A lactic acid bacteria antibacterial composition, antibacterial tablets and applications

Abstract

The present invention discloses a lactic acid bacteria antibacterial composition, antibacterial tablets and applications. The lactic acid bacteria antibacterial composition comprises the following raw materials in parts by weight: 15-20 parts of lactic acid bacteria powder, 10-15 parts of gastrodia extract, 4-8 parts of borneol, 5-10 parts of mint extract, 2-5 parts of honeysuckle extract, 1-3 parts of cnidium monnieri extract, 0.8-1 part of polyhexamethylene biguanide hydrochloride, 10-22 parts of stabilizer, and 10-20 parts of auxiliary agent. The lactic acid bacteria antibacterial composition provided by the present invention has a strong antibacterial effect, can adjust the vaginal environment, damage the cell membrane and cell wall structure, interfere with energy metabolism and effectively inhibit the growth of bacteria and fungi. At the same time, it can dose-dependently mediate the levels of inflammatory mediators and the expression of signal pathway proteins, so as to achieve the purpose of antibacterial and anti-inflammatory.

Description

Technical Field

[0001] The present invention relates to the field of medical pharmaceuticals, and particularly to a lactic acid bacteria antibacterial composition, antibacterial tablets and applications thereof. Background Art

[0002] Gynecological inflammation refers to the inflammation of the female reproductive system. Inflammation is a reaction of the body's struggle against pathogens invading the body, and it is a common and multiple disease in gynecology. Clinically common gynecological inflammations mainly include vulvitis, vaginitis, cervicitis, Bartholin gland inflammation, adnexitis, pelvic inflammation, etc. The main manifestations are lower abdominal pain, distension, increased leucorrhea with abnormal odor, abdominal pain during menstruation and increased menstrual volume, etc. Acute inflammation will also present systemic infection symptoms such as persistent high fever and pelvic abscess.

[0003] For the treatment of gynecological inflammation, for example, the treatment of vaginitis, usually antibiotics are used for treatment, but it is prone to rebound phenomenon, with large adverse reactions and increasing drug resistance. Therefore, agents derived from natural products with broad-spectrum antibacterial activity and low drug resistance have gradually become a research hotspot. In daily life, common ones include Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, etc. Among them, Candida albicans is the main cause of female vaginitis. The pathogen Staphylococcus aureus can secrete enterotoxin that can cause acute gastroenteritis. Escherichia coli is a common bacterium living in our intestines and is a source of human infection, while Pseudomonas aeruginosa is a common bacterial infection in wounds.

[0004] Lactic acid bacteria are a general term for a class of bacteria that can utilize fermentable carbohydrates to produce a large amount of lactic acid. These bacteria are widely distributed in nature and have rich species diversity. They are not only ideal materials for studying classification, biochemistry, genetics, molecular biology and genetic engineering, and have important academic value in theory, but also have extremely high application value in important fields closely related to human life such as industry, agriculture, animal husbandry, food and medicine. Regarding the antibacterial performance of lactic acid bacteria, many organic acids will be produced during their metabolism, mainly including lactic acid and acetic acid, as well as some short-chain fatty acids. When lactic acid bacteria produce a large amount of organic acids, the pH value of the whole system will decrease, and a large amount of H + will enter the cell membrane of pathogenic bacteria, acidifying their cytoplasm, thereby causing pathogenic bacteria to be unable to grow normally, thus achieving the inhibition of the growth of pathogenic bacteria. At the same time, lactic acid bacteria can also produce some bacteriocins. Bacteriocins are some peptide substances with the ability to antagonize or kill pathogenic bacteria. And lactic acid bacteria can produce H2O2 under specific conditions. When H2O2 exists in the environment, superoxide anions will form destructive hydroxyl free radicals. This process can cause the oxidation of lipids on the cell membrane, thereby increasing the permeability of the cell membrane, thus achieving the purpose of sterilization. However, for pure lactic acid bacteria, its antibacterial effect is not strong and may not achieve the expected antibacterial effect.

[0005] Therefore, there is an urgent need for a lactic acid bacteria antibacterial composition to enhance the antibacterial effect through this composition, ensure the safety of the vaginal environment, and be less likely to generate drug resistance. Summary of the Invention

[0006] In view of the problems of the prior art, the present invention provides a lactic acid bacteria antibacterial composition, antibacterial tablets and applications thereof. The lactic acid bacteria antibacterial composition has strong antibacterial effects, can improve gynecological inflammation induced by Candida albicans, and can also protect the balance of the vaginal environment. The lactic acid bacteria antibacterial composition can damage the cell membrane and cell wall structures, interfere with energy metabolism, and effectively inhibit the growth of bacteria and fungi. At the same time, it can dose-dependently mediate the levels of inflammatory mediators and the expression of signal pathway proteins, thereby producing an anti-inflammatory effect, so as to achieve the purpose of antibacterial and anti-inflammatory.

[0007] A lactic acid bacteria antibacterial composition, comprising the following raw materials in parts by weight: 15-20 parts of lactic acid bacteria powder, 10-15 parts of Gastrodia elata extract, 4-8 parts of Dryobalanops aromatica, 5-10 parts of Mentha haplocalyx extract, 2-5 parts of Lonicera japonica extract, 1-3 parts of Cnidium monnieri extract, 0.8-1 part of polyhexamethylene biguanide hydrochloride, 10-22 parts of stabilizer, and 10-20 parts of auxiliary agent.

[0008] According to the lactic acid bacteria antibacterial composition provided by the present invention, the lactic acid bacteria antibacterial composition has strong antibacterial effects, can improve gynecological inflammation induced by Candida albicans, and can also protect the balance of the vaginal environment. The lactic acid bacteria antibacterial composition can damage the cell membrane and cell wall structures, interfere with energy metabolism, and effectively inhibit the growth of bacteria and fungi. At the same time, it can dose-dependently mediate the levels of inflammatory mediators and the expression of signal pathway proteins, thereby producing an anti-inflammatory effect, so as to achieve the purpose of antibacterial and anti-inflammatory.

[0009] In addition, according to the lactic acid bacteria antibacterial composition provided by the present invention, it may also have the following additional technical features:

[0010] Preferably, the lactic acid bacteria antibacterial composition comprises the following raw materials in parts by weight: 18 parts of lactic acid bacteria powder, 12 parts of Gastrodia elata extract, 5.5 parts of Dryobalanops aromatica, 6 parts of Mentha haplocalyx extract, 3 parts of Lonicera japonica extract, 1.5 parts of Cnidium monnieri extract, 1 part of polyhexamethylene biguanide hydrochloride, 19 parts of stabilizer, and 15 parts of auxiliary agent.

[0011] Preferably, the stabilizer comprises 8-16 parts of sorbitol, 1-3 parts of fructooligosaccharide, and 1-3 parts of silicon dioxide.

[0012] Preferably, the auxiliary agent comprises 1-3 parts of glidant, 1-3 parts of anticaking agent, 3-6 parts of diluent, and 5-8 parts of binder.

[0013] Preferably, the glidant is magnesium stearate, the anti-caking agent is microcrystalline cellulose, the diluent is mannitol, and the binder is ethanol.

[0014] The present invention also provides a lactic acid bacteria antibacterial tablet, which comprises the above lactic acid bacteria antibacterial composition and excipients.

[0015] In addition, the lactic acid bacteria antibacterial tablet provided by the present invention may further have the following additional technical features:

[0016] Preferably, the excipients include tartaric acid and sodium bicarbonate.

[0017] The present invention also provides an application of the above lactic acid bacteria antibacterial composition in the preparation of drugs for inhibiting gynecological inflammation.

[0018] The additional aspects and advantages of the present invention will be partly given in the following description, partly will become obvious from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 It is the target cell morphology diagrams of the first to sixth groups provided by the embodiments of the present invention;

[0021] Figure 2 It is the target cell proliferation diagrams of the first to sixth groups provided by the embodiments of the present invention;

[0022] Figure 3 It is the NO level diagrams of the first to sixth groups provided by the embodiments of the present invention;

[0023] Figure 4 It is the content level diagrams of interleukin-1β, prostaglandin E2 and intercellular adhesion molecule-1 of the first to sixth groups provided by the embodiments of the present invention;

[0024] Figure 5 It is the content level diagrams of malondialdehyde, total superoxide dismutase, glutathione and catalase of the first to sixth groups provided by the embodiments of the present invention;

[0025] Figure 6 It is the expression level diagrams or phosphorylation level diagrams of monocyte chemoattractant protein-1, nuclear factor erythroid 2-related factor 2, heme oxygenase-1 and extracellular regulated protein kinases of the first to sixth groups provided by the embodiments of the present invention;

[0026] Figure 7 Phosphorylation level diagrams of c-Jun N-terminal kinase, P38 protein, and p65 protein for the first to sixth groups provided by the embodiments of the present invention;

[0027] Figure 8 ROS content diagrams for the first to sixth groups provided by the embodiments of the present invention;

[0028] Figure 9 Inhibition result diagrams of the first to fourth mouse groups against Candida albicans provided by the embodiments of the present invention;

[0029] Figure 10 Lactobacillus colony diagrams of the first to fourth mouse groups provided by the embodiments of the present invention;

[0030] Figure 11 Mouse pathological section diagrams of the first to fourth mouse groups provided by the embodiments of the present invention;

[0031] Figure 12 Growth curve diagrams of the experimental group and the control group corresponding to four strains respectively provided by the embodiments of the present invention;

[0032] Figure 13 Scanning electron microscope diagrams of the experimental group and the control group corresponding to four strains respectively provided by the embodiments of the present invention;

[0033] Figure 14 Diagrams of the activity levels of extracellular β-galactosidase, Na + / K + -ATPase, and Ca 2+ -ATPase for the experimental group and the control group corresponding to four strains respectively provided by the embodiments of the present invention;

[0034] Figure 15 Diagrams of the activity levels of adenosine triphosphatase, malate dehydrogenase, and succinate dehydrogenase for the experimental group and the control group corresponding to four strains respectively provided by the embodiments of the present invention.

[0035] The embodiments of the present invention will be further described below with reference to the accompanying drawings. Detailed implementation manners

[0036] To make the objectives, features, and advantages of the present invention more obvious and understandable, the following describes the specific implementation manners of the present invention in detail with reference to the embodiments. Several embodiments of the present invention are given in the embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0037] Embodiment 1

[0038] In Example 1 of the present invention, a lactic acid bacteria antibacterial composition is provided. The lactic acid bacteria antibacterial composition comprises the following raw materials in parts by weight: 15-20 parts of lactic acid bacteria powder, 10-15 parts of gastrodia extract, 4-8 parts of dryobalanops aromatica, 5-10 parts of mint extract, 2-5 parts of honeysuckle extract, 1-3 parts of cnidium monnieri extract, 0.8-1 part of polyhexamethylene biguanide hydrochloride, 10-22 parts of stabilizer, and 10-20 parts of auxiliary agent;

[0039] Specifically, the lactic acid bacteria antibacterial composition comprises the following raw materials in parts by weight: 18 parts of lactic acid bacteria powder, 12 parts of gastrodia extract, 5.5 parts of dryobalanops aromatica, 6 parts of mint extract, 3 parts of honeysuckle extract, 1.5 parts of cnidium monnieri extract, 1 part of polyhexamethylene biguanide hydrochloride, 19 parts of stabilizer, and 15 parts of auxiliary agent.

[0040] Among them, the lactic acid bacteria in the lactic acid bacteria powder are specifically lactobacilli;

[0041] Among them, the extraction process of the gastrodia extract is as follows: Select a certain amount of dry gastrodia, dry it at 60°C for 2h and then crush it. Under the condition of constant temperature at 80°C, use 0.8% NaCl aqueous solution to extract for 2h, filter and take the supernatant. Add papain to the collected supernatant, and then put the supernatant into a water bath at 60°C and heat it in a water bath for 30min. Heat it at 100°C for 40min to inactivate papain and terminate hydrolysis. Filter it with a 10kD ultrafiltration membrane and concentrate the sample with a rotary evaporator to obtain the gastrodia extract.

[0042] Among them, the extraction process of the mint extract is as follows: Dissolve mint with an equal amount of anhydrous ethanol solution, continuously stir it at a constant temperature of 40°C in a magnetic stirrer for 75 min, place it at 4°C for 24h and then filter it by suction. Wash the filter cake 5 times with 15 times the amount of distilled water and 8 times the amount of petroleum ether respectively, and dry it to obtain the mint extract;

[0043] Among them, the extraction process of the honeysuckle extract is as follows: Weigh a certain amount of honeysuckle, reflux and extract it with 10, 8, and 6 times the amount of 75% ethanol for 1h each time. After each extraction, quickly filter and combine the filtrates, filter by suction, concentrate, and dry it in a vacuum oven at 60°C to obtain the honeysuckle extract.

[0044] Among them, the extraction process of the cnidium monnieri extract is as follows: Weigh a certain amount of cnidium monnieri, reflux and extract it with 12 times the amount of 65% ethanol for 1h, repeat it 2 times. After each extraction, quickly filter and combine the two filtrates, filter by suction, concentrate, and dry it in a vacuum oven at 60°C to obtain the cnidium monnieri extract.

[0045] In this example, the stabilizer comprises 8-16 parts of sorbitol, 1-3 parts of fructooligosaccharide, and 1-3 parts of silicon dioxide.

[0046] In this embodiment, the auxiliary agents include 1-3 parts of glidant, 1-3 parts of anticaking agent, 3-6 parts of diluent, and 5-8 parts of binder.

[0047] In this embodiment, the glidant is magnesium stearate, the anticaking agent is microcrystalline cellulose, the diluent is mannitol, and the binder is ethanol.

[0048] Example 2

[0049] Example 2 of the present invention provides a lactic acid bacteria antibacterial tablet, which comprises the lactic acid bacteria antibacterial composition as described in Example 1 and excipients;

[0050] In this embodiment, the excipients include tartaric acid and sodium bicarbonate;

[0051] The preparation method of the lactic acid bacteria antibacterial tablet is as follows: First, the lactic acid bacteria antibacterial composition described in Example 1 is evenly divided into two parts, then 12 g of tartaric acid and 4 g of sodium bicarbonate are added to the two parts of raw materials respectively, and after stirring, two parts of soft materials are made respectively. Then the two parts of soft materials are mixed evenly, and after mixing, a lactic acid bacteria antibacterial tablet can be obtained by using a rotary tablet press.

[0052] Example 3

[0053] Example 3 of the present invention provides an application of a lactic acid bacteria antibacterial composition in the preparation of drugs for inhibiting gynecological inflammation.

[0054] In order to further illustrate the performance of the lactic acid bacteria antibacterial composition and the lactic acid bacteria antibacterial tablet provided in the above embodiments of the present invention, a series of experiments are carried out on them, and the experiments are as follows:

[0055] I. Antibacterial ability experiment

[0056] 1. Experimental materials

[0057] The experimental cells selected are mouse vaginal macrophages, and the experimental solution selected is LPS solution. The LPS solution can stimulate mouse vaginal macrophages to produce various inflammatory mediators and inflammatory chemotactic factors, etc., and then promote the occurrence of inflammatory reactions and aggravate inflammation. Therefore, the LPS solution and mouse vaginal macrophages are selected as the experimental solution and experimental cells respectively, and the antibacterial solution selected is the lactic acid bacteria antibacterial composition described in Example 1. At the same time, for the convenience of the experiment, the lactic acid bacteria antibacterial composition is dissolved in PBS buffer solutions with different volumes, and then antibacterial solutions with different concentrations are prepared. For the convenience of description, it is assumed that 1 mg of the lactic acid bacteria antibacterial composition can be dissolved in A ml of PBS buffer solution, and at this time, the maximum concentration is reached and recorded as A -1 mg / ml, and this solution is recorded as the high-concentration antibacterial solution. The solution with a concentration of (2A) -1 mg / ml is recorded as the medium-concentration antibacterial solution, and the solution with a concentration of (4A) -1The solution of mg / ml is recorded as a low-concentration antibacterial solution. In actual situations, the low-, medium-, and high-concentration antibacterial solutions are 0.25 mg / ml, 0.5 mg / ml, and 1 mg / ml respectively. At the same time, mouse vaginal macrophages are cultured, and the culture process is as follows: First, the mouse vaginal macrophages are resuscitated. After cell resuscitation, they are transferred to a culture flask and cultured in an incubator at a temperature of 37°C and a CO2 concentration of 5%. A tray containing saturated copper sulfate solution is placed on the bottom layer of the incubator, which can provide the humidity required for cell growth and also play a bactericidal role. When the cell density reaches about 90%, it is digested with 0.25% trypsin at 37°C for 30 s, and then gently pipetted with a Pasteur pipette and passaged at a ratio of 1:3 to obtain the target cells.

[0058] 2. Cell viability detection

[0059] The target cells are evenly plated in a six-well plate (1×10 6 / well), and six groups are set up to facilitate the observation of the viability of the target cells. 4 ml of complete medium is added to the first group, 2 ml of 1.2 μg / ml LPS solution and 2 ml of complete medium are added to the second group, 2 ml of 1.2 μg / ml LPS solution and 2 ml of 0.26 mg / ml dexamethasone solution are added to the third group, 2 ml of 1.2 μg / ml LPS solution and 2 ml of low-concentration antibacterial solution are added to the fourth group, 2 ml of 1.2 μg / ml LPS solution and 2 ml of medium-concentration antibacterial solution are added to the fifth group, and 2 ml of 1.2 μg / ml LPS solution and 2 ml of high-concentration antibacterial solution are added to the sixth group. The above groups are cultured for one day, and the morphology of the target cells after culture is observed, as specifically shown in Figure 1 shown;

[0060] It can be seen from Figure 1 that most of the cells in the first group have normal morphology, and a few cells are deformed with short pseudopods. After the cells in the second group are stimulated by LPS, the cells grow longer antennae and have irregular shapes, and some cells show vacuolation. Most of the cells in the third group are round, and a few have short pseudopods, and the cell morphology has been greatly improved. The cell morphology in the fourth group has been slightly improved, and there are still many deformed cells, but there are more normal round cells compared with the second group. The cell morphology in the fifth group is more regular than that in the second and fourth groups, and the number of cell deformations and cell vacuoles has decreased significantly. The cell morphology in the sixth group is close to normal cells, with a few cell vacuoles, and the number of cell deformations has decreased significantly.

[0061] 3. Cell proliferation experiment

[0062] Take 50 mg of MTT powder and dilute it with PBS solution in the dark to a final concentration of 5 mg / mL. Add the sterilized PBS solution to the edge wells around the 96-well plate. Add 100 μL of the cell suspension of the target cells to each well. After the cells adhere, administer the drugs according to the six groups in the above-mentioned cell viability detection experiment procedure. After co-culturing for 24 h, add 20 μL of MTT solution to each well, co-culture for 4 h, then discard the supernatant. Subsequently, add 150 μL of dimethyl sulfoxide to each well, shake gently at low speed for 10 min, read the absorbance value of each well at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader and analyze the results. The results are as Figure 2 shown, where ** p < 0.01 vs. the first group, ## p < 0.01 vs. the second group, && p < 0.01 vs. the fourth group;

[0063] It can be seen from Figure 2 that compared with the first group, the treatment with LPS solution significantly reduced the cell viability (p < 0.01). After the treatment of the third group and the fourth, fifth, and sixth groups, that is, after adding dexamethasone solution or antibacterial solutions with different concentrations, the cell viability could be significantly up-regulated (p < 0.01), and with the increase in the concentration of the antibacterial solution, the effect of increasing the cell viability was more obvious. Among them, the cell viability of the fifth and sixth groups increased significantly compared with the fourth group, and the cell viability of the sixth group increased most significantly.

[0064] 4. Detection of NO level

[0065] Divide the wells in the 96-well plate into blank wells, control wells, and experimental wells. Add the chromogenic agent to all wells, add double-distilled water to the blank wells, add sodium nitrite standard solution to the control wells, and add the target cells to the experimental wells. Then, administer the drugs to the experimental wells according to the six groups in the above-mentioned cell viability detection experiment procedure. Use an enzyme-linked immunosorbent assay (ELISA) analyzer to measure the OD value of the wells. The results are Figure 3 shown;

[0066] According to Figure 3 it can be known that compared with the first group, the NO level in the second group increased significantly (p < 0.01). Compared with the second group, the third, fourth, fifth, and sixth groups could all significantly reduce the NO level (p < 0.01). Among them, the NO content in the fifth and sixth groups decreased significantly compared with the fourth group.

[0067] 5. Detection of factor content

[0068] Add the standard solutions of each factor to the standard well plate, and add the cell culture supernatant of the target cells to the wells respectively. Then, administer the drugs according to the methods of the six groups in the above cell viability detection experiment steps, place at 7°C for 30 min, centrifuge and discard the supernatant after washing with the washing solution, add 50 μL of the enzyme-labeled reagent to each well, place at 37°C for 30 min, centrifuge and discard the supernatant after washing with the washing solution, add the chromogenic solution to each well and develop color for 10 min at 37°C in the dark, then terminate the reaction, and detect the absorbance of each well with an enzyme-labeled instrument at a wavelength of 450 nm to obtain the detection results. The detection results are as Figure 4 shown, Figure 4 where a, b, and c in

[0069] respectively represent the content level diagrams of interleukin-1β, prostaglandin E2, and intercellular adhesion molecule-1; Figure 4 As can be seen from a, b, and c in

[0070] 6. Detection of the lysate of the target cells

[0071] Administer the drugs according to the methods of the six groups in the above cell viability detection experiment steps. After culturing for 1 day, remove the culture medium and lyse the target cells at low temperature. Then, centrifuge and retain the supernatant, and detect malondialdehyde, total superoxide dismutase, glutathione, and catalase in the supernatant. The detection results are as Figure 5 shown, Figure 5 where a, b, c, and d in

[0072] respectively represent the content level diagrams of malondialdehyde, total superoxide dismutase, glutathione, and catalase; Figure 5As can be seen from a, b, c, and d in [the relevant content], the induction of the LPS solution significantly up-regulated the level of malondialdehyde in the lysate, and significantly decreased the contents of total superoxide dismutase, glutathione, and catalase (p<0.01). Compared with the second group, there were no significant differences in the changes of malondialdehyde, total superoxide dismutase, glutathione, and catalase in the fourth group (p>0.05). However, the content of malondialdehyde in the third, fifth, and sixth groups was significantly down-regulated, and the contents of total superoxide dismutase, glutathione, and catalase were significantly up-regulated (p<0.05 or p<0.01). Moreover, compared with the fourth group, there were obvious statistical differences in the changes of malondialdehyde, total superoxide dismutase contents in the fifth group, and malondialdehyde, total superoxide dismutase, glutathione, and catalase in the sixth group.

[0073] 7. Detection of the levels of each protein

[0074] Administer drugs in the same way as the six groups in the above-mentioned cell viability detection experiment steps. After culturing for 1 day, remove the culture medium, perform low-temperature lysis on the target cells, scrape off the protein and centrifuge, take the supernatant, add buffer, boil and denature, add polyacrylamide gel electrophoresis, transfer the membrane at a constant voltage of 110V, block with 5% skim milk at room temperature for 2 h, incubate with the corresponding primary antibody overnight at 4°C, incubate with the secondary antibody at room temperature for 2 h, after washing, add ECL for development and exposure, and then use professional software to count the results. The results are as Figure 6 , Figure 7 shown. Figure 6 a, b, c, and d in [the relevant content] respectively represent the expression level diagram of monocyte chemoattractant protein-1, the expression level diagram of transcription factor E2-related factor 2, the expression level diagram of heme oxygenase-1, and the phosphorylation level diagram of extracellular regulated protein kinase. Figure 7 a, b, and c in [the relevant content] respectively represent the phosphorylation level diagrams of c-Jun N-terminal kinase, P38 protein, and p65 protein.

[0075] From Figure 6 a in [the relevant content], it can be seen that for monocyte chemoattractant protein-1, compared with the first group, the content of monocyte chemoattractant protein-1 in the second group increased more significantly (p<0.01). The third, fourth, fifth, and sixth groups could all significantly reduce the expression level of monocyte chemoattractant protein-1 (p<0.01), and the decrease in the expression level of monocyte chemoattractant protein-1 in the fifth and sixth groups was more significant than that in the fourth group (p<0.01).

[0076] From Figure 6From b in [reference], it can be seen that for nuclear factor erythroid 2-related factor 2, compared with the first group, the expression level of nuclear factor erythroid 2-related factor 2 in the second group of cells was significantly up-regulated (p<0.01). The expression levels of nuclear factor erythroid 2-related factor 2 in the third, fourth, fifth, and sixth groups showed significant increases to varying degrees (p<0.01), and the increases in the expression levels of nuclear factor erythroid 2-related factor 2 in the fifth and sixth groups were significant compared with the fourth group;

[0077] From Figure 6 From c in [reference], it can be seen that for heme oxygenase-1, compared with the first group, the expression level of heme oxygenase-1 in the second group of cells was significantly up-regulated (p<0.01). The expression levels of heme oxygenase-1 in the third, fourth, fifth, and sixth groups showed significant increases to varying degrees (p<0.01), and the increases in the expression levels of heme oxygenase-1 in the fifth and sixth groups were significant compared with the fourth group;

[0078] From Figure 6 From d in [reference] and Figure 7 From a and b in [reference], it can be seen that for extracellular regulated protein kinases, c-Jun N-terminal kinases, and P38 protein, compared with the first group, the increase in the phosphorylation levels of extracellular regulated protein kinases, c-Jun N-terminal kinases, and P38 protein in the second group of cells was significant (p<0.05 or p<0.01). The phosphorylation levels of extracellular regulated protein kinases, c-Jun N-terminal kinases, and P38 protein in the third, fourth, fifth, and sixth groups of cells showed significant decreases to varying degrees (p<0.01), and the phosphorylation levels of extracellular regulated protein kinases, c-Jun N-terminal kinases, and P38 protein in the fifth and sixth groups of cells were significantly lower than those in the fourth group;

[0079] From Figure 7 From c in [reference], it can be seen that for p65 protein, compared with the first group, the phosphorylation level of p65 protein in the second group increased significantly. Compared with the second group, the phosphorylation levels of p65 protein in the third, fourth, fifth, and sixth groups decreased significantly. Compared with the fourth group, the phosphorylation level of p65 protein in the fifth group was significantly down-regulated, and the phosphorylation level of p65 protein in the sixth group was significantly down-regulated.

[0080] 8. Detection of ROS expression level

[0081] Administer drugs according to the six groups in the above-mentioned cell viability detection experimental steps. After culturing for 1 day, remove the culture medium. Then, add 2′,7′-dichlorodihydrofluorescein diacetate to each well and incubate at 37°C for 1 h. Wash the cells and add PBS solution, and count the ROS expression rate to obtain the results. The results are as Figure 8 shown;

[0082] From Figure 8It can be seen that, compared with the first group, after LPS stimulation, the ROS content in the second group was significantly up-regulated (p<0.01), while the ROS contents in the third, fourth, fifth, and sixth groups were significantly decreased (p<0.01). Compared with the fourth group, the fifth and sixth groups could significantly inhibit the overexpression of ROS.

[0083] Therefore, it can be known that the lactic acid bacteria antibacterial composition provided by the present application can mediate the expression of inflammatory mediators and inflammatory signaling pathway proteins and oxidative stress damage in a dose-dependent manner, and relieve the LPS-induced gynecological inflammatory response.

[0084] II. Mouse experiment

[0085] 1. Mouse grouping

[0086] Forty-eight female mice were taken. During the experiment, 36 of the female mice were subcutaneously injected with 0.1 mL of 0.2 mg / m estradiol valerate every two days to induce pseudopregnancy in the mice. After 6 days of pseudopregnancy, 10 μL of Candida albicans suspension with a concentration of 5.0x10 6 CFU / mL was inoculated intravaginally for 5 consecutive days. After inoculation, the mice were tilted backward for 5 minutes to ensure that the suspension remained in the vagina without flowing out. During the whole experiment, 12 other mice were subcutaneously injected with 0.1 mL of 0.9% normal saline every two days, and 0.9% normal saline was continuously inoculated intravaginally for 5 days. In this experiment, the 48 mice were divided into four groups. The administration process of the first mouse group was: inoculating normal saline for 5 days and treating with 2.5% hydroxypropyl methylcellulose 5 days later; the administration process of the second mouse group was: inoculating Candida albicans suspension for 5 days and treating with 2.5% hydroxypropyl methylcellulose 5 days later; the administration process of the third mouse group was: inoculating Candida albicans suspension for 5 days and treating with 20 mg / kg miconazole nitrate and 2.5% hydroxypropyl methylcellulose 5 days later; the administration process of the fourth mouse group was: inoculating Candida albicans suspension for 5 days and treating with high-concentration antibacterial liquid and 2.5% hydroxypropyl methylcellulose 5 days later.

[0087] 2. Mouse body weight detection

[0088] During the administration period, the body weight of the mice was recorded every 5 days. The difference in body weight between the first mouse group and the second mouse group was not significant, indicating that Candida albicans did not affect the body weight of the mice. The difference in body weight change between the third mouse group and the first mouse group was not significant, indicating that the high-concentration antibacterial liquid had no obvious side effects on the mice, and the organ indices of the kidneys and spleens of the mice did not increase or decrease significantly, and no organ edema or hypertrophy was observed, proving that the administration of the high-concentration antibacterial liquid did not affect the organs of the mice.

[0089] 3. Candida albicans detection

[0090] An equal number of mice were selected from each group of mice. After administration for a certain period of time, the administration was stopped, and lavage was performed on the 5th and 7th days after the cessation of administration. The vaginas of the mice in each group were lavaged with PBS solution. The lavage fluids of the same group were mixed and diluted with PBS solution. A certain amount of the diluted solution was cultured using Sabouraud dextrose agar medium to obtain the results, as shown in Figure 9 shown;

[0091] From Figure 9 it can be seen that compared with the first group of mice, the high-concentration antibacterial solution in the fourth group of mice effectively inhibited the reproduction of Candida albicans 5 days after the cessation of administration (P<0.05). The inhibitory effect on Candida albicans was still significant 9 days after the cessation of administration, and there was no rebound growth.

[0092] 4. Detection of Lactobacillus

[0093] The diluted solution was taken and placed in a culture dish for cultivation. After the Lactobacillus grew sufficiently, the number of colonies was calculated to obtain the results, as shown in Figure 10 shown;

[0094] From Figure 10 it can be seen that the number of Lactobacillus colonies in the lavage fluid of the second group of mice decreased, the number of colonies in the fourth group of mice increased significantly compared with the second group, and the number of colonies in the third group of mice decreased compared with the second group of mice. This indicates that miconazole nitrate can inhibit the growth of Lactobacillus. The number of Lactobacillus in the second group of mice decreased significantly compared with the first group of mice, indicating that Candida albicans can damage the microenvironment in the vagina, resulting in a decrease in the content of Lactobacillus. The content of Lactobacillus in the vaginas of the mice in the third group was lower than that in the second group, indicating that miconazole nitrate may have a certain degree of side effect on Lactobacillus while inhibiting the growth of Candida albicans. The number of Lactobacillus in the fourth group was the largest, indicating that the antibacterial solution does not inhibit Lactobacillus while inhibiting the growth of Candida albicans in the vaginas of mice and protects the microenvironment in the vagina during the treatment process.

[0095] 5. Pathological observation

[0096] The vaginal tissues of the mice in each group were collected and observed by pathological section staining. The results are shown in Figure 11 shown, Figure 11 The magnification of Figure 11 is 200. From

[0097] it can be seen that a large number of inflammatory cells such as neutrophils were infiltrated under the vaginal epithelium of the mice in the first and second groups of mice. The inflammatory infiltration was reduced under the vaginal epithelium in the fourth group of mice. Therefore, the antibacterial solution can inhibit inflammatory strains and relieve vaginal inflammation.

[0098] 1. Bacterial and fungal culture

[0099] Candida albicans, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were cultured using their respective culture media. After culturing, PBS solution was added to the cultured strains as the control group, and a high-concentration antibacterial solution was added as the experimental group. Then, the mixture was taken out, centrifuged, and filtered. The absorbance at 600 nm was measured using a Synergy HT multimode microplate reader. The results are as Figure 12 shown, Figure 12 In , a, b, c, and d represent the growth curves of Candida albicans, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, respectively;

[0100] From Figure 12 a, b, c, and d in , it can be seen that compared with the control group, the number of the four types of bacteria after treatment in the experimental group decreased significantly. As time increased, after 9 h, the growth of the control group entered the stationary phase and the growth curve tended to be flat, indicating that the bacterial concentration reached the peak. The high-concentration antibacterial solution almost completely inhibited the growth of Escherichia coli and Pseudomonas aeruginosa. After 20 h of culture, the growth curve of the control group tended to be flat, indicating that the bacterial concentration reached the peak. The inhibitory effect of the high-concentration antibacterial solution on Candida albicans and Staphylococcus aureus was significant. Therefore, the high-concentration antibacterial solution has an obvious inhibitory effect on Candida albicans, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0101] 2. Electron microscopy analysis

[0102] The suspensions of the control group and the experimental group were taken respectively. The precipitates were collected by washing with PBS solution, dehydrated step by step, and analyzed by SEM. The results are as Figure 13 shown, Figure 13 The magnification of is 10,000. From Figure 13 it can be seen that the Candida albicans and [not clear what is missing here] treated with PBS in the control group were smooth and plump spheres with complete cell morphology. The surface of the strains in the experimental group was wrinkled and discontinuous, and showed irregular or irregular protrusion structures. For Escherichia coli and Pseudomonas aeruginosa, compared with the control group, the cell surface in the experimental group showed obvious damage, with cell wall adhesion forming irregular bacterial clusters, and there were uneven spots and curvatures. Some visible cell debris could be observed in Escherichia coli and Staphylococcus aureus in the experimental group, and there were many vesicular structures outside the membrane. Therefore, the high-concentration antibacterial solution inhibited the growth of the strains and caused certain damage to the integrity of the cell wall.

[0103] 3. Content determination of extracellular β-galactosidase and ion detection

[0104] Take the suspensions of the control group and the experimental group, add them to 400 μL of 0.05 mol / L o-nitrophenyl-β-D-galactopyranoside, and then incubate the mixture in a 37 °C water bath for 40 minutes. After mixing in 500 μL of 0.5 mol / L sodium bicarbonate for 1 minute, measure the absorbance at 420 nm using an enzyme-linked immunosorbent assay (ELISA) reader. At the same time, extract the proteins of each fungus and bacterium in the suspension and detect the enzyme activity in the proteins. The results are as Figure 14 shown, Figure 14 where a, b, and c in + / K + -ATPase, Ca 2+ -ATPase activity level diagrams of four strains;

[0105] From Figure 14 a in

[0106] it can be seen that compared with the control group, the treatment of the experimental group for 4 h led to an increase in the extracellular β-galactosidase activity of Candida albicans, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa by 28.3%, 7.6%, 8.1%, and 7.1% respectively, indicating that the high-concentration antibacterial solution can cause the leakage of intracellular enzymes by enhancing the cell membrane permeability; Figure 14 From + / K + -ATPase activity decreased by 88.3% in Candida albicans, 16.9% in Escherichia coli, 64.7% in Staphylococcus aureus, and 51.3% in Pseudomonas aeruginosa compared with the control group. Compared with the control group, the Ca 2+ -ATPase activity decreased by 73.5% in Candida albicans, 65.5% in Escherichia coli, 36.9% in Staphylococcus aureus, and 22.7% in Pseudomonas aeruginosa, indicating that the high-concentration antibacterial solution can inhibit the growth of bacteria and fungi by inhibiting the activity of transport enzymes on the cell membrane.

[0107] 4. Detection of intracellular enzyme activity

[0108] Extract the proteins of each fungus and bacterium in the suspension and detect the extracellular protease activity. The results are as Figure 15 shown, Figure 15 where a, b, and c in

[0109] From Figure 15As can be seen from a in the figure, compared with the control group, the adenosine triphosphatase activities in the experimental group decreased by 100.0%, 39.4%, 93.0% and 87.1% in Candida albicans, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa respectively, indicating that the high-concentration bacteriostatic solution reduced the ability of adenosine triphosphatase to hydrolyze ATP, thereby reducing the energy metabolism of microorganisms and promoting cell death;

[0110] From Figure 15 As can be seen from b and c in the figure, compared with the control group, the treatment in the experimental group led to a 78.4% decrease in malate dehydrogenase activity in Candida albicans, a 76.6% decrease in Escherichia coli, an 89.4% decrease in Staphylococcus aureus, and a 50.2% decrease in Pseudomonas aeruginosa. From Figure 15 As can be seen from the figure, compared with the control group, the experimental group inhibited 21.1% of the succinate dehydrogenase activity in Candida albicans, 48.6% in Escherichia coli, 25.1% in Staphylococcus aureus, and 70.7% in Pseudomonas aeruginosa, indicating that the high-concentration bacteriostatic solution disrupted the energy metabolism of microorganisms, especially the tricarboxylic acid cycle.

[0111] IV. Component Experiment

[0112] Meanwhile, in order to further illustrate the effectiveness of the prescription provided in Example 1 of the present invention, the following experimental groups and control group are provided:

[0113] Experimental Group 1: The prescription provided by Experimental Group 1 is exactly the same as the prescription provided by Example 1;

[0114] Experimental Group 2: The prescription provided by Experimental Group 2 is roughly the same as the prescription provided by Example 1, except that the lactic acid bacteria powder in Experimental Group 2 is 15 parts;

[0115] Experimental Group 3: The prescription provided by Experimental Group 3 is roughly the same as the prescription provided by Example 1, except that the lactic acid bacteria powder in Experimental Group 3 is 20 parts;

[0116] Experimental Group 4: The prescription provided by Experimental Group 4 is roughly the same as the prescription provided by Example 1, except that the gastrodia extract in Experimental Group 4 is 10 parts;

[0117] Experimental Group 5: The prescription provided by Experimental Group 5 is roughly the same as the prescription provided by Example 1, except that the gastrodia extract in Experimental Group 5 is 15 parts;

[0118] Experimental Group 6: The prescription provided by Experimental Group 6 is roughly the same as the prescription provided by Example 1, except that the dryobalanops aromatica in Experimental Group 6 is 4 parts;

[0119] Test Group 7: The prescription provided by Test Group 7 is roughly the same as the prescription provided in Example 1, except that the amount of Dryobalanops aromatica in Test Group 7 is 8 parts;

[0120] Test Group 8: The prescription provided by Test Group 8 is roughly the same as the prescription provided in Example 1, except that the amount of Cnidium monnieri extract in Test Group 8 is 1 part;

[0121] Test Group 9: The prescription provided by Test Group 9 is roughly the same as the prescription provided in Example 1, except that the amount of Cnidium monnieri extract in Test Group 9 is 3 parts;

[0122] Test Group 10: The prescription provided by Test Group 10 is roughly the same as the prescription provided in Example 1, except that the amount of lactic acid bacteria powder in Test Group 10 is 15 parts, the amount of Gastrodia elata extract is 10 parts, the amount of Dryobalanops aromatica is 4 parts, and the amount of Cnidium monnieri extract is 1 part;

[0123] Test Group 11: The prescription provided by Test Group 11 is roughly the same as the prescription provided in Example 1, except that the amount of lactic acid bacteria powder in Test Group 11 is 20 parts, the amount of Gastrodia elata extract is 15 parts, the amount of Dryobalanops aromatica is 8 parts, and the amount of Cnidium monnieri extract is 3 parts;

[0124] Control Group 1: The prescription provided by Control Group 1 is roughly the same as the prescription provided in Example 1, except that Control Group 1 does not contain lactic acid bacteria powder;

[0125] Control Group 2: The prescription provided by Control Group 2 is roughly the same as the prescription provided in Example 1, except that Control Group 2 does not contain Gastrodia elata extract;

[0126] Control Group 3: The prescription provided by Control Group 3 is roughly the same as the prescription provided in Example 1, except that Control Group 3 does not contain Dryobalanops aromatica;

[0127] Control Group 4: The prescription provided by Control Group 4 is roughly the same as the prescription provided in Example 1, except that Control Group 4 does not contain Cnidium monnieri extract;

[0128] Control Group 5: The prescription provided by Control Group 5 is roughly the same as the prescription provided in Example 1, except that Control Group 5 does not contain lactic acid bacteria powder and Gastrodia elata extract;

[0129] Control Group 6: The prescription provided by Control Group 6 is roughly the same as the prescription provided in Example 1, except that Control Group 6 does not contain Dryobalanops aromatica and Cnidium monnieri extract;

[0130] Control Group 7: The prescription provided by Control Group 7 is roughly the same as the prescription provided in Example 1, except that Control Group 7 does not contain Mentha haplocalyx Briq. extract and Lonicera japonica Thunb. extract;

[0131] Control group 8: The prescription provided by control group 8 is substantially the same as the prescription provided in Example 1, except that control group 8 does not contain lactic acid bacteria powder, gastrodia extract, dryobalanops aromatica, and cnidium monnieri extract;

[0132] Specifically, the above experimental groups and control groups were respectively prepared into corresponding finished products, and they were used on candida albicans for a cell bacteriostatic experiment. The experimental process is as follows: The above experimental groups and control groups were prepared into corresponding samples. Take candida albicans, put it into a culture dish and use Sabouraud dextrose agar medium to carry out activation culture at 28 °C until candida albicans covers 80% of the entire culture dish area. Then take a certain amount of cultured candida albicans and put it into a container containing a certain amount of sterile sodium chloride solution, and dilute it to a candida albicans suspension with a concentration of 5.0x10 6 CFU / mL. The samples prepared from the above experimental groups and control groups were incorporated into PBS buffer to prepare corresponding high-concentration bacteriostatic solutions. Then, 20 μL of the high-concentration bacteriostatic solutions corresponding to each group were pipetted and placed on a plate containing the candida albicans suspension, evenly spread with a bacterium spreading rod and left to stand for 15 min. The culture medium plate was inverted and cultured in an incubator at 28 °C for 1 d. Then, colony counting was performed on each group, and the corresponding bacteriostatic results were determined according to the number of surviving colonies and the number of colonies before adding the bacteriostatic solution. The experimental results are shown in Table 1 below:

[0133] Table 1: Results of cell bacteriostatic test

[0134]

[0135] According to the above experimental results, the highest antibacterial rate of the prescription provided in Example 1 of the present application against Candida albicans is 91%. From the perspective of experimental groups 2-9, control groups 5, 6, and 8, an increase or decrease in the content of each component in the lactic acid bacteria powder, gastrodia extract, dryobalanops aromatica, and cnidium monnieri extract will affect the antibacterial rate. At the same time, it can be seen that the effect of the content change of lactic acid bacteria powder and gastrodia extract on the antibacterial rate is greater than that of the content change of dryobalanops aromatica and cnidium monnieri extract on the antibacterial rate. This is because lactic acid bacteria powder can adjust the vaginal environment and effectively inhibit the growth of inflammatory bacteria, and gastrodia extract can effectively damage the cell structure of bacteria and interfere with their energy metabolism to achieve the purpose of antibacterial. Moreover, dryobalanops aromatica is mainly used to activate the anti-inflammatory pathway and inhibit the protein expression of bacteria, and cnidium monnieri extract is mainly used to inhibit the level of inflammatory mediators and oxidative stress, so as to exert an anti-inflammatory effect. At the same time, if the content of lactic acid bacteria powder, gastrodia extract, dryobalanops aromatica, and cnidium monnieri extract is too small, the best antibacterial effect cannot be achieved. If the content of lactic acid bacteria powder, gastrodia extract, dryobalanops aromatica, and cnidium monnieri extract is too large, it will affect the vaginal environment and then affect the antibacterial rate. From the perspective of experimental groups 10-11, control groups 1, 2, 3, and 4, there is a synergistic effect among lactic acid bacteria powder, gastrodia extract, dryobalanops aromatica, and cnidium monnieri extract. Compared with the prescription of a single component, the mixture of lactic acid bacteria powder, gastrodia extract, dryobalanops aromatica, and cnidium monnieri extract can improve the corresponding inhibitory effect. From the perspective of control group 7, the influence of mint extract and honeysuckle extract on the antibacterial effect is weakened compared with other control groups. This is because mint extract and honeysuckle extract do not have the corresponding antibacterial effect. The role of mint extract is to stimulate capillaries to promote the penetration of other drugs, and the role of honeysuckle extract is to use the heat-clearing and detoxifying effect of honeysuckle to achieve the effect of assisting anti-inflammatory and antibacterial. From the perspective of control group 8, its antibacterial rate is 31%, which is because of the antibacterial effect brought by polyhexamethylene biguanide hydrochloride itself;

[0136] In summary, the lactic acid bacteria antibacterial composition provided by the present invention can exert an anti-inflammatory effect in a dose-dependent manner within a certain concentration range. Its mechanism is to activate the transcription factor E2-related factor 2 and heme oxygenase-1 pathways, inhibit the inflammation-related pathways, reduce the content of NO and ROS, and at the same time inhibit the level of inflammatory mediators and oxidative stress, and mediate the protein expression of the transcription factor E2-related factor 2 and heme oxygenase-1 pathways, so as to exert an anti-inflammatory effect, and reduce the proliferation of Candida albicans and effectively inhibit its growth, reduce the inflammatory infiltration in the vagina of mice to relieve the symptoms of vulvar swelling and redness, have a protective effect on the vaginal flora, and help create a favorable vaginal environment, so that the level of lactobacilli returns to normal, that is, it can target and inhibit harmful pathogenic bacteria. This lactic acid bacteria antibacterial composition destroys the cell wall and cell membrane of harmful strains, resulting in serious changes in cell morphology and even fragmentation, and inhibits mitochondrial electron transport and oxidative respiration, resulting in ATP consumption, thus hindering the TCA cycle and energy metabolism, and then the antibacterial effect.

[0137] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims.

Claims

1. A lactic acid bacteria antibacterial composition, characterized in that, The lactic acid bacteria antibacterial composition comprises the following raw materials in parts by weight: 15-20 parts of lactic acid bacteria powder, 10-15 parts of gastrodia extract, 4-8 parts of dryobalanops aromatica, 5-10 parts of mint extract, 2-5 parts of honeysuckle extract, 1-3 parts of cnidium monnieri extract, 0.8-1 part of polyhexamethylene biguanide hydrochloride, 10-22 parts of stabilizer, 10-20 parts of auxiliary agent, and the auxiliary agent comprises 1-3 parts of glidant, 1-3 parts of anticaking agent, 3-6 parts of diluent, and 5-8 parts of binder.

2. The lactic acid bacteria antibacterial composition according to claim 1, wherein The lactic acid bacteria antibacterial composition comprises the following raw materials in parts by weight: 18 parts of lactic acid bacteria powder, 12 parts of gastrodia extract, 5.5 parts of dryobalanops aromatica, 6 parts of mint extract, 3 parts of honeysuckle extract, 1.5 parts of cnidium monnieri extract, 1 part of polyhexamethylene biguanide hydrochloride, 19 parts of stabilizer, and 15 parts of auxiliary agent.

3. The lactic acid bacteria antibacterial composition according to claim 1, characterized in that, The stabilizer comprises 8-16 parts of sorbitol, 1-3 parts of fructooligosaccharide, and 1-3 parts of silicon dioxide.

4. The lactic acid bacteria antibacterial composition according to claim 1, characterized in that, The glidant is magnesium stearate, the anticaking agent is microcrystalline cellulose, the diluent is mannitol, and the binder is ethanol.

5. A lactic acid bacteria antibacterial tablet, which comprises the lactic acid bacteria antibacterial composition as described in any one of claims 1-3 and excipients.

6. The lactic acid bacteria antibacterial tablet according to claim 5, wherein The excipients comprise tartaric acid and sodium bicarbonate.

7. Use of the lactic acid bacteria antibacterial composition as described in any one of claims 1-3 in the preparation of a drug for inhibiting gynecological inflammation.

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