 -10 11 cfu / mL, centrifuge, collect the bacterial sludge, perform freeze-drying to obtain probiotics, and perform freeze-drying on the supernatant to obtain probiotic secretion;

[0090] S4. Preparation of inclusion complex / gold nanocage / probiotic secretion complex: Add 7 g of inclusion complex, 4 g of gold nanocage, 11 g of probiotic secretion, 6 g of phytic acid, and 1.5 g of dipotassium hydrogen phosphate to 500 mL of water, heat to 95 °C, stir for 1.5 h, perform hydrothermal reaction at 155 °C for 9 h, adjust the pH value to neutral, centrifuge, wash, and dry to obtain the inclusion complex / gold nanocage / probiotic secretion complex;

[0091] S5. Preparation of Lactobacillus drug-loading: Add 10 g of the inclusion complex / gold nanocage / probiotic secretion complex to 200 mL of water, add 3 g of EDC and 3 g of NHS, stir and activate at 2°C for 35 min, add the activated seed solution of Lactobacillus salivarius LS02 strain, with an inoculation amount of 4 v / v%, at 36°C, with a CO2 content of 5 v / v%, stir and react for 11 h, centrifuge, wash, and freeze-dry to obtain Lactobacillus drug-loading;

[0092] S6. Preparation of the composition for inhibiting Streptococcus anginosus: Stir and mix 10 g of Lactobacillus drug-loading, 4 g of the probiotic in step S3, and 2.5 g of the polysaccharide in step S1 for 15 min to obtain the composition for inhibiting Streptococcus anginosus.

[0093] Comparative Example 5

[0094] Compared with Example 3, the difference lies in that magnetic gold nanocages were not added in step S5.

[0095] Specifically as follows:

[0096] S5. Preparation of the inclusion complex / probiotic secretion complex: Add 7 g of the inclusion complex, 11 g of probiotic secretion, 6 g of phytic acid, and 1.5 g of dipotassium hydrogen phosphate to 500 mL of water, heat to 95°C, stir for 1.5 h, perform a hydrothermal reaction at 155°C for 9 h, adjust the pH value to neutral, centrifuge, wash, and dry to obtain the inclusion complex / probiotic secretion complex.

[0097] Comparative Example 6

[0098] Compared with Example 3, the difference lies in that probiotic secretion was not added in step S5.

[0099] Specifically as follows:

[0100] Specifically as follows:

[0101] S5. Preparation of the inclusion complex / magnetic gold nanocage complex: Add 7 g of the inclusion complex, 4 g of magnetic gold nanocages, 6 g of phytic acid, and 1.5 g of dipotassium hydrogen phosphate to 500 mL of water, heat to 95°C, stir for 1.5 h, perform a hydrothermal reaction at 155°C for 9 h, adjust the pH value to neutral, centrifuge, wash, and dry to obtain the inclusion complex / magnetic gold nanocage complex.

[0102] Comparative Example 7

[0103] Compared with Example 3, the difference lies in that step S6 was not carried out.

[0104] Specifically as follows:

[0105] S1. Preparation of anti-inflammatory composition and polysaccharide: Wash 4 g of purslane and 8 g of ginseng, dry them, crush them, add them to 200 mL of water, heat to boiling and extract for 1.5 h, filter, add ethanol to the filtrate until the ethanol content in the system is 90 wt%, precipitate for 1 h, wash the solid, dry it to obtain polysaccharide, recover ethanol from the liquid, dialyze with a dialysis bag with a pore size of 4 kDa for 18 h, dry the undialyzed liquid to obtain the anti-inflammatory composition;

[0106] S2. Preparation of inclusion complex: Dissolve 11 g of β-cyclodextrin in 200 mL of 35 wt% ethanol aqueous solution, add 5.5 g of anti-inflammatory composition and 1.5 g of antibiotic mixture to the above solution, stir and mix for 1.5 h, and freeze-dry to obtain the inclusion complex;

[0107] The antibiotic is a mixture of penicillin and azithromycin, and the mass ratio is 1:1.5;

[0108] S3. Preparation of magnetic gold nanocages: Disperse 5.5 g of gold nanocages in 200 mL of PBS buffer solution with pH = 7, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride, under nitrogen protection, dropwise add ammonia water to adjust the pH value to 10.5, heat to 90 °C, stir and react for 4 h, centrifuge, wash, and dry to obtain magnetic gold nanocages;

[0109] S4. Preparation of probiotic secretions: Inoculate 5.5 g of Lactobacillus reuteri FPHC2951 and 3 g of Lactobacillus acidophilus LA16 into 1 L of liquid Gao's medium, ferment and culture at 36 °C and 150 r / min for 30 h to obtain a mixed solution with a bacterial content of 10 10 -10 11 cfu / mL, centrifuge, collect the bacterial sludge, freeze-dry to obtain probiotics, and freeze-dry the supernatant to obtain probiotic secretions;

[0110] S5. Preparation of inclusion complex / magnetic gold nanocage / probiotic secretion complex: Add 7 g of inclusion complex, 4 g of magnetic gold nanocages, 11 g of probiotic secretions, 6 g of phytic acid, and 1.5 g of dipotassium hydrogen phosphate to 500 mL of water, heat to 95 °C, stir for 1.5 h, perform hydrothermal reaction at 155 °C for 9 h, adjust the pH value to neutral, centrifuge, wash, and dry to obtain the inclusion complex / magnetic gold nanocage / probiotic secretion complex;

[0111] S6. Preparation of the composition for inhibiting Streptococcus anginosus: Stir and mix 10 g of inclusion complex / magnetic gold nanocage / probiotic secretion complex, 4 g of the probiotics in step S4, and 2.5 g of the polysaccharide in step S1 for 15 min to obtain the composition for inhibiting Streptococcus anginosus.

[0112] Test Example 1

[0113] Take Streptococcus anginosus (ATCC 33397, MBL Microbiologics, USA) out of the -70°C refrigerator and thaw it in a 37°C constant temperature water bath. Preheat the sterilized THB medium (from Medium Company) to 37°C. Aseptically pipette 50 mL of THB medium into a 250 mL conical flask in a biosafety cabinet, and then add 0.5 mL of Streptococcus anginosus suspension into the conical flask. Seal the flask mouth, and then place the conical flask in a 37°C constant temperature shaker at 200 r / min. Take 1 mL of the seed liquid at 16 h in the late logarithmic phase and transfer it into a 500 mL conical flask, and culture it at 37°C with constant shaking until the bacterial liquid concentration is about 10 8 cfu / mL, and then dilute it 300 times.

[0114] Adopt the single-layer agar plate diffusion method to control the number of bacteria in the dilution at 10 6 cfu / mL. Pipette 0.2 mL of Streptococcus anginosus bacterial liquid onto a solid medium plate, spread it evenly, and let it stand for 1 h. After the surface bacterial liquid is fixed on the surface of the plate, punch holes with a diameter of 8 mm and a depth of 3 mm. Immerse the holes in the aqueous solution (prepared with sterile water) of the composition for inhibiting Streptococcus anginosus prepared in Examples 1-3 or Comparative Examples 1-7 at 1 mg / mL, and then inject it into the holes. Culture at 37°C for 8 h, and apply near-infrared light (808 nm) and a 0.05 T magnetic field for 10 min during this period, applying it once every 1 h. Observe the results and measure the diameter of the inhibition zone. The results are shown in Table 1. There are three holes on each plate, and two parallel plates are made. Take the average value.

[0115] Table 1

[0116]

[0117]

[0118] As can be seen from the above table, the compositions for inhibiting Streptococcus anginosus prepared in Examples 1-3 of the present invention have good antibacterial effects on Streptococcus anginosus.

[0119] Test Example 2

[0120] Gastrically intubate SD rats in other groups except the blank group with a 2 wt% indomethacin aqueous solution at 3 mL / kg once a day for a total of 2 times. On the 6th day, gastrically intubate 2 mL of saturated sodium bicarbonate, and 15 min later, gastrically intubate 1 mL of Streptococcus anginosus bacterial liquid (with a bacterial content of 10 11 -10 12 cfu / mL), once every 2 days for a total of 4 times.

[0121] SD rats, half male and half female, were randomly divided into 12 groups, with 8 rats in each group, including a blank group (equal amount of normal saline), a model group (equal amount of normal saline), groups of Examples 1 - 3, groups of Comparative Examples 1 - 7 (the prepared compositions for inhibiting Streptococcus anginosus, 1 g / kg), and a positive group (penicillin V potassium tablets, ground into powder, 0.5 g / kg). Near-infrared light (808 nm) and a 0.05 T magnetic field were applied to the stomach for 10 min, and this was repeated every two days.

[0122] After successful modeling, the mice were given drug intervention for 7 days, then sacrificed, and the gastric tissues of the mice were excised. The ulcer area was measured under a microscope, and the total ulcer area of each mouse was divided into 5 grades, which was used as the ulcer index for evaluating the degree of ulcer. The scoring was as follows: ulcer area 1 - 12 mm 2 , ulcer index 1; ulcer area 13 - 25 mm 2 , ulcer index 2; ulcer area 26 - 37 mm 2 , ulcer index 3; ulcer area 38 - 50 mm 2 , ulcer index 4; ulcer area > 50 mm 2 or perforation, ulcer index 1.

[0123] Ulcer inhibition rate (%) = (ulcer index of the model group - ulcer index of the model group) / ulcer index of the model group × 100%

[0124] After sacrificing the rats, the gastric mucosa tissue was homogenized, and the contents of TNF-α and IL-8 were detected according to the ELISA method. The results are shown in Table 2.

[0125] Table 2

[0126] Group Ulcer inhibition rate (%) TNF-α (pg / mL) IL-8 (pg / mL) Blank group / 0.69±0.11 11.62±1.09 Model group / 1.75±0.24* 25.77±5.10* Positive drug group 77.8 0.79±0.06# 14.21±0.97# Example 1 94.5 0.90±0.12# 14.01±1.21# Example 2 94.9 0.92±0.14# 13.95±1.17# Example 3 95.2 0.87±0.10# 13.78±1.20# Comparative example 1 90.1 1.08±0.20 15.55±1.52 Comparative example 2 89.5 1.13±0.19 15.49±1.39 Comparative example 3 87.6 1.21±0.16 16.08±1.46 Comparative example 4 85.9 1.01±0.18 15.03±1.33 Comparative example 5 82.1 1.04±0.15 15.17±1.41 Comparative example 6 86.2 1.31±0.24 16.59±1.56 Comparative example 7 78.9 1.19±0.21 15.33±1.48

[0127] Note: * P < 0.05 compared with the blank group; # P < 0.05 compared with the model group.

[0128] As can be seen from the above table, the compositions for inhibiting Streptococcus anginosus prepared in Examples 1 - 3 of the present invention have a good inhibitory effect on gastric ulcers caused by Streptococcus anginosus, and at the same time, have a good anti-inflammatory effect.

[0129] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a composition for inhibiting Streptococcus anginosus, characterized in that, Extract the polysaccharide and anti-inflammatory composition from purslane and ginseng by water extraction. The anti-inflammatory composition is mixed with antibiotics and embedded in β-cyclodextrin. The prepared inclusion complex forms a complex with the magnetic gold nanocage and the probiotic secretion complex, and is loaded on Lactobacillus salivarius LS02. It is mixed evenly with Lactobacillus reuteri FPHC2951, Lactobacillus acidophilus LA16, and the polysaccharide to prepare a composition for inhibiting Streptococcus anginosus.

2. The preparation method according to claim 1, wherein, It includes the following steps: S1. Preparation of the anti-inflammatory composition and the polysaccharide: Wash, dry, and pulverize purslane and ginseng, add them to water, heat to boiling for extraction, filter, add ethanol to the filtrate for precipitation, wash the solid, dry it to obtain the polysaccharide, recover ethanol from the liquid, dialyze, and dry the non-permeated liquid to obtain the anti-inflammatory composition; S2. Preparation of the inclusion complex: Dissolve β-cyclodextrin in an ethanol aqueous solution, add the anti-inflammatory composition and antibiotics to the solution, stir and mix, and freeze-dry to obtain the inclusion complex; S3. Preparation of the magnetic gold nanocage: Disperse the gold nanocage in PBS buffer solution, add ferric chloride and ferrous chloride, under the protection of inert gas, dropwise add ammonia water, heat and stir for reaction, centrifuge, wash, and dry to obtain the magnetic gold nanocage; S4. Preparation of the probiotic secretion: Inoculate Lactobacillus reuteri FPHC2951 and Lactobacillus acidophilus LA16 in a culture medium, ferment and culture, centrifuge, collect the bacterial sludge, freeze-dry to obtain the probiotic, and freeze-dry the supernatant to obtain the probiotic secretion; S5. Preparation of the inclusion complex / magnetic gold nanocage / probiotic secretion complex: Add the inclusion complex, magnetic gold nanocage, probiotic secretion, phytic acid, and dipotassium hydrogen phosphate to water, heat and stir, carry out a hydrothermal reaction, adjust the pH value to neutral, centrifuge, wash, and dry to obtain the inclusion complex / magnetic gold nanocage / probiotic secretion complex; S6. Preparation of the drug-loaded lactobacillus: Add the inclusion complex / magnetic gold nanocage / probiotic secretion complex to water, add EDC and NHS, stir for activation, add the activated seed solution of Lactobacillus salivarius LS02 strain, stir for reaction, centrifuge, wash, and freeze-dry to obtain the drug-loaded lactobacillus; S7. Preparation of the composition for inhibiting Streptococcus anginosus: Mix the drug-loaded lactobacillus, the probiotic in step S4, and the polysaccharide in step S1 evenly to prepare the composition for inhibiting Streptococcus anginosus.

3. The preparation method according to claim 2, characterized in that, In step S1, the mass ratio of purslane to ginseng is 3-5:7-10, the extraction time is 1-2 h, the pore size of the dialysis bag used for dialysis is 3-5 kDa, and the dialysis time is 12-24 h.

4. The preparation method according to claim 2, characterized in that, In step S2, the ethanol content in the ethanol aqueous solution is 30-40 wt%, the mass ratio of β-cyclodextrin, the anti-inflammatory composition, and the antibiotic is 10-12:4-7:1-2, the antibiotic is selected from at least one of penicillin, cefixime, cefprozil, cefdinir, erythromycin, azithromycin, vancomycin, preferably, a mixture of penicillin and azithromycin with a mass ratio of 1:1-2, and the stirring and mixing time is 1-2 h.

5. The preparation method according to claim 2, characterized in that, In step S3, the mass ratio of the gold nanocage, ferric chloride, and ferrous chloride is 4-7: 3.24:1.26, the pH value of the solution is adjusted to 10-11 by dropping ammonia water, the temperature of the heating and stirring reaction is 85-95 °C, and the time is 3-5 h.

6. The preparation method according to claim 2, characterized in that, The culture medium described in step S4 is a liquid Gause's medium, and the conditions for fermentation culture are 35 - 37°C, 100 - 200 r / min, and culture for 24 - 36 h to obtain a mixed solution with a bacterial content of 10 10 -10 11 cfu / mL, and the mass ratio of Lactobacillus reuteri FPHC2951 to Lactobacillus acidophilus LA16 is 4 - 7:2 - 4.

7. The preparation method according to claim 2, characterized in that, In step S5, the mass ratio of the clathrate, magnetic gold nanocage, probiotic secretion, phytic acid, and dipotassium hydrogen phosphate is 6-8:3-5:10-12:5-7:1-2. The temperature of the heating and stirring is 90-100 °C, and the time is 1-2 h. The temperature of the hydrothermal reaction is 150-160 °C, and the time is 8-10 h.

8. The preparation method according to claim 2, characterized in that, In step S6, the mass ratio of the clathrate / magnetic gold nanocage / probiotic secretion complex, EDC, and NHS is 10:2-4:2-4. The inoculation amount of the activated Lactobacillus salivarius LS02 strain seed liquid is 3-5 v / v%. The bacterial content of the strain seed liquid is 10 8 -10 9 cfu / mL. The temperature for stirring activation is 0-4 °C, and the time is 30-40 min. The temperature for stirring reaction is 35-37 °C, the CO2 content is 4-6 v / v%, and the time is 10-12 h.

9. The preparation method according to claim 2, wherein In step S7, the mass ratio of the drug-loaded Lactobacillus, the probiotic in step S4, and the polysaccharide in step S1 is 10:3-5:2-3.

10. A composition for inhibiting Streptococcus anginosus prepared by the preparation method according to any one of claims 1-9.

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