Vagina flora repairing liquid and preparation method thereof

Abstract

The invention provides a vaginal flora repairing liquid and a preparation method thereof. Specifically, the repair liquid prepared from the lactobacillus paracasei CGMCC No. 1.12731, the lactobacillus rhamnosus CGMCC 24282 and the lactobacillus gasseri CGMCC No. 22909 in a specific mass ratio can be used for well killing pathogenic bacteria, has no obvious toxic or side effect and is low in cost. The broad-spectrum vaginitis bactericidal peptide YDG-C screened by the invention is further added into the repair liquid, so that the treatment effect on bacterial vaginitis can be further improved. The preparation method is simple, low in cost and suitable for large-scale popularization and application.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to a vaginal flora repair liquid and a preparation method thereof. Background Art

[0002] The female vagina is a complex microecological system, which is composed of the vaginal anatomical structure, microecological flora, local immunity and endocrine regulation function of the body. There are more than 50 kinds of microorganisms parasitizing in the vagina of healthy women, including: Lactobacillus, Bifidobacterium, Bacteroides, Enterococcus, Staphylococcus epidermidis, Streptococcus, Corynebacterium, Escherichia coli, Veillonella, Peptostreptococcus, Gardnerella, as well as protozoa, viruses, mycoplasma and Candida albicans other than bacteria, among which obligate and facultative anaerobes are the main ones. These microorganisms mainly live on the side wall mucosa around the vagina. They restrict and interact with each other, colonize in the vaginal mucosal epithelium in a hierarchical and orderly manner, and form a biofilm together. The vaginal bacteria parasitizing in the biofilm will continue to evolve with the changes in physiological state and local environment. Lactobacillus is dominant in the vagina of healthy women of childbearing age, which plays an important role in maintaining the health of the vaginal microecology and preventing the occurrence of vaginal infections.

[0003] Studies have shown that menstruation, vaginal douching, contraceptives, vaginal medication, number of sexual partners, frequency of sexual intercourse, and the way of using sanitary products during menstruation can affect the composition of vaginal flora. The structure of vaginal epithelial cells and the composition of vaginal flora change significantly from the neonatal period to postmenopause in women's lives. Newborns are affected by the estrogen in their mothers, and the vaginal epithelial cells are rich in glycogen. Therefore, within 24 hours of birth, the dominant bacteria colonizing the vagina of newborns are lactobacilli, which are mainly obtained from the mother's birth canal. After a few weeks, when the estrogen level in the baby's body decreases, the vaginal epithelium begins to become thin and atrophy due to the decrease in glycogen levels. At this time, Gram-positive cocci and other bacteria other than lactobacilli dominate, and this situation continues until puberty. During menstruation, other bacteria other than lactobacilli multiply in large numbers, while the number of lactobacilli decreases or basically remains the same. During pregnancy, due to the increase in estrogen levels, lactobacilli will occupy a dominant position in the vaginal flora. But at the same time, the incidence of vulvovaginal candidiasis is also increased compared to the non-pregnancy period. Some studies have suggested that cell-mediated immunity is suppressed during pregnancy for some reason, leading to susceptibility to pathogenic microorganisms, such as Candida albicans. During menopause, the decrease in estrogen levels leads to a decrease in the number of vaginal lactobacilli and an increase in vaginal pH. The composition of vaginal flora during menopause is similar to that during adolescence. The number of lactobacilli, candida, and bacteria associated with bacterial vaginosis is reduced compared to the reproductive period, while the number of Escherichia coli is dominant. Therefore, estrogen levels have a decisive influence on the composition of vaginal microbial flora. Antibiotic use can have an adverse effect on vaginal lactobacilli. Lactobacilli have different sensitivities to different cephalosporin antibiotics, but are very sensitive to penicillin. In contrast, vancomycin, doxycycline, and metronidazole have no significant inhibitory effect on lactobacilli. Clindamycin ointment used to treat bacterial vaginosis has an inhibitory effect on vaginal lactobacilli. There are currently conflicting reports on the effects of contraceptives on vaginal flora. Some studies have shown that the use of oral contraceptives or intrauterine devices does not significantly change the number of aerobic and anaerobic bacteria in the vagina. On the contrary, other studies have shown that the number of anaerobic bacteria in the vagina of women with intrauterine devices increases significantly. The results of culturing samples of removed intrauterine devices showed that coagulase-negative Staphylococci, Escherichia coli, and actinomycetes-like bacteria grew. The colonization of vaginal lactobacilli in postmenopausal women using hormone replacement is the same as that of women of childbearing age. Comparison of vaginal flora in women using tampons and sanitary napkins showed that the number of coagulase-negative Staphylococci in the vagina of women using tampons during menstruation increased. There are also many reports showing that vaginal douching increases the incidence of pelvic inflammatory disease, ectopic pregnancy, premature birth and other gynecological diseases.

[0004] Bacterial vaginosis (BV) is a disease of vaginal microbial flora imbalance. It is the most common type of vaginitis in women of childbearing age. Its incidence is related to factors such as age group. The incidence in my country ranges from 4.96% to 36.00%. The common characteristics of BV patients are the decrease of Lactobacillus bacteria and the increase of several pathogenic bacteria, most of which are strict anaerobes. At present, the main treatments for BV include oral or intravaginal metronidazole or clindamycin administration, but the recurrence rate of BV is very high. The recurrence rate is 40% after 3 months of traditional antibiotic treatment and increases to 60% after 6 months. The high recurrence rate may be due to the formation of multiple bacterial biofilms, which adhere to vaginal epithelial cells, resulting in the inability of antibiotics to completely kill pathogens. The main pathogens are G. vaginalis and A. vaginae. The high recurrence rate leads to the repeated use of antibiotics, which makes bacteria resistant. Studies have shown that vaginal pathogens, especially Trichomonas and anaerobic bacteria, are becoming more and more resistant. The emergence of resistant strains urgently requires new antibacterial agents. Summary of the invention

[0005] The inventors have found through research that when antibiotics are used to treat bacterial vaginitis, pathogens are quickly killed, but non-pathogenic bacteria are also killed at the same time, forming a sterile environment for a short time, which leads to a relative weakening of vaginal immunity and the ability to resist exogenous bacteria. The present invention uses specific probiotics combined with broad-spectrum bactericidal peptides to specifically kill pathogens without harming non-pathogenic bacteria. At the same time, the addition of probiotics can more effectively consolidate the therapeutic effect of bacterial vaginitis.

[0006] In one aspect, the present invention provides a broad-spectrum vaginitis bactericidal peptide YDG-C, whose amino acid sequence is shown in SEQ ID NO: 1.

[0007] Specifically, PFAAPKKKKRKRGXGKKDFLR, N-terminal acetylation and C-terminal amidation.

[0008] The polypeptide of the present invention is preferably the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80, 85, 90, 95, 96, 97, 98, 99, 99.5 or 100% amino acid sequence identity thereto, or a fragment thereof.

[0009] The broad-spectrum vaginitis bactericidal peptide YDG-C of the present invention has very significant antibacterial activity against Gardnerella vaginalis ATCC14018, Candida albicans ATCC10231 and Staphylococcus aureus ATCC6538.

[0010] The bactericidal peptide of the present invention is a broad-spectrum vaginitis bactericidal peptide obtained by entrusting KAMD Biotechnology to develop customized peptide drugs based on several common pathogenic bacteria of vaginitis.

[0011] In one aspect, the present invention provides a vaginal flora repair liquid, characterized in that the repair liquid contains the broad-spectrum vaginitis bactericidal peptide of the present invention and suitable vaginal probiotics.

[0012] Another aspect of the present invention provides a use of the broad-spectrum vaginitis bactericidal peptide and vaginal probiotics of the present invention in the preparation of a medicament for treating vaginitis.

[0013] Furthermore, the present invention also provides a probiotic composition, specifically, the composition is a bacterial powder and a pharmaceutically acceptable carrier in a mass ratio of Lactobacillus paracasei CGMCC No.1.12731: Lactobacillus rhamnosus CGMCC 24282: Lactobacillus gasseri CGMCC No.22909 = 2:2:1. The bacterial concentration in the probiotic composition is 10 9 -10 10 CFU / g.

[0014] Furthermore, the present invention also provides a vaginal flora repair liquid, which is prepared by dissolving 1 g of a probiotic composition in 1 mL of water. Optionally, a broad-spectrum vaginitis bactericidal peptide YDG-C is also added to the repair liquid.

[0015] Furthermore, the present invention also provides a polypeptide-containing vaginal flora repair liquid, which is prepared by dissolving 1 g of a probiotic composition and 100 mg of a broad-spectrum vaginitis bactericidal peptide YDG-C in 1 mL of water.

[0016] Specifically, the vaginal flora repair solution of the present invention further contains another therapeutic agent.

[0017] Specifically, additional therapeutic agents may include one or more antimicrobial agents and / or one or more conventional antibiotics. In order to accelerate the treatment of infection, additional therapeutic agents may include agents that can enhance the antimicrobial or bactericidal activity of polypeptides. Antimicrobial agents interfere with the structure or function of bacterial cells mainly by inhibiting cell wall synthesis, inhibiting cell membrane function and / or inhibiting metabolic function. Antibiotics can be broadly divided into antibiotics that affect cell wall peptidoglycan biosynthesis and antibiotics that affect gram-positive bacteria DNA or protein synthesis. Cell wall synthesis inhibitors, including penicillin and similar antibiotics, destroy the rigid outer cell wall, thereby causing relatively unsupported cells to swell and eventually rupture. Antibiotics that affect cell wall peptidoglycan biosynthesis include glycopeptides, which inhibit peptidoglycan synthesis by preventing N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) peptide subunits from being incorporated into the peptidoglycan matrix. Available glycopeptides include vancomycin and teicoplanin. Penicillin works by inhibiting the formation of peptidoglycan crosslinks. The functional group of penicillin - the lactam part - binds to and inhibits the D,D-transpeptidase that connects the peptidoglycan molecules in bacteria. The hydrolase proceeds to break down the cell wall, causing the cell to lyse or die due to osmotic pressure. Common penicillins include oxacillin, ampicillin, and cloxacillin.

[0018] The polypeptides or compositions described herein can be administered topically or systemically, preferably vaginally or orally, preferably vaginally. The therapeutically effective dose is usually initially estimated in mice, rabbits, dogs or pigs in cell culture assays or animal models. Animal models are also used to obtain the desired concentration range and route of administration. Such information can then be used as a basis for determining useful doses and routes of administration in humans through clinical studies.

[0019] Treatment duration and topical therapy will be selected by the physician based in part on these scientifically robust clinical studies, but also on a number of factors including, but not limited to: severity of BV and related conditions as scored by the NugentScore / Amstel criteria, the patient's weight, co-morbidities, diet, desired duration of treatment, method of administration, timing and frequency of administration, drug combinations, reaction sensitivities, and tolerance / response to treatment.

[0020] The polypeptide can be delivered during or near menstruation. Depending on the pharmaceutical composition, the present invention can be in various time periods (e.g., several times a day, every day, alternating days or every week), depending on the half-life and clearance rate of the specific formulation. The total duration of treatment can be a single dose, or it can last for several days or weeks when multiple doses are given. For example, a 400-500 mg dose can be administered orally twice a day for 5-7 days, or a 1-2 g single dose can be administered orally. Alternatively, it can be topically applied in the vagina 1-2 times a day for 3-7 days, and in some embodiments, it is preferably applied at night. Vaginal suppositories can also be used under similar dosage regimens as described in the above-mentioned mixture or repair solution, cream and lotion.

[0021] Beneficial Effects

[0022] The invention provides a vaginal flora repair liquid. Specifically, the repair liquid prepared by using Lactobacillus paracasei CGMCC No.1.12731, Lactobacillus rhamnosus CGMCC 24282 and Lactobacillus gasseri CGMCC No.22909 in a specific mass ratio can effectively kill pathogenic bacteria without obvious toxic side effects, and the cost is low, and the synergistic effect of the three is obvious.

[0023] In addition, further adding the broad-spectrum vaginitis bactericidal peptide YDG-C screened by the present invention to the repair solution can further improve the therapeutic effect of bacterial vaginitis. The present invention is simple to prepare, low in cost and suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Treatment score results of vaginal flora repair solution for bacterial vaginosis. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial sources.

[0026] and materials

[0027] Example 1 Verification of the bactericidal effect of the broad-spectrum vaginitis bactericidal peptide YDG-C

[0028] The broad-spectrum vaginitis bactericidal peptide YDG-C is artificially synthesized and prepared into a concentration of 1 mg / mL for use.

[0029] Inoculate 1 mL of the bacterial solution from a frozen tube of Gardnerella vaginalis ATCC14018 into 100 mL of TSB medium + 5% defibrinated sheep blood and culture in a micro-anaerobic environment (5% CO2) at 37°C for 24 hours.

[0030] Candida albicans ATCC10231 was cultured on sand agar medium and Staphylococcus aureus ATCC6538 was cultured on nutrient agar medium for 24 h.

[0031] The above bacteria were inoculated into fresh culture for 3 h, centrifuged at 4000 r / min for 10 min, washed 3 times with phosphate buffered saline (PBS, pH 7.4), and resuspended with TSB to adjust the bacterial concentration to 1 × 10 6 CFU / mL, take 50μL of bacterial suspension and add it to a 96-well bacterial culture plate, then add 50μL of broad-spectrum vaginitis bactericidal peptide YDG-C solution (concentrations are 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625 mg / L), act at 37℃ for 24h, observe the clarity, and the concentration corresponding to the clear well with the largest dilution is judged as the MIC of the antimicrobial peptide. The results are shown in Table 1.

[0032] Table 1 Antibacterial activity of broad-spectrum vaginitis bactericidal peptide YDG-C

[0033]

[0034]

[0035] As can be seen from Table 1, the broad-spectrum vaginitis bactericidal peptide YDG-C of the present invention has very significant antibacterial activity against Gardnerella vaginalis ATCC14018, Candida albicans ATCC10231 and Staphylococcus aureus ATCC6538.

[0036] Example 2 Preparation of probiotic composition and efficacy verification

[0037] MRS medium (g / L): peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast powder 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, cysteine ​​hydrochloride 0.5 g / L.

[0038] Lactobacillus paracasei CGMCC No.1.12731, Lactobacillus rhamnosus CGMCC 24282, and Lactobacillus gasseri CGMCC No.22909 are inoculated into MRS liquid culture medium respectively, cultured at 37°C for 24 hours for activation, and activated twice continuously to obtain activation solution; the activation solution is inoculated into MRS liquid culture medium at an inoculum amount of 5% (v / v), and cultured at 37°C for 30 hours to obtain bacterial liquid; the bacterial liquid is centrifuged at 4°C and 4500 rpm for 10 minutes, filtered to obtain bacterial cells, and mixed according to a weight ratio of Lactobacillus paracasei CGMCC No.1.12731: Lactobacillus rhamnosus CGMCC 24282: Lactobacillus gasseri CGMCC No.22909=1:1:1 or 2:1:1 or 2:2:1 or 1:1:2 or 1:2:1, and 1 g of the mixed powder of each group is dissolved in 1 mL of water respectively.

[0039] Staphylococcus aureus ATCC6538 was inoculated on a nutrient agar plate, Candida albicans ATCC10231 was inoculated on a Sabouraud agar plate, and Gardnerella vaginalis ATCC14018 was inoculated on a TSB medium + 5% defibrinated sheep blood agar plate, and placed in an Oxford cup, and 100 μL of the above bacterial solution was added to the Oxford cup; placed at 4°C for 4 hours, and then cultured at 37°C for 24 hours, each repeated 3 times, and the diameter of the inhibition zone was measured with a vernier caliper. The inhibition zone is directly shown in Table 2 below.

[0040] Table 2 Size of inhibition zone

[0041]

[0042]

[0043] As can be seen from Table 2, when the weight ratio is Lactobacillus paracasei CGMCC No.1.12731: Lactobacillus rhamnosus CGMCC 24282: Lactobacillus gasseri CGMCC No.22909=2:2:1, the antibacterial effect on Staphylococcus aureus, Candida albicans and Gardnerella vaginalis is the best, and this mass ratio can achieve better antibacterial effect.

[0044] Example 3 Preparation of vaginal flora repair solution

[0045] According to the bacterial preparation method of Example 2, bacterial powders with a mass ratio of Lactobacillus paracasei CGMCC No.1.12731: Lactobacillus rhamnosus CGMCC 24282: Lactobacillus gasseri CGMCC No.22909=2:2:1 were prepared, and 1 g of the mixed powders of each group were dissolved in 1 mL of water to prepare vaginal flora repair liquid.

[0046] Further, according to the bacterial preparation method of Example 2, bacterial powder with a mass ratio of Lactobacillus paracasei CGMCC No.1.12731: Lactobacillus rhamnosus CGMCC 24282: Lactobacillus gasseri CGMCC No.22909 = 2:2:1 was prepared, and 1 g of the mixed powder of each group and 100 mg of the broad-spectrum vaginitis bactericidal peptide YDG-C were dissolved in 1 mL of water to prepare a vaginal flora repair liquid containing polypeptides.

[0047] Example 4 Application of vaginal flora repair solution in the treatment of vaginitis

[0048] Preparation of bacterial vaginosis model: Staphylococcus aureus ATCC6538, Candida albicans ATCC10231, and Gardnerella vaginalis ATCC140183 were revived and passaged, and then prepared with sterile saline to a concentration of 1.0×10 9 / mL of bacterial solution, and then mixed in a ratio of 1:1:1 to make the infection solution. First, rinse the rat vagina with sterile PBS solution (pH=8.5) 3 times (5 minutes each time), then use a No. 5 scalp needle silicone tube coated with sterile paraffin oil, slowly insert it into the rat vagina about 1-1.5cm, and slowly inject the infection solution from the tube. The injection amount of each bacteria is 0.025mL / 100g, once a day. After inoculation of bacteria, observe the vaginal lesions of the rats every day, and take vaginal secretion smears for staining and microscopic examination. When the rat vagina is obviously congested, red and swollen with large and purulent secretions, take secretion smears, and a large number of infected bacteria and necrotic cells can be seen under the microscope, indicating that the bacterial vaginitis model of rats has been successfully prepared.

[0049] Female rats were randomly divided into 5 groups: normal control group, the aforementioned model group, positive drug group, vaginal flora repair liquid group, and vaginal flora repair liquid group containing peptides, with 10 rats in each group. Rats in the normal control group were not inoculated with bacteria. Rats in the model group and each drug-treated group all showed symptoms of vaginitis 3-4 days after the bacteria were injected, proving that the model was successfully established. Drug treatment began on the 5th day after the model preparation experiment.

[0050] The positive control group (Xiao Mi vaginal effervescent tablets (Shu Yan / Dong Tai), national medicine standard Z20090857) was administered intravaginally at a dose of 0.2 g / kg;

[0051] Vaginal flora repair liquid group: 0.2 g (i.e. 0.2 ml) / kg was administered intravaginally (prepared in Example 3);

[0052] Vaginal flora repairing solution containing polypeptide group: 0.2 g (i.e. 0.2 ml) / kg was administered intravaginally (prepared in Example 3);

[0053] The normal control group and the model group were given equal weight / volume of excipients into the vagina and the drugs were administered continuously for 7 days.

[0054] Smear examination and pathological examination analysis 2h after the last administration, the vagina of each group of rats was photographed locally, and the efficacy was evaluated according to the following standards. Cured: The vagina had no congestion, redness and swelling, no purulent secretions, no infection bacteria and necrotic cells were found in the vaginal secretion smear microscopy, and the vaginal mucosa was intact and the submucosal tissue was basically normal. Effective: The vagina had mild congestion, redness and swelling, a small amount of purulent secretions, a small amount of infection bacteria and a small amount of necrotic cells were found in the vaginal secretion smear microscopy, and the vaginal mucosa had slight defects, capillary dilation and erythrocytes were increased in the submucosal tissue, and a small amount of neutrophil infiltration was found in the tissue. Ineffective: The vagina had congestion, redness and swelling, a large amount of purulent secretions, a large amount of infection bacteria and necrotic cells were found in the vaginal secretion smear microscopy, and the vaginal tissue section microscopy showed that the mucosa had defects, a large amount of capillary dilation and erythrocytes were increased in the submucosal tissue, and a large amount of neutrophil infiltration was found in the tissue. Vaginal tissue scoring criteria: Vaginal specimens were scored based on four basic indicators: congestion, edema, bleeding, and infiltration. No congestion, edema, bleeding, or infiltration was scored as 0 points, mild as 1 point, moderate as 2 points, and severe as 3 points. Figure 1 shown.

[0055] from Figure 1 It can be seen that the effective rates of bacterial vaginitis treatment in each treatment group were higher than those in the model control group, with a very significant difference (P<0.01). Specifically, the total effective rates of the positive drug group, the vaginal flora repair liquid group, and the vaginal flora repair liquid group containing polypeptides were 70%, 90%, and 100%, respectively. This also shows that a specific amount of Lactobacillus paracasei CGMCC No.1.12731, Lactobacillus rhamnosus CGMCC 24282, and Lactobacillus gasseri CGMCC No.22909 can be significantly used to treat bacterial vaginitis, and after further adding polypeptides, the therapeutic effect is further enhanced.

[0056] The mice in each group were dissected, and compared with the normal group, the experimental groups had no obvious organ damage, indicating that the repair solution of the present invention has good biosafety.

[0057] The above is only an illustrative embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and supplements made without departing from the method of the present invention should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, using the technical content disclosed above to make some changes, modifications and evolutions of equivalent changes, are all equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any and equivalent changes made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention. All parts not fully disclosed in the present invention are prior art.

Claims

1. A broad-spectrum vaginitis bactericidal peptide YDG-C, whose amino acid sequence is shown in SEQ ID NO: 1; wherein the N-terminus of the amino acid sequence is acetylated and the C-terminus is amidated.

2. A vaginal flora repair solution, characterized in that The probiotic powder is composed of probiotic powders with a mass ratio of Lactobacillus paracasei CGMCC No.1.12731: Lactobacillus rhamnosus CGMCC 24282: Lactobacillus gasseri CGMCC No.22909=2:2:1, and the broad-spectrum vaginitis bactericidal peptide YDG-C described in claim 1 is also added, and the weight ratio of the bactericidal peptide to the powder is 1:

10.

3. Use of probiotic powder and the broad-spectrum vaginitis bactericidal peptide YDG-C described in claim 1 in preparing a vaginal flora repair solution for treating bacterial vaginitis, wherein the probiotic powder is composed of probiotic powders with a mass ratio of Lactobacillus paracasei CGMCC No.1.12731: Lactobacillus rhamnosus CGMCC 24282: Lactobacillus gasseri CGMCC No.22909=2:2:1, the weight ratio of the bactericidal peptide to the powder is 1:10, and the repair solution is a water-soluble type.

4. The use according to claim 3, characterized in that A pharmaceutically acceptable excipient may also be added to the repair solution.

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