Lactic acid bacteria bacteriostatic composition, bacteriostatic tablet and application

Abstract

The invention discloses a lactic acid bacteria bacteriostatic composition, a bacteriostatic tablet and application. The lactic acid bacteria bacteriostatic composition is prepared from the following raw materials in parts by weight: 15 to 20 parts of lactic acid bacteria powder, 10 to 15 parts of rhizoma gastrodiae extract, 4 to 8 parts of kapur, 5 to 10 parts of herba menthae extract, 2 to 5 parts of honeysuckle flower extract, 1 to 3 parts of fructus cnidii extract, 0.8 to 1 part of polyhexamethylene biguanide hydrochloride, 10 to 22 parts of stabilizer and 10 to 20 parts of additive. The lactobacillus bacteriostatic composition provided by the invention has a strong bacteriostatic effect, adjusts the vaginal environment, damages cell membrane and cell wall structures, interferes energy metabolism, effectively inhibits the growth of bacteria and fungi, and can dose-dependently mediate the inflammatory mediator level and the expression of signaling pathway protein so as to achieve the purposes of bacteriostasis and inflammation diminishing.

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 the 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 inflammatory disease, 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, has 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. For 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 antagonistic or bactericidal effects on pathogenic bacteria, and lactic acid bacteria can produce H 2 O 2 under specific conditions. When H 2 O 2When superoxide anions are present, destructive hydroxyl free radicals are formed. This process can cause the oxidation of lipids on the cell membrane, thereby increasing the permeability of the cell membrane and achieving the purpose of sterilization. However, for simple 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 produce drug resistance. Summary of the Invention In view of the problems of the prior art, the present invention provides a lactic acid bacteria antibacterial composition, antibacterial tablets and applications. The lactic acid bacteria antibacterial composition has a strong antibacterial effect, 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, effectively inhibit the growth of bacteria and fungi, and at the same time 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.

[0006] A lactic acid bacteria antibacterial composition, 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.

[0007] According to the lactic acid bacteria antibacterial composition provided by the present invention, the lactic acid bacteria antibacterial composition has a strong antibacterial effect, 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, effectively inhibit the growth of bacteria and fungi, and at the same time 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.

[0008] In addition, according to the lactic acid bacteria antibacterial composition provided by the present invention, the following additional technical features may also be included: 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 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, 15 parts of auxiliary agent.

[0009] Preferably, the stabilizer comprises 8-16 parts of sorbitol, 1-3 parts of fructooligosaccharide, 1-3 parts of silicon dioxide.

[0010] Preferably, the adjuvant includes 1-3 parts of glidant, 1-3 parts of anticaking agent, 3-6 parts of diluent, and 5-8 parts of binder.

[0011] Preferably, the glidant is magnesium stearate, the anticaking agent is microcrystalline cellulose, the diluent is mannitol, and the binder is ethanol.

[0012] The present invention also provides a lactic acid bacteria antibacterial tablet, which includes the above lactic acid bacteria antibacterial composition and excipients.

[0013] In addition, the lactic acid bacteria antibacterial tablet provided by the present invention may further have the following additional technical features: Preferably, the excipients include tartaric acid and sodium bicarbonate.

[0014] The present invention also provides an application of the above lactic acid bacteria antibacterial composition in the preparation of drugs for inhibiting gynecological inflammation.

[0015] The additional aspects and advantages of the present invention will be partially given in the following description, partially become obvious from the following description, or be understood through the practice of the present invention. Description of the Drawings

[0016] 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, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 Morphological diagrams of target cells of the first to sixth groups provided in the embodiments of the present invention; Figure 2 Proliferation diagrams of target cells of the first to sixth groups provided in the embodiments of the present invention; Figure 3 NO level diagrams of the first to sixth groups provided in the embodiments of the present invention; Figure 4 Content level diagrams of interleukin-1β, prostaglandin E 2 and intercellular adhesion molecule-1 of the first to sixth groups provided in the embodiments of the present invention; Figure 5 Content level diagrams of malondialdehyde, total superoxide dismutase, glutathione, and catalase of the first to sixth groups provided in the embodiments of the present invention; Figure 6 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 in the embodiments of the present invention; Figure 7 Phosphorylation level diagrams of c-Jun N-terminal kinase, P38 protein, and p65 protein for the first to sixth groups provided in the embodiments of the present invention; Figure 8 ROS content diagrams for the first to sixth groups provided in the embodiments of the present invention; Figure 9 Inhibitory effect diagrams of the first to fourth mouse groups on Candida albicans provided in the embodiments of the present invention; Figure 10 Lactobacillus colony diagrams of the first to fourth mouse groups provided in the embodiments of the present invention; Figure 11 Mouse pathological section diagrams of the first to fourth mouse groups provided in the embodiments of the present invention; Figure 12 Growth curve diagrams of the experimental group and the control group corresponding to four strains respectively provided in the embodiments of the present invention; Figure 13 Scanning electron microscope diagrams of the experimental group and the control group corresponding to four strains respectively provided in the embodiments of the present invention; Figure 14 Extracellular β-galactosidase, Na + / K + -ATPase, Ca 2+ -ATPase activity level diagrams of the experimental group and the control group corresponding to four strains respectively provided in the embodiments of the present invention; Figure 15 Adenosine triphosphatase, malate dehydrogenase, and succinate dehydrogenase activity level diagrams of the experimental group and the control group corresponding to four strains respectively provided in the embodiments of the present invention.

[0018] The embodiments of the present invention will be further described below with reference to the accompanying drawings. Detailed implementation manners

[0019] To make the objectives, features, and advantages of the present invention more apparent and understandable, the following will describe in detail the specific implementation manners of the present invention 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, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0020] Example 1 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 elata 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; 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 elata 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.

[0021] Among them, the lactic acid bacteria in the lactic acid bacteria powder are specifically lactobacilli; Among them, the extraction process of the gastrodia elata extract is as follows: Select a certain amount of dry gastrodia elata, dry it at 60°C for 2 h and then crush it. Under the condition of constant temperature at 80°C, use 0.8% NaCl aqueous solution to extract for 2 h, filter to obtain the supernatant, add papain to the collected supernatant, then put the supernatant into a water bath at 60°C, heat it in a water bath for 30 min, heat it at 100°C for 40 min to inactivate papain and terminate hydrolysis, filter with a 10 kD ultrafiltration membrane and concentrate the sample with a rotary evaporator to obtain the gastrodia elata extract.

[0022] Among them, the extraction process of the mint extract is as follows: Dissolve mint with an equal amount of absolute ethanol solution, continuously stir at a constant temperature of 40°C in a magnetic stirrer for 75 min, place it at 4°C for 24 h and then filter by suction, and 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; 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 1 h each time. After each extraction, quickly filter and combine the filtrates, filter by suction, concentrate, and dry in a vacuum oven at 60°C to obtain the honeysuckle extract.

[0023] 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 1 h, repeat 2 times. After each extraction, quickly filter and combine the two filtrates, filter by suction, concentrate, and dry in a vacuum oven at 60°C to obtain the cnidium monnieri extract.

[0024] In this example, the stabilizer comprises 8-16 parts of sorbitol, 1-3 parts of fructooligosaccharide, and 1-3 parts of silicon dioxide.

[0025] In this example, 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.

[0026] In this embodiment, the glidant is magnesium stearate, the anti-caking agent is microcrystalline cellulose, the diluent is mannitol, and the binder is ethanol.

[0027] Example 2 Embodiment 2 of the present invention provides a lactic acid bacteria antibacterial tablet, which comprises the lactic acid bacteria antibacterial composition as described in Embodiment 1 and excipients; In this embodiment, the excipients include tartaric acid and sodium bicarbonate; The preparation method of the lactic acid bacteria antibacterial tablet is as follows: First, divide the lactic acid bacteria antibacterial composition described in Embodiment 1 into two equal parts, then add 12 g of tartaric acid and 4 g of sodium bicarbonate to the two parts of raw materials respectively, stir to make two parts of soft materials respectively, then mix the two parts of soft materials, and use a rotary tablet press after mixing to obtain the lactic acid bacteria antibacterial tablet.

[0028] Example 3 Embodiment 3 of the present invention provides an application of a lactic acid bacteria antibacterial composition in the preparation of a drug for inhibiting gynecological inflammation.

[0029] 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 were conducted on them, and the experiments are as follows: I. Antibacterial ability experiment 1. Experimental materials 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 chemokines, etc., thereby promoting the occurrence of inflammatory reactions and aggravating inflammation. Therefore, 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 Embodiment 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 to prepare antibacterial solutions with different concentrations. 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 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) -1 mg / ml is recorded as the 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, the 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. Under the conditions of a temperature of 37 °C and CO2 Cultivate in an incubator with a concentration of 5%. Place a tray at the bottom layer of the incubator, which contains saturated copper sulfate solution, providing the humidity required for cell growth and also playing a bactericidal role. When the cell density reaches about 90%, digest with 0.25% trypsin at 37°C for 30 s, then gently pipette with a Pasteur pipette and passage at a ratio of 1:3 to obtain the target cells.

[0030] 2. Cell viability detection Evenly seed the target cells in a six-well plate (1×10 6 / well), and set six groups to facilitate the observation of the viability of the target cells. Add 4 ml of complete medium to the first group, add 2 ml of 1.2 μg / ml LPS solution and 2 ml of complete medium to the second group, add 2 ml of 1.2 μg / ml LPS solution and 2 ml of 0.26 mg / ml dexamethasone solution to the third group, add 2 ml of 1.2 μg / ml LPS solution and 2 ml of low-concentration antibacterial solution to the fourth group, add 2 ml of 1.2 μg / ml LPS solution and 2 ml of medium-concentration antibacterial solution to the fifth group, add 2 ml of 1.2 μg / ml LPS solution and 2 ml of high-concentration antibacterial solution to the sixth group. Culture the above groups for one day and observe the morphology of the target cells after culture, as specifically shown in Figure 1 the following; 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 pseudopodia. 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 pseudopodia, 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.

[0031] 3. Cell proliferation experiment 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 outside the 96-well plate, and add 100 μL of the cell suspension of the target cells to each well. After the cells adhere, administer drugs according to the six groups in the above cell viability detection experiment steps. After co-culturing for 24 h, add 20 μL of MTT solution to each well and 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, and read the absorbance value of each well at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader and analyze the results. The results are as shown in Figure 2as shown, where, ** p < 0.01 vs. the first group, ## p < 0.01 vs. the second group, && p < 0.01 vs. the fourth group; From Figure 2 it can be seen that compared with the first group, the treatment with LPS solution significantly reduced the cell survival rate (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 survival rate 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 survival rate was more obvious. Among them, the cell survival rates of the fifth and sixth groups were significantly higher than that of the fourth group, and the cell survival rate of the sixth group increased most significantly.

[0032] 4. Detection of NO level The wells in the 96-well plate were divided into blank wells, control wells, and experimental wells. Color reagent was added to all wells, double-distilled water was added to the blank wells, sodium nitrite standard solution was added to the control wells, and target cells were added to the experimental wells. Then, drugs were administered to the experimental wells according to the six groups in the above-mentioned cell viability detection experiment steps. The OD values of the wells were measured using an enzyme-linked immunosorbent assay analyzer, and the results Figure 3 are shown; According to Figure 3 it can be known that compared with the first group, the NO level in the second group was significantly increased (p < 0.01). Compared with the second group, the NO levels in the third, fourth, fifth, and sixth groups were all significantly decreased (p < 0.01). Among them, the NO contents in the fifth and sixth groups were significantly lower than that in the fourth group.

[0033] 5. Detection of factor content The standard solutions of each factor were added to the standard well plate, and the cell culture supernatants of the target cells were respectively added to the wells. Then, drugs were administered according to the six groups in the above-mentioned cell viability detection experiment steps, and the plate was placed at 7 °C for 30 min. After washing with the washing solution and centrifuging to dry, 50 μL of enzyme-labeled reagent was added to each well, and the plate was placed at 37 °C for 30 min. After washing with the washing solution and centrifuging to dry, the chromogenic solution was added to each well and the reaction was terminated after 10 min of color development in the dark at 37 °C. The absorbance of each well was detected using an enzyme-linked immunosorbent assay analyzer at a wavelength of 450 nm to obtain the detection results. The detection results are as Figure 4 shown, Figure 4 in which a, b, and c respectively represent the content level diagrams of interleukin-1β, prostaglandin E 2 and intercellular adhesion molecule-1; From Figure 4 a, b, and c in it can be seen that compared with the first group, the interleukin-1β and prostaglandin E in the second group 2and the levels of interleukin-1β and intercellular adhesion molecule-1 were significantly increased. Compared with the second group, the third, fourth, fifth, and sixth groups could significantly reduce the levels of interleukin-1β and prostaglandin E in the supernatant 2 and intercellular adhesion molecule-1 (p < 0.01). Among them, the content of interleukin-1β in the fifth and sixth groups was significantly lower than that in the fourth group (p < 0.01), and the levels of prostaglandin E 2 and intercellular adhesion molecule-1 in the sixth group were also significantly lower than those in the fourth group (p < 0.05 or p < 0.01).

[0034] 6. Detection of the lysate of target cells Administer drugs according to the methods of the six groups in the above-mentioned cell viability detection experiment steps. After culturing for 1 day, remove the culture medium and lyse the target cells at low temperature. Then centrifuge to 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 In a, b, c, and d in respectively represent the content level diagrams of malondialdehyde, total superoxide dismutase, glutathione, and catalase; Figure 5 It can be seen from a, b, c, and d in

[0035] 7. Detection of the levels of each protein Administer drugs according to the methods of the six groups in the above-mentioned cell viability detection experiment steps. After culturing for 1 day, remove the culture medium and lyse the target cells at low temperature. Scrape the protein and centrifuge it. Take the supernatant, add buffer and boil for denaturation, add polyacrylamide gel electrophoresis, transfer the membrane at a constant voltage of 110V, block it 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, wash it, 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 6In it, a, b, c, and d respectively represent the expression level graph of monocyte chemoattractant protein-1, the expression level graph of transcription factor E2-related factor 2, the expression level graph of heme oxygenase-1, and the phosphorylation level graph of extracellular regulated protein kinases, Figure 7 In it, a, b, and c respectively represent the phosphorylation level graphs of c-Jun N-terminal kinase, P38 protein, and p65 protein; From Figure 6 As can be seen from a in For monocyte chemoattractant protein-1, compared with the first group, the content of monocyte chemoattractant protein-1 in the second group increased significantly (p < 0.01). The third, fourth, fifth, and sixth groups could 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); Figure 6 As can be seen from b in For transcription factor E2-related factor 2, compared with the first group, the expression level of transcription factor E2-related factor 2 in the second group was significantly up-regulated (p < 0.01). The expression levels of transcription factor E2-related factor 2 in the third, fourth, fifth, and sixth groups increased significantly to varying degrees (p < 0.01), and the increase in the expression level of transcription factor E2-related factor 2 in the fifth and sixth groups was significant compared with that in the fourth group; Figure 6 As can be seen from c in For heme oxygenase-1, compared with the first group, the expression level of heme oxygenase-1 in the second group was significantly up-regulated (p < 0.01). The expression levels of heme oxygenase-1 in the third, fourth, fifth, and sixth groups increased significantly to varying degrees (p < 0.01), and the increase in the expression level of heme oxygenase-1 in the fifth and sixth groups was significant compared with that in the fourth group; Figure 6 As can be seen from d in Figure 7 and a, b in For extracellular regulated protein kinases, c-Jun N-terminal kinase, and P38 protein, compared with the first group, the phosphorylation levels of extracellular regulated protein kinases, c-Jun N-terminal kinase, and P38 protein in the second group increased significantly (p < 0.05 or p < 0.01). The phosphorylation degrees of extracellular regulated protein kinases, c-Jun N-terminal kinase, and P38 protein in the third, fourth, fifth, and sixth groups decreased to varying degrees (p < 0.01), and the phosphorylation levels of extracellular regulated protein kinases, c-Jun N-terminal kinase, and P38 protein in the fifth and sixth groups were significantly lower than those in the fourth group; Figure 7As can be seen from c in [context], for the p65 protein, compared with the first group, the phosphorylation level of the p65 protein in the second group increased significantly. Compared with the second group, the phosphorylation levels of the p65 protein in the third, fourth, fifth, and sixth groups decreased significantly. Compared with the fourth group, the phosphorylation level of the p65 protein in the fifth group was significantly down-regulated, and the phosphorylation level of the p65 protein in the sixth group was significantly down-regulated.

[0036] 8. Detection of ROS Expression Level Administer 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. Then, add 37℃ incubated 1h of dichlorodihydrofluorescein diacetate to each well, wash the cells and add PBS solution, and count the ROS expression rate to obtain the results, as shown in Figure 8 shown; From Figure 8 it 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.

[0037] Therefore, it can be known that the lactic acid bacteria antibacterial composition provided by this 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.

[0038] II. Mouse Experiment 1. Grouping of Mice Take 48 female mice. 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 uL of Candida albicans suspension with a concentration of 5.0x10 6 CFU / mL was intravaginally inoculated 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 intravaginally inoculated with 0.9% normal saline 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 after 5 days; the administration process of the second mouse group was: inoculating Candida albicans suspension for 5 days and treating with 2.5% hydroxypropyl methylcellulose after 5 days; 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 after 5 days; 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 after 5 days.

[0039] 2. Mouse body weight detection During the drug 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 does 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 bacteriostatic solution has no obvious side effects on the mice. Moreover, 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 bacteriostatic solution does not affect the organs of the mice.

[0040] 3. Candida albicans detection The same number of mice were selected from each group of mice. After a certain period of drug administration, the drug administration was stopped, and lavage was performed on the 5th and 7th days after the drug administration was stopped. The vagina of each group of mice was lavaged with PBS solution, and the lavage fluids of the same group were mixed and diluted with PBS solution. A certain amount of the diluted solution was taken and cultured using Sabouraud dextrose agar medium to obtain the results. The results are as Figure 9 shown; From Figure 9 it can be seen that compared with the first mouse group 5 days after the drug administration was stopped, the high-concentration bacteriostatic solution in the fourth mouse group effectively inhibited the reproduction of Candida albicans (P<0.05). The inhibitory effect on Candida albicans was still significant 9 days after the drug administration was stopped, and there was no rebound growth.

[0041] 4. Lactobacillus detection The diluted solution was taken and placed in a culture dish for culture. After the Lactobacillus grew sufficiently, the number of colonies was calculated to obtain the results. The results are as Figure 10 shown; From Figure 10 it can be seen that the number of Lactobacillus colonies in the lavage fluid of the second mouse group decreased, the number of colonies in the fourth mouse group increased significantly compared with the second group, and the number of colonies in the third mouse group decreased compared with the second mouse group, indicating that miconazole nitrate inhibits the growth of Lactobacillus. The number of Lactobacillus in the second mouse group decreased significantly compared with the first mouse group, indicating that Candida albicans can destroy the microenvironment in the vagina, resulting in a decrease in the content of Lactobacillus. The content of Lactobacillus in the vagina of the mice in the third mouse group was lower than that in the second mouse group, indicating that miconazole nitrate may have certain side effects on Lactobacillus while inhibiting the growth of Candida albicans. The number of Lactobacillus in the fourth mouse group was the largest, indicating that the bacteriostatic solution does not inhibit Lactobacillus while inhibiting the growth of Candida albicans in the vagina of the mice and protects the microenvironment in the vagina during the treatment process.

[0042] 5. Pathological observation The vaginal tissues of each group of mice were collected and observed by pathological section staining. The results are as Figure 11 shown,Figure 11 The magnification is 200. As can be seen from Figure 11 , a large number of inflammatory cells such as neutrophils can be seen infiltrating under the vaginal epithelium of mice in the first mouse group and the second mouse group. In the fourth mouse group, the inflammatory infiltration under the vaginal epithelium can be seen to decrease. Therefore, the bacteriostatic liquid can inhibit inflammatory strains and relieve vaginal inflammation.

[0043] III. Bacteriostatic mechanism experiment 1. Bacterial and fungal culture Take Candida albicans, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, and culture them using the corresponding culture media respectively. Add PBS solution to the cultured strains as the control group and add high-concentration bacteriostatic liquid as the experimental group. Then take out the mixture, centrifuge and filter it. Use a Synergy HT multi-detection microplate reader to detect its absorbance at 600 nm. The results are as Figure 12 shown. Figure 12 In , a, b, c, and d respectively represent the growth curves of Candida albicans, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa; As can be seen from Figure 12 a, b, c, and d in , compared with the control group, the numbers of the four kinds of bacteria after treatment in the experimental group are significantly reduced. As time goes by, after 9 h, the growth of the control group enters the stationary phase and the growth curve tends to be flat, indicating that the bacterial concentration reaches the peak. The high-concentration bacteriostatic liquid almost completely inhibits the growth of Escherichia coli and Pseudomonas aeruginosa. After 20 h of culture, the growth curve of the control group tends to be flat, indicating that the bacterial concentration reaches the peak. The inhibitory effect of the high-concentration bacteriostatic liquid on Candida albicans and Staphylococcus aureus is significant. Therefore, the high-concentration bacteriostatic liquid has an obvious inhibitory effect on Candida albicans, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.

[0044] 2. Electron microscopy analysis Take the suspensions of the control group and the experimental group respectively, wash and collect the precipitates with PBS solution, perform gradient dehydration on the precipitates and conduct SEM analysis. The results are as Figure 13 shown. Figure 13 The magnification is 10,000. As can be seen from Figure 13 , the Candida albicans and treated with PBS in the control group are smooth and plump spheres in shape, and the cell morphology is complete. The strains in the experimental group have wrinkled and discontinuous surfaces and show irregular or irregular protrusion structures. For Escherichia coli and Pseudomonas aeruginosa, compared with the control group, the cell surfaces in the experimental group show obvious damage, with cell wall adhesion to form irregular bacterial clusters, and there are uneven spots and bends. Some visible cell debris can be observed in Escherichia coli and Staphylococcus aureus in the experimental group, and there are many vesicular structures outside the membrane. Therefore, the high-concentration bacteriostatic liquid inhibits the growth of the strains and causes certain damage to the integrity of the cell wall.

[0045] 3. Extracellular β-galactosidase content and ion detection 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 water bath at 37 °C 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 In a, b, and c of + / K + -ATPase, Ca 2+ -ATPase activity level diagrams of four strains; As can be seen from Figure 14 a in , compared with the control group, the extracellular β-galactosidase activities of Candida albicans, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in the experimental group increased by 28.3%, 7.6%, 8.1%, and 7.1% respectively after 4 h of treatment, indicating that the high-concentration antibacterial liquid can enhance the cell membrane permeability and cause the leakage of intracellular enzymes; Figure 14 As can be seen from + / K + -ATPase activities 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 activities 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 liquid can inhibit the growth of bacteria and fungi by inhibiting the activity of transport enzymes on the cell membrane.

[0046] 4. Intracellular enzyme activity detection 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 In a, b, and c of As can be seen from Figure 15As can be seen from a in the figure, compared with the control group, the activities of adenosine triphosphatase 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; 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 resulted in a 78.4% decrease in the 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.

[0047] IV. Component Experiment 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 groups are provided: Experimental Group 1: The prescription provided in Experimental Group 1 is exactly the same as the prescription provided in Example 1; Experimental Group 2: The prescription provided in Experimental Group 2 is roughly the same as the prescription provided in Example 1, except that the lactic acid bacteria powder in Experimental Group 2 is 15 parts; Experimental Group 3: The prescription provided in Experimental Group 3 is roughly the same as the prescription provided in Example 1, except that the lactic acid bacteria powder in Experimental Group 3 is 20 parts; Experimental Group 4: The prescription provided in Experimental Group 4 is roughly the same as the prescription provided in Example 1, except that the gastrodia elata extract in Experimental Group 4 is 10 parts; Experimental Group 5: The prescription provided in Experimental Group 5 is roughly the same as the prescription provided in Example 1, except that the gastrodia elata extract in Experimental Group 5 is 15 parts; Experimental Group 6: The prescription provided in Experimental Group 6 is roughly the same as the prescription provided in Example 1, except that the dryobalanops aromatica in Experimental Group 6 is 4 parts; Experimental Group 7: The prescription provided in Experimental Group 7 is roughly the same as the prescription provided in Example 1, except that the dryobalanops aromatica in Experimental Group 7 is 8 parts; Experimental Group 8: The prescription provided by Experimental Group 8 is substantially the same as the prescription provided in Example 1, except that the cnidium fruit extract in Experimental Group 8 is 1 part; Experimental Group 9: The prescription provided by Experimental Group 9 is substantially the same as the prescription provided in Example 1, except that the cnidium fruit extract in Experimental Group 9 is 3 parts; Experimental Group 10: The prescription provided by Experimental Group 10 is substantially the same as the prescription provided in Example 1, except that in Experimental Group 10, the lactic acid bacteria powder is 15 parts, the gastrodia elata extract is 10 parts, the dryobalanops aromatica is 4 parts, and the cnidium fruit extract is 1 part; Experimental Group 11: The prescription provided by Experimental Group 11 is substantially the same as the prescription provided in Example 1, except that in Experimental Group 11, the lactic acid bacteria powder is 20 parts, the gastrodia elata extract is 15 parts, the dryobalanops aromatica is 8 parts, and the cnidium fruit extract is 3 parts; Control Group 1: The prescription provided by Control Group 1 is substantially the same as the prescription provided in Example 1, except that Control Group 1 does not contain lactic acid bacteria powder; Control Group 2: The prescription provided by Control Group 2 is substantially the same as the prescription provided in Example 1, except that Control Group 2 does not contain gastrodia elata extract; Control Group 3: The prescription provided by Control Group 3 is substantially the same as the prescription provided in Example 1, except that Control Group 3 does not contain dryobalanops aromatica; Control Group 4: The prescription provided by Control Group 4 is substantially the same as the prescription provided in Example 1, except that Control Group 4 does not contain cnidium fruit extract; Control Group 5: The prescription provided by Control Group 5 is substantially 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; Control Group 6: The prescription provided by Control Group 6 is substantially the same as the prescription provided in Example 1, except that Control Group 6 does not contain dryobalanops aromatica and cnidium fruit extract; Control Group 7: The prescription provided by Control Group 7 is substantially the same as the prescription provided in Example 1, except that Control Group 7 does not contain mentha haplocalyx extract and honeysuckle flower extract; 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 elata extract, dryobalanops aromatica, and cnidium fruit extract; Specifically, the above experimental group and control group were respectively prepared into corresponding finished products and applied to Candida albicans for a cell antibacterial experiment. The experimental process is as follows: The above experimental group and control group were prepared into corresponding samples. Candida albicans was taken and placed in a culture dish, and activated and cultured in an environment of 28 °C using Sabouraud dextrose agar medium until Candida albicans covered 80% of the entire culture dish area. Then, a certain amount of the cultured Candida albicans was taken and placed in a container containing a certain amount of sterile sodium chloride solution, and it was diluted to a Candida albicans suspension with a concentration of 5.0x10 6 CFU / mL by the 10-fold serial dilution method. The samples prepared from the above experimental group and control group were incorporated into PBS buffer to prepare corresponding high-concentration antibacterial solutions. Then, 20 μL of the high-concentration antibacterial solutions corresponding to each group was aspirated and placed in a plate containing the Candida albicans suspension, evenly spread with a bacterium spreading rod and left standing 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 antibacterial results were determined based on the number of surviving colonies and the number of colonies before adding the antibacterial solution. The experimental results are shown in Table 1 below: Table 1: Results of cell antibacterial test

[0048] 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 and 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 the lactic acid bacteria powder and gastrodia extract on the antibacterial rate is greater than the effect of the content change of dryobalanops aromatica and cnidium monnieri extract on the antibacterial rate. This is because the lactic acid bacteria powder can adjust the vaginal environment and effectively inhibit the growth of inflammatory pathogens. The gastrodia extract can effectively damage the cell structure of the pathogens and interfere with their energy metabolism to achieve the purpose of antibacterial. And dryobalanops aromatica is mainly used to activate the anti-inflammatory pathway and inhibit the protein expression of pathogens. The cnidium monnieri extract is mainly used to inhibit the levels of inflammatory mediators and oxidative stress, thereby exerting an anti-inflammatory effect. At the same time, if the content of the 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 the lactic acid bacteria powder, gastrodia extract, dryobalanops aromatica, and cnidium monnieri extract is too much, it will affect the vaginal environment and thus affect the antibacterial rate. From the perspective of experimental groups 10-11 and control groups 1, 2, 3, and 4, there is a synergistic effect among the lactic acid bacteria powder, gastrodia extract, dryobalanops aromatica, and cnidium monnieri extract. Compared with the prescription of a single component, the mixture of the 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 the mint extract and honeysuckle extract on the antibacterial effect is weakened compared with other control groups. This is because the mint extract and honeysuckle extract do not have the corresponding antibacterial effect. The role of the mint extract is to stimulate capillaries to promote the penetration of other drugs. The role of the honeysuckle extract is to utilize the heat-clearing and detoxifying effect of honeysuckle to achieve the effect of assisting in 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; 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 inflammation-related pathways, reduce the contents of NO and ROS, at the same time inhibit the levels of inflammatory mediators and oxidative stress, and mediate the protein expression of the transcription factor E2-related factor 2 and heme oxygenase-1 pathways, thereby exerting an anti-inflammatory effect, as well as reducing the proliferation of Candida albicans and effectively inhibiting its growth, reducing the inflammatory infiltration in the vagina of mice to relieve the symptoms of vulvar swelling and redness, having a protective effect on the vaginal flora, and helping to create a favorable vaginal environment to restore the normal level of lactobacilli, that is, it can target and inhibit harmful pathogenic bacteria. The lactic acid bacteria antibacterial composition destroys the cell walls and cell membranes of harmful strains, resulting in serious changes in cell morphology and even fragmentation, as well as inhibiting mitochondrial electron transport and oxidative respiration, leading to ATP consumption, thereby hindering the TCA cycle and energy metabolism, and further exerting an antibacterial effect.

[0049] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on 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 belong to 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 elata extract, 4-8 parts of borneol, 5-10 parts of peppermint extract, 2-5 parts of honeysuckle extract, 1-3 parts of cnidium monnieri extract, 0.8-1 parts of polyhexamethylene biguanide hydrochloride, 10-22 parts of stabilizer, and 10-20 parts of auxiliary agent.

2. The lactic acid bacteria antibacterial composition according to claim 1, characterized in that 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 borneol, 6 parts of peppermint 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 oligofructose and 1-3 parts of silicon dioxide.

4. The lactic acid bacteria antibacterial composition according to claim 1, characterized in that The auxiliary agent comprises 1 to 3 parts of a glidant, 1 to 3 parts of an anti-caking agent, 3 to 6 parts of a diluent, and 5 to 8 parts of a binder.

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

6. A lactic acid bacteria antibacterial tablet, comprising the lactic acid bacteria antibacterial composition according to any one of claims 1 to 3 and auxiliary materials.

7. The lactic acid bacteria antibacterial tablet according to claim 6, characterized in that: The auxiliary materials include tartaric acid and sodium bicarbonate.

8. Use of the lactic acid bacteria antibacterial composition according to any one of claims 1 to 3 in the preparation of a drug for inhibiting gynecological inflammation.

Images