TWI754126B - Use of probiotic composition for improving microflora of female vagina and external hygiene products - Google Patents

Abstract

The present disclosure provides a use of a combination of 3-phenyllactic acid and prebiotics for improving microflora.

Description

Use and external use of probiotic composition for improving bacterial phase in female vagina hygiene products

The present invention relates to the use of a combination of 3-phenyllactic acid and prebiotics for improving bacterial phase.

Under normal circumstances, a woman's vagina will secrete a little secretion, which has the function of lubricating the vagina, which is indispensable for the vagina. However, in the case of poor hygiene or decreased resistance, vaginal secretions may sometimes increase, change in color and have a peculiar smell, that is, vaginitis. These abnormal secretions can cause extreme itching or pain in the affected part of the patient, thus causing great psychological and physical discomfort to the patient. The secretions produced by vaginitis may still cause redness, swelling and itching of the vulva, which is called vulvitis. Vaginitis and vulvitis can be collectively referred to as genital infection. The common causes of female genital infection include: 1. Candida spp., such as Candida albicans ( Candida albicans ) infection, Candida albicans is easy to grow in a warm and humid environment, when the individual's resistance is insufficient , prone to repeated infections (such as lack of sleep, colds, before and after menstruation and during pregnancy), and the symptoms after infection include itching, burning, red and swollen genitals, cheese-like white discharge, sore, red and swollen urethra, painful urination, etc., Especially before and after menstruation can be particularly uncomfortable. 2. Bacterial infection, common symptoms include a large amount of gray-white or yellow secretions after sexual intercourse, and a smell similar to fishy smell, most patients do not itch. Too frequent sexual intercourse or too frequent douching of the vagina can lead to an increase in vaginal pH, which is the main cause of bacterial infection, because the bacteria that cause the infection are adapted to live in an alkaline environment (pH>4.5). Bacteria that cause bacterial infection include Escherichia coli ( Escherichia coli ), Gardnerella vaginalis ( Gardnerella vaginalis ) and Streptococcus ( Streptococcus ).

The normal vaginal environment is not sterile, it contains about six species of bacteria, including Staphylococcus, Escherichia coli, Diphtheria-like bacteria, Candida species and Lactobacillus spp. Among them, the most abundant Lactobacillus species are probiotics, while Staphylococcus, Escherichia coli, Diphtheria-like bacteria and Candida species are pathogenic bacteria, but when the amount of pathogenic bacteria is small, it will not have adverse effects on vaginal health , and as long as the vagina maintains a normal bacterial phase, that is, when probiotics are dominant, pathogenic bacteria will not breed in large quantities, so there will be no such infections. In addition, the normal pH value of the vagina is about pH 3.8~4.2, which is relatively weakly acidic. If the pH value of the vagina changes to more neutral or alkaline, infection will occur. Lactobacillus species can maintain the pH value of the vagina at pH 3.8~4.2, and in this weakly acidic environment, most of the pathogenic bacteria that cause vaginal infections cannot grow, so the vagina can be maintained healthy.

However, when the bacterial phase in the vagina is destroyed, the aforementioned infection occurs. The existing methods of treating female genital infection are generally the use of antibiotics, steroids and other drugs for treatment, but this often kills probiotics and pathogenic bacteria together, thus causing the vaginal environment to be sterile at the initial stage of treatment, and probiotics and pathogenic bacteria compete with each other. However, as a result of competition, pathogenic bacteria often outgrow probiotics, so re-infections are prone to occur, requiring repeated treatments. However, repeated use of antibiotics and steroids can easily make pathogenic bacteria resistant to drugs, and even damage the human immune system. In addition, most of the vaginal care products currently on the market are chemical agents containing iodine. Although they can alleviate the symptoms of infection in the short term, due to their neutral pH value, the pH value of the vagina will rise after use, which is suitable for the growth of pathogenic bacteria. environment, so the symptoms are prone to recurrence, making the symptoms difficult to cure.

In order to solve the above problems, those skilled in the art urgently need to develop novel pharmaceutical compositions with the effect of improving bacterial phase to benefit the vast population in need.

In view of this, the purpose of the present invention is to provide a combination of 3-phenyllactic acid (3-phenyllactic acid) and prebiotics for preparing a composition for improving bacterial phase.

In one embodiment of the present invention, the prebiotic is lactitol.

In one embodiment of the present invention, the bacterial phase is a bacterial phase in a female vagina.

In an embodiment of the present invention, the bacterial phase includes Lactobacillus spp., Gardnerella vaginalis , Candida spp. and Escherichia coli .

In one embodiment of the present invention, the combination of 3-phenyllactic acid and prebiotics increases the number of Lactobacillus sp., and reduces the number of Gardnerella vaginalis, Candida sp. and E. coli.

In an embodiment of the present invention, the volume ratio of 3-phenyllactic acid to lactitol is between 1:10 and 25.

In one embodiment of the present invention, the composition is a pharmaceutical composition.

In one embodiment of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the pharmaceutical composition is in a form for topical administration.

In one embodiment of the present invention, the pharmaceutical composition is in the form of a sanitary napkin or a spray.

In an embodiment of the present invention, 3-phenyllactic acid is purified by high performance liquid chromatography (high performance liquid chromatography, HPLC).

To sum up, the effect of the combination of 3-phenyllactic acid and probiotics of the present invention is that the growth of pathogenic bacteria can be reduced by regulating and improving the bacterial phase in the vagina of women, thereby achieving the effect of health care of women's private parts.

The embodiments of the present invention will be further described below. The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Anyone who is familiar with this technique, without departing from the spirit and scope of the present invention, Some changes and modifications can be made, so the protection scope of the present invention should be determined by the scope of the appended patent application.

Figure 1 is a data graph of the efficacy of the combination of 3-phenyllactic acid and prebiotics of the present invention in improving the bacterial phase of the first subject.

Figure 2 is a data graph of the efficacy of the combination of 3-phenyllactic acid and prebiotics of the present invention in improving the bacterial phase of the second subject.

Figure 3 is a data graph of the efficacy of the combination of 3-phenyllactic acid and prebiotics of the present invention in improving the bacterial phase of the third subject.

Figure 4 is a data graph of the efficacy of the combination of 3-phenyllactic acid and prebiotics of the present invention in improving the bacterial phase of the fourth subject.

Figure 5 is a data graph of the efficacy of the combination of 3-phenyllactic acid and prebiotics of the present invention in improving the average bacterial phase of 4 subjects.

definition

Numerical values used herein are approximations and all experimental data are expressed within 20%, preferably within 10%, and most preferably within 5%.

According to the present invention, 3-phenyllactic acid has the following chemical formula (I):

As used herein, the term "prebiotics" means substances that exert biological effects on an individual by selectively stimulating the growth or biological activity of beneficial microorganisms.

According to the present invention, lactitol has the following formula (II):

In accordance with the present invention, pharmaceutical compositions can be manufactured in a form suitable for topical administration using techniques well known to those skilled in the art, including, but not limited to, sprays, gels, gels, lotions, Creams, ointments, suppositories, powders, tissues, cotton swabs and pads.

According to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of: solvent, buffer, emulsifier, suspending agent, decomposer ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. The selection and quantity of these reagents are within the scope of the expertise and routine skills 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, phosphate buffered saline (PBS), Aqueous solutions containing alcohol and combinations thereof.

Example 1. Preparation of 3-phenyllactic acid and a combination of prebiotics

First, the isolation process of Lactobacillus plantarum TCI378 is as follows:

(1) Screening

The homogeneous material of kimchi (including kimchi and sauce) and MRS broth (MRS broth) were mixed in a volume ratio of 1:100, and placed at 37 ° C for anaerobic cultivation (oxygen concentration below 5%), which lasted for a period of time. 18 hours. After that, the aforementioned culture solution was spread on MRS solid medium (MRS agar) and placed at 37° C. for anaerobic culture to generate different colonies. Next, in order to ensure the singleness of the strains, a sterile inoculation loop was used to pick a colony, streaked on the solid medium, and then cultured in an anaerobic incubator at 37°C until a single colony appeared.

(2) Identification

The strain of the single colony isolated by the above screening process was taken, and phylogenetic analysis was performed to confirm that the strain had the 16S ribosomal ribonucleic acid (16S rRNA) fragment shown in SEQ ID NO: 11. Using the NCBI (National Center for Biotechnology Information) online database for comparison, it was confirmed that the SEQ ID NO: 11 line and the 16S ribosomal ribonucleic acid (16S rRNA) fragment of the Lactobacillus plantarum strain have about 99% of the Due to the similarity, the target strain was identified as Prospermum spp., and named as Prospermum spp. TCI378, which was deposited in the Biological Resource Preservation of the Institute of Food Industry Development on December 27, 2016. and Research Center, the deposit number is BCRC910760, see the patent case TWI645854 of the Republic of China.

(3) Save

The single strain obtained by the above screening process was cultivated in the MRS medium by liquid culture to provide a bacterial solution, then 25% glycerol was added to the bacterial solution, and the resulting mixture was transferred to a cryopreservation tube. After that, store at -80°C.

Next, after the Lactobacillus plantarum TCI378 strain storage tube was activated once, it was cultured in the MRS medium at 1% to 10% of the bacteria and cultured at 37° C. for 18 hours. Next, the bacterial liquid was centrifuged at 5,000 rpm for 20 minutes, and then the supernatant was collected, and the supernatant was taken as an analysis sample.

After that, take 2L of the supernatant and concentrate under reduced pressure to a volume of 300mL, then carry out column chromatography with a macroporous resin (Diaion HP-20, 100cm x 8cm) with a gradient from water to methanol to obtain 5 layers ( Layer 1: 0% methanol, Layer 2: 20% methanol, Layer 3: 40% methanol, Layer 4: 60% methanol, Layer 5: 100% methanol). Next, take layer 3 for RP-C18 flash column chromatography (HPLC column: Luna 5u RP-C18, 250 x 10 mm, Phenomenex, USA), with a linear gradient from 20% methanol to 100% methanol, through thin layer chromatography Thin Layer Chromatography (TLC) (Silica gel 60 F ₂₅₄ ; RP-18 F ₂₅₄ -S, Merck, EMD Millopore Co., Germany) resulted in 8 sub-layers. After that, sublayer 7 was subjected to column chromatography on Sephadex LH-20 gel (Pharmacia, Piscataway, NJ, USA), and 8 sublayers were obtained by thin layer chromatography. The compound was obtained by purifying the sub-layer 5 by reversed phase-high performance liquid chromatography (RP-HPLC) (methanol/water=2/3).

Then, the compound was subjected to 1H and 13C nuclear magnetic resonance (Nuclear Magnetic Resonance, NMR) (Ascend 400'54 400MHz, Bruker Co., Germany), heteronuclear single quantum correlation (heteronuclear single quantum correlation, HSQC), heteronuclear multiple bond Correlation spectrum The chemical structure was confirmed to be 3-phenyllactate by heteronuclear multiple bond correlation (HMBC), Correlation Spectroscopy (COSY) and electrospray ionization mass spectrometry (ESIMS) analysis. Next, 1-5% (w/w) lactitol was added to the fermentation broth of Lactobacillus blastobacterium TCI378 containing 3-phenyllactic acid to obtain a combination of 3-phenyllactic acid and prebiotics (ie, lactitol).

Example 2. Evaluation of the effect of the combination of 3-phenyllactic acid and probiotics in improving bacterial phase

In this example, a combination of 3-phenyllactic acid and a prebiotic (eg, lactitol) was used to evaluate the efficacy of bacterial phase improvement in the vagina of women.

The combination of 3-phenyllactic acid and prebiotics was applied in the form of feminine sanitary pads at a rate of 4 per day. First, 4 adult females were recruited as subjects, and then a sanitary napkin containing a combination of 3-phenyllactic acid and prebiotics was replaced after three meals a day and before bedtime. Next, the vaginal opening was smeared and sampled with a cotton swab before bathing, and the sampling time points were 0, 3, 7 and 14 days. The sampling process is as follows: take a sterilized cotton swab and smear the upper part of the female perineum back and forth 5~6 times, and then put the smeared cotton swab into the preservative solution containing 0.5mL (17mM sodium citrate (sodium citrate), 13mM EDTA, 46.7% ( w/v) ammonium sulfate (pH 5.2) in the sampling tube. Next, break the upper part of the cotton swab, leaving the cotton swab head in the preservation solution, then screw the cap of the sampling tube tightly, and store it at room temperature until it is sent to the laboratory for analysis.

After that, take the sampled cotton swabs, and then extract and purify the microbial samples in the cotton swabs with Presto Buccal Swab gDNA Extraction kit (Geneaid, Cat. No. GSK300-DG), then extract and purify them. The concentration and purity of the extracted DNA samples were measured. Next, 16S ribosomes were performed against Lactobacillus spp. as probiotics and Gardnerella vaginalis , Candida spp. and Escherichia coli as pathogens Quantitative real-time polymerase chain reaction (qRT-PCR) detection of gene (16S ribosomal gene). The bacterial phase composition in 4 ng DNA samples was analyzed by qRT-PCR, and each sample was tested in triplicate. The reaction conditions for qRT-PCR are shown in Table 1.

Target microorganisms, primer sequences and amplicon sizes used for qRT-PCR analysis are shown in Table 2.

The results of this example are shown in FIGS. 1 to 5 . Figure 1 is a data graph of the efficacy of the combination of 3-phenyllactic acid and prebiotics of the present invention in improving the bacterial phase of the first subject. Figure 2 is a data graph of the efficacy of the combination of 3-phenyllactic acid and prebiotics of the present invention in improving the bacterial phase of the second subject. Figure 3 is a data graph of the efficacy of the combination of 3-phenyllactic acid and prebiotics of the present invention in improving the bacterial phase of the third subject. Figure 4 is a data graph of the efficacy of the combination of 3-phenyllactic acid and prebiotics of the present invention in improving the bacterial phase of the fourth subject. Figure 5 is a data graph of the efficacy of the combination of 3-phenyllactic acid and prebiotics of the present invention in improving the average bacterial phase of 4 subjects. As can be seen from Figures 1 to 5, compared to day 0, the number of probiotics (i.e. Lactobacillus spp.) after administration of the tampon comprising the combination of 3-phenyllactic acid and prebiotics was higher. increased, while the number of pathogenic bacteria (including Gardnerella vaginalis, Candida spp. and Escherichia coli) decreased. Because there is no pathogenic infection in itself, there is no significant difference.

To sum up, the combination of 3-phenyllactic acid and probiotics of the present invention can regulate and improve the bacterial phase in women's vagina, reduce the growth of pathogenic bacteria, reduce inflammation, and achieve the effect of health care of women's private parts.

The above description is exemplary only, not limiting. Any equivalent modifications or changes that do not depart from the spirit and scope of the present invention shall be included in the appended patent application scope.

<110> Dajiang Biomedical Co., Ltd.

<120> Use of probiotic composition for improving bacterial phase in female vagina and external hygiene product

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Claims (7)

Use of a probiotic composition for preparing a composition for improving bacterial phase in a female vagina, wherein the composition is an external hygiene product for applying to a female subject, the external hygiene product having The prebiotic composition and the prebiotic composition is composed of 99% (w/w) of Lactobacillus embryonica TCI378 fermentation broth with the 3-phenyllactic acid and 1% (w/w) of the lactitol, the The bacterial phase in the vagina of a female subject includes Lactobacillus having 16S rRNA defined by SEQ ID NOs: 1 and 2, Gardnerella vaginalis having 16S rRNA defined by SEQ ID NO: 3 and 4 ) and Candida having 16S rRNA as defined by SEQ ID NOs: 5 and 6, the topical hygiene product was administered in an amount of 4 tablets/day, and the female subject's The increased bacterial count percentage of the Lactobacillus in the female vagina is between 70% and 99.9% of the bacterial phase, and the reduced bacterial count percentage of the G. vaginalis and the Candida in the female vagina Below 30% of the bacterial phase. The use as described in item 1 of the patented scope of application, wherein the bacterial phase further comprises Escherichia coli . The use as described in claim 2, wherein the Gardnerella vaginalis, the Candida spp. and the Escherichia coli in the female vagina of the female subject are reduced 3 days after the external hygiene product is applied The percentage of bacterial counts is less than 30% of the bacterial phase. The use as described in item 1 of the scope of the application, wherein the external hygiene product is a feminine hygiene pad. The use according to claim 1, wherein the 3-phenyllactic acid is purified by high performance liquid chromatography (HPLC). A kind of external hygiene product, it is characterized in that, this external use hygiene product has 99% (w/w) the probiotic that the Lactobacillus prosperus TCI378 fermentation broth with 3-phenyllactic acid and the lactitol of 1% (w/w) are formed A composition such that after 3 days of administration of the external hygiene product to a female subject at 4 tablets/day, the bacterial phase in the female vagina of the female subject is defined by SEQ ID NOs: 1 and 2 of 16S rRNA. The increased bacterial count percentage of Lactobacillus was between 70% and 99.9% of the bacterial phase, and Gardnerella vaginalis by the 16S rRNA defined by SEQ ID NOs: 3 and 4 in the female vagina and by SEQ ID NO: 3 and 4. The percentage of Candida with 16S rRNA defined by ID NO: 5 and 6 was lower than 30% of the bacterial phase. The sanitary product according to item 6 of the scope of the application, wherein the external sanitary product is a feminine sanitary napkin.

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