CN111135157B - Use of a fermentation broth of Lactobacillus plantarum TCI378 containing 3-phenyllactic acid and a combination of probiotics for improving the bacterial phase - Google Patents
Use of a fermentation broth of Lactobacillus plantarum TCI378 containing 3-phenyllactic acid and a combination of probiotics for improving the bacterial phase Download PDFInfo
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Abstract
The invention relates to the field of novel application of 3-phenyllactic acid, in particular to application of a combination of fermentation culture solution of lactobacillus plantarum TCI378 containing 3-phenyllactic acid and probiotics to improvement of bacterial phases, and the combination of the fermentation culture solution of lactobacillus plantarum TCI378 containing 3-phenyllactic acid and the probiotics has the following effects: can reduce the growth of pathogenic bacteria by adjusting and improving the bacterial phase in the vagina of a female, thereby achieving the effect of protecting the private parts of the female.
Description
Technical Field
The invention relates to the field of novel application of 3-phenyllactic acid, in particular to application of a combination of 3-phenyllactic acid (3-phenyllactic acid) and probiotics (prespecies) in improving bacterial phases.
Background
Under normal circumstances, the vagina of a woman secretes a small amount of secretions, which have the effect of lubricating the vagina and are essential for the vagina. However, in the case of poor hygiene or decreased resistance, vaginal secretion is sometimes increased, color is changed and there is an odor, i.e., vaginitis is suffered. These abnormal secretions can cause extreme itching or pain in the affected area of the patient, thus causing a great psychological and physiological discomfort to the patient. The secretions from vaginitis may cause redness, swelling and itching of the vulva, so-called vulvitis, which may be known as pudendum infections. Common causes of female pudendum infections include: 1. candida species (Candida spp.), such as Candida albicans (Candida albicans) infections, tend to grow in warm and humid environments, and in individuals with insufficient resistance, infections (such as hyposleep, cold, pre-menstrual and post-pregnancy) tend to repeatedly occur, and post-infection symptoms include itching, burning, vulvar redness, white secretion of cheese cake, urethral pain and redness, pain in urination, etc., and especially pre-and post-menstrual discomfort can occur. 2. Bacterial infections, common symptoms include a large amount of off-white or yellow secretions after sexual intercourse, and a fishy smell-like smell, most patients do not have itching. Too frequent intercourse or too frequent vaginal irrigation results in an increase in the vaginal pH, which is a major cause of bacterial infection, since the bacteria responsible for the infection are suitable for growing in a more alkaline environment (pH > 4.5). Bacteria responsible for bacterial infection include Escherichia coli (Escherichia coli), gardnerella vaginalis (Gardnerella vaginalis), streptococcus (Streptococcus), and the like.
The normal vaginal environment is not sterile and contains about six bacteria, among which are staphylococci, coliforms, diphtheria-like species, candida species (Lactobacillus spp). The lactobacillus species with the largest content is probiotics, and staphylococcus, escherichia coli, diphtheria-like bacteria, candida species and the like are pathogenic bacteria, but when the pathogenic bacteria are small in quantity, the health of the vagina is not affected adversely, and as long as the vagina is maintained in a normal bacterial phase, namely when the probiotics are dominant, the pathogenic bacteria are not bred in a large quantity, so that the infection is avoided. In addition, the normal pH of the vagina is about pH 3.8-4.2, i.e., less acidic, and if the pH of the vagina is changed to more neutral or basic, infection may occur. The lactobacillus species can maintain the pH value of the vagina at 3.8-4.2, and most pathogenic bacteria causing vaginal infection cannot grow in the weak acidic environment, so that the vagina can be kept healthy.
However, when the bacterial phase in the vagina is destroyed, the aforementioned infection occurs. The existing method for treating female pudendum infection generally uses medicines such as antibiotics, steroid and the like for treatment, but the probiotics and pathogenic bacteria are killed together, so that the initial vaginal environment of treatment is aseptic, the probiotics and the pathogenic bacteria compete with each other, and as a result of competition, the pathogenic bacteria grow better than the probiotics, so that repeated infection is easy to occur, and repeated treatment is needed. However, repeated use of antibiotics and steroids is likely to cause drug resistance to pathogenic bacteria and even to cause systemic impairment of human immunity. In addition, most of the currently marketed vaginal care products are iodine-containing chemical agents, which can alleviate the symptoms of infection in a short period of time, but since the pH value is neutral, the pH value of the vagina is raised after use and changed into an environment suitable for the growth of pathogenic bacteria, the symptoms are easy to relapse, and the symptoms are difficult to radically cure.
In order to solve the above problems, it is highly desirable for those skilled in the art to develop novel pharmaceutical compositions having the efficacy of improving bacterial phase to benefit a broad group of people in need thereof.
Disclosure of Invention
Accordingly, the present invention provides a use of a combination of 3-phenyllactic acid (3-phenyllactic acid) and a probiotic for preparing a composition for improving bacterial phase.
In one embodiment of the invention, the probiotic is lactitol.
In one embodiment of the invention, the bacterial phase is a bacterial phase in a woman's vagina.
In one embodiment of the invention, the bacterial phase comprises Lactobacillus species (Lactobacillus spp.), gardnerella vaginalis (Gardnerella vaginalis), candida species (Candida spp.), and Escherichia coli.
In one embodiment of the invention, the combination of 3-phenyllactic acid and the probiotic increases the number of Lactobacillus species and decreases the number of Gardnerella vaginalis, candida species and Escherichia coli.
In one embodiment of the invention, the volume ratio of 3-phenyllactic acid to lactitol is between 1:10 and 25.
In one embodiment of the invention, the composition is a pharmaceutical composition.
In one embodiment of the invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.
In one embodiment of the invention, the pharmaceutical composition is in a form for topical administration.
In one embodiment of the invention, the pharmaceutical composition is in the form of a sanitary napkin or a spray.
In one embodiment of the invention, 3-phenyllactic acid is purified by high performance liquid chromatography (high performance liquid chromatography, HPLC).
In summary, the efficacy of the combination of 3-phenyllactic acid and the prebiotics of the present invention is: can reduce the growth of pathogenic bacteria by adjusting and improving the bacterial phase in the vagina of a female, thereby achieving the effect of protecting the private parts of the female.
The following examples are set forth to illustrate the present invention and are not intended to limit the scope of the invention, as many variations and modifications are possible without departing from the spirit and scope of the invention as defined in the appended claims.
Drawings
FIG. 1 is a graph of data showing the efficacy of a combination of 3-phenyllactic acid and a probiotic of the present invention in improving the bacterial phase of subject 1;
FIG. 2 is a graph of data showing the efficacy of the combination of 3-phenyllactic acid and a probiotic of the present invention in improving the bacterial phase in subject 2;
FIG. 3 is a graph of data showing the efficacy of the combination of 3-phenyllactic acid and a probiotic of the present invention in improving the bacterial phase of subject 3;
FIG. 4 is a graph of data showing the efficacy of the combination of 3-phenyllactic acid and the probiotic of the present invention in improving the bacterial phase of subject 4. The method comprises the steps of carrying out a first treatment on the surface of the
Figure 5 is a graph of data showing the efficacy of the combination of 3-phenyllactic acid and the probiotic of the present invention in improving the average bacterial phase in 4 subjects.
Detailed Description
Definition of the definition
The values used herein are approximations, and all experimental data are presented in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.
According to the present invention, 3-phenyllactic acid (3-phenyllactic acid) has the following chemical formula (I):
as used herein, the term "probiotics" means substances that exert a biological effect on a subject by selectively stimulating the growth or biological activity of beneficial microorganisms.
According to the invention, lactitol has the following formula (II):
in accordance with the present invention, the pharmaceutical compositions may be manufactured in a form suitable for topical (topicaly) application using techniques well known to those skilled in the art, including, but not limited to, sprays, gels, emulsions, creams, ointments, tampons, powders, tissues, sticks, and pads.
In accordance with the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of: solvents (solvents), buffers (buffers), emulsifiers (dispersing agents), disintegrants (decomponents), disintegrants (disintegrating agent), dispersants (dispersing agents), binders (binding agents), excipients (excipients), stabilizers (stabilizing agent), chelating agents (chelating agents), diluents (diluents), gelling agents, preservatives (solvents), wetting agents (lubricants), lubricants (absorption delaying agent), liposomes (lipo-agents), and the like. The choice and quantity of such agents is within the skill and routine skill of those skilled in the art.
According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of: water, normal saline (normal saline), phosphate buffered saline (phosphate buffered saline, PBS), an aqueous solution containing alcohol (aqueous solution containing alcohol), and combinations thereof.
Example 1.3 preparation of combinations of phenyllactic acid (3-phenyllactic acid) and prebiotics
First, the isolation procedure of lactobacillus plantarum (Lactobacillus plantarum) TCI378 is as follows:
(1) Screening
The kimchi homogenate (containing kimchi and sauce) and MRS broth (MRS broth) were mixed in a volume ratio of 1:100 and subjected to anaerobic culture (oxygen concentration of less than 5%) at 37 deg.c for 18 hours. Thereafter, the aforementioned culture solution was spread on an MRS solid medium (MRS agar) and subjected to anaerobic culture at 37℃to generate different colonies. Next, to ensure the single nature of the strain, a colony was selected using a sterile inoculating loop, streaked onto a solid medium, and cultured in an anaerobic incubator at 37℃until a single colony (single colony) appeared.
(2) Authentication
The single colony strain isolated by the screening procedure above was taken and subjected to genetic genealogy analysis (phylogenetic analysis) to confirm that the strain had the sequence as set forth in SEQ ID NO:11 (16S ribosomal ribonucleic acid (16S rRNA) fragment). Alignment was performed using NCBI (National Center for Biotechnology Information) on-line database, confirming the SEQ ID NO:11 has about 99% similarity with the 16S ribosomal ribonucleic acid (16S rRNA) fragment of the Lactobacillus plantarum (Lactobacillus plantarum) strain, so that the target strain is identified as Lactobacillus plantarum by genetic relationship and named Lactobacillus plantarum TCI378, wherein the Lactobacillus plantarum TCI378 has been deposited in the food industry institute of development and biological resource preservation and research center at 12 and 27 of 2016 under deposit number BCRC910760, see Taiwan patent TWI645854.
(3) Preservation of
The strain with singleness obtained in the above screening procedure is cultivated in MRS culture solution by liquid cultivation to provide bacterial solution, 25% glycerol is added into the bacterial solution, and the obtained mixed solution is transferred into a cryopreservation tube and then stored at-80 ℃.
Next, after once activating the Lactobacillus plantarum (Lactobacillus plantarum) TCI378 strain preservation tube, it is cultured in MRS medium in an amount of 1% -10% of the amount of germ and cultured at 37℃for 18 hours. Next, the bacterial liquid was centrifuged at 5,000rpm for 20 minutes, and then the supernatant was collected and taken as an analysis sample.
Thereafter, 2L of the supernatant was taken and concentrated under reduced pressure to a volume of 300mL, followed by column chromatography with macroporous resin (Diaion HP-20, 100cm x 8 cm) gradient from water to methanol, giving 5 layers (1:0% methanol, 2:20% methanol, 3:40% methanol, 4:60% methanol, 5:100% methanol). Next, the layer 3 was taken and subjected to RP-C18 flash column chromatography (HPLC column: luna 5u RP-C18, 250X10mm, phenomenex, USA) with a linear gradient from 20% methanol to 100% methanol, followed by thin layer chromatography (Thin Layer Chromatography, TLC) (Silica gel 60F) 254 ；RP-18F 254 -S, merck, EMD Millopore co., germany) gives 8 sub-layers. Thereafter, sub-fraction 7 was subjected to column chromatography with Sephadex LH-20 gel (Pharmacia, piscataway, NJ, USA), and 8 sub-fractions were obtained by thin layer chromatography. Purification by reverse phase-high performance liquid chromatography (reversed phase-high performance liquid chromatography, RP-HPLC) from secondary partition 5 (methanol/water=2/3) afforded the compound.
The chemical structure was then confirmed to be 3-phenyllactic acid by 1H and 13C nuclear magnetic resonance (Nuclear Magnetic Resonance, NMR) (Ascend 400'54 400MHz,Bruker Co, germany), heteronuclear single quantum correlation spectroscopy (heteronuclear single quantum correlation, HSQC), heteronuclear multiple bond correlation spectroscopy (heteronuclear multiple bond correlation, HMBC), correlated magnetic resonance spectroscopy (Correlation Spectroscopy, COSY) and electrospray ionization mass spectrometry (electrospray ionization mass spectrometry, ESIMS) analysis of the compounds. Next, 1-5% (w/w) lactitol is added to the fermentation broth of Lactobacillus plantarum TCI378 containing 3-phenyllactic acid to obtain a combination of 3-phenyllactic acid and a probiotic (i.e., lactitol).
Example 2.3 evaluation of the effectiveness of combinations of phenyllactic acid and Probiotics in improving bacterial phase
In this example, a combination of 3-phenyllactic acid and a probiotic (e.g., lactitol) was taken for evaluation of the utility of improved bacterial phase in the female vagina.
The combination of 3-phenyllactic acid and the prebiotic is applied in the form of a feminine hygiene pad in an amount of 4 tablets per day. First, 4 adult females were recruited as subjects, and then a piece of sanitary napkin containing a combination of 3-phenyllactic acid and probiotics was replaced after three meals a day and before sleep. Next, the vaginal opening was smeared with a cotton swab before bathing for 0, 3, 7 and 14 days. The sampling flow is as follows: the sterilized cotton stick is smeared on the upper part of the female perineum for 5-6 times, and then the smeared cotton stick head is placed into a sampling tube containing 0.5mL of preservation solution (17 mM sodium citrate, 13mM EDTA, 46.7% (w/v) ammonium sulfate, pH 5.2). Then, the upper part of the cotton stick is broken off, the cotton stick head in the preservation solution is left, then the sampling tube cover is screwed tightly, and the cotton stick is preserved at normal temperature until the cotton stick head is delivered to laboratory for analysis.
Thereafter, the sampled cotton swab was taken, and then the microbial phase sample in the cotton swab was extracted and purified by a Presto Buccal Swab gDNA extraction kit (Presto Buccal Swab gDNA Extraction kit) (Geneaid, cat. No. gsk 300-DG), followed by measuring the concentration and purity of the extracted DNA sample. Next, quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction, qRT-PCR) detection of 16S ribosomal gene (16S ribosomal gene) was performed against Lactobacillus species (Lactobacillus spp.) as a probiotic and Gardner vaginalis (Gardnerella vaginalis), candida spp.) and Escherichia coli (Escherichia coli) as pathogens. Bacterial phase composition was analyzed in 4ng of DNA samples by qRT-PCR, and triplicate experiments were performed for each sample. The reaction conditions for qRT-PCR are shown in Table 1.
TABLE 1
The target microorganism, primer sequences and amplicon (amplicon) sizes for qRT-PCR analysis are shown in table 2.
TABLE 2
The results of this example are shown in fig. 1 to 5. FIG. 1 is a graph of data showing the efficacy of a combination of 3-phenyllactic acid and a probiotic of the present invention in improving the bacterial phase of subject 1. Figure 2 is a graph of data showing the efficacy of the combination of 3-phenyllactic acid and a probiotic of the present invention in improving the bacterial phase of subject 2. FIG. 3 is a graph of data showing the efficacy of the combination of 3-phenyllactic acid and the probiotic of the present invention in improving the bacterial phase of subject 3. FIG. 4 is a graph of data showing the efficacy of the combination of 3-phenyllactic acid and the probiotic of the present invention in improving the bacterial phase of subject 4. Figure 5 is a graph of data showing the efficacy of the combination of 3-phenyllactic acid and the probiotic of the present invention in improving the average bacterial phase in 4 subjects. As can be seen from FIGS. 1 to 5, the number of probiotics (i.e., lactobacillus species) was increased and the number of pathogenic bacteria (including Gardnerella vaginalis, candida species and Escherichia coli) was decreased in the subjects after administration of the sanitary napkin sheet comprising the combination of 3-phenyllactic acid and the probiotics, compared to day 0, wherein the improvement in the bacterial phase was most remarkable in the subject 1, and the subject 2 was not significantly different because of the absence of pathogenic bacterial infection itself.
In conclusion, the combination of the 3-phenyllactic acid and the probiotics can reduce pathogenic bacteria growth and inflammation by adjusting and improving the bacterial phase in the vagina of a female, so that the effect of protecting the private parts of the female is achieved.
The foregoing is by way of example only and is not intended as limiting. Any equivalent modifications or variations to the present invention without departing from the spirit and scope of the present invention shall be included in the appended claims.
Claims (8)
1. Use of a combination of a fermentation broth of lactobacillus plantarum TCI378 comprising 3-phenyllactic acid (3-phenyllactic acid) and a probiotic, said probiotic being lactitol (lactitol), for the preparation of a composition for improving the bacterial phase in the vagina of a female.
2. The use according to claim 1, wherein the bacterial phases comprise Lactobacillus species (Lactobacillus spp.), gardnerella vaginalis (Gardnerella vaginalis), candida species (Candida spp.), and Escherichia coli.
3. The use according to claim 2, characterized in that the combination of fermentation broth of lactobacillus plantarum TCI378 comprising 3-phenyllactic acid and a probiotic increases the number of lactobacillus species and decreases the number of gardnerella vaginalis, candida species and escherichia coli.
4. The use according to claim 1, characterized in that the volume ratio of the fermentation broth of lactobacillus plantarum TCI378 containing 3-phenyllactic acid to the lactitol is between 1:10 and 25.
5. The use according to claim 1, wherein the composition is a pharmaceutical composition.
6. The use according to claim 5, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.
7. The use according to claim 5, wherein the pharmaceutical composition is in a form for topical administration.
8. The use according to claim 5, wherein the pharmaceutical composition is in the form of a sanitary napkin or a spray.
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| US201862700497P | 2018-07-19 | 2018-07-19 | |
| US62/700,497 | 2018-07-19 | ||
| TW108103154A TWI754126B (en) | 2018-07-19 | 2019-01-28 | Use of probiotic composition for improving microflora of female vagina and external hygiene products |
| TW108103154 | 2019-01-28 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557288A (en) * | 2004-02-10 | 2004-12-29 | 周世兰 | Lactic acid , glycerin formulation for female vagina acidity, humidity improvement |
| TW200806303A (en) * | 2006-07-18 | 2008-02-01 | Tensall Bio Tech Co Ltd | Composition applied to female pudendum |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20060106117A1 (en) * | 2004-11-12 | 2006-05-18 | Kimberly-Clark Worldwide, Inc. | Compound and method for prevention and/or treatment of vaginal infections |
| CA2609754C (en) * | 2005-04-27 | 2012-01-10 | Shenzhen Phlora Biotechnology Limited | Compositions of benzoic acid and saccharides for regulating and maintaining bacterial flora and acidity in the vagina |
| SG182959A1 (en) * | 2007-03-30 | 2012-08-30 | Suntory Holdings Ltd | Medicinal composition, food or drink having effect of enhancing parasympathetic nervous activity |
| KR101860552B1 (en) * | 2015-04-16 | 2018-07-02 | 주식회사 고바이오랩 | Lactobacilli having inhibitory effect against pathogenic microorganisms in vagina |
| AU2015410635B2 (en) * | 2015-09-29 | 2021-08-19 | Kimberly-Clark Worldwide, Inc. | Synergistic composition for maintenance of healthy balance of microflora |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557288A (en) * | 2004-02-10 | 2004-12-29 | 周世兰 | Lactic acid , glycerin formulation for female vagina acidity, humidity improvement |
| TW200806303A (en) * | 2006-07-18 | 2008-02-01 | Tensall Bio Tech Co Ltd | Composition applied to female pudendum |
Non-Patent Citations (2)
| Title |
|---|
| "Evaluation of lactitol, 1 lactulose, raffinose, and oligofructose for maintenance of a Lactobacillus-dominated vaginal microbiota establishes a promising prebiotic candidate";Collins, Stephanie L.等;《Applied and Environmental Microbiology》;20171221;1-37 * |
| "Isolation and Selection of Anti-Candida albicans Metabolites Producing Lactic Acid Bacteria from Various Sources";Tanes Sungsri等;《KKU Res. J.》;20121231;第17卷(第4期);630-638 * |
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