TWI451871B - Anti-inflammatory and anti-vaginitis food composition and pharmaceutical composition containing lactobacillus - Google Patents

Description

Lactic acid-containing food composition and pharmaceutical composition for inhibiting inflammatory reaction and anti-vaginitis

The present invention relates to a food composition and a pharmaceutical composition, and more particularly to a food composition comprising a lactic acid bacteria strain and a pharmaceutical composition for inhibiting an inflammatory reaction.

Probiotics are defined as active microorganisms that promote the health of the host, as discussed in international publications. Probiotics have the ability to promote human health, have been discovered at the end of the last century, and gradually carry out many scientifically validated studies. It was first discovered that probiotics have a soothing ability for the symptoms of gastrointestinal discomfort. Until today, adjusting gastrointestinal function is still an important part of probiotics in product application.

However, in addition to the function of the gastrointestinal tract, the function of probiotics in immunomodulation has begun to emerge in recent years. Many literatures on probiotics and immunity have been published, and the results of the research have sprung up. The ability of probiotics to regulate immune regulation. Can be seen. At present, in the immune function of probiotics, it has improved immunity, adjusted allergies, assisted in alleviating autoimmune diseases and anti-inflammatory, etc., all in the research field. Generally, the products containing Lactic acid bacteria (LAB) only have the health effect of regulating the intestinal tract. Although there are tens of thousands of lactic acid bacteria strains in nature, only a few lactic acid bacteria strains have the characteristics of regulating immunity and even anti-inflammatory reaction.

To date, only a few strains of lactic acid bacteria have been identified that have been shown to have a health-improving immune effect, and the function of lactic acid bacteria in terms of body health lies in the specificity of the strain rather than the species. The guidelines for the evaluation of probiotics in food; Report of joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food; London Ontario, Canada April 30 and May 1, 2002: 1-7).

Anti-inflammatory probiotic strains have the effect of soothing symptoms for vaginal inflammation, especially through oral route, which can accelerate the relief of vaginal inflammation. There are many reports of probiotics and anti-vaginitis in the literature at home and abroad. In vitro, Lactobacillus crispatus and Lactobacillus jensenii have been found to produce H ₂ O ₂ and bacteriostatic compounds, which are effective in inhibiting and killing pathogenic strains in the vagina (Jasadee et al. , 2006). Lactobacillus acidophilus , Lactobacillus rhamnosus , Lactobacillus plantarum and Lactobacillus fermentum strains were co-cultured with vaginal candida and found to be effective in inhibiting the growth of vaginal candida (Magdalena et al. , 2005). The vaginitis pathogenic strains Gardnerella vaginalis and Prevotella bivia were co-cultured with HeLa cells (simulating vaginal mucosal endothelial cells), and the pathogenic strains were adsorbed on the cells, and then Lactobacillus gasseri was co-cultured. It was found that Lactobacillus gasseri could destroy Gardnerella vaginalis and Prevotella bivia. In addition to the adsorption of HeLa cells, the pH value between 4.5 and 5.5 has the best destructive effect, indicating that probiotics can inhibit and kill vaginitis-causing strains, and maintain the weak acidity of vaginal mucosa, for inhibiting pathogenic strains. Both the activity and the activity of probiotics are greatly helpful (Fabrice et al. , 2006). These documents confirm that in the in vitro simulation of vaginitis flora, probiotics can indeed fight the pathogenic flora of vaginitis, thereby improving the symptoms of vaginitis.

Lactobacillus acidophilus is fermented in Lactobacillus acidophilus in clinical trials. The number of bacteria is more than 10 ⁸ and the vaginitis patients take 2 months. The improvement of patients is 60%, which is much larger than that of the placebo group. 25% (Shalve et al. , 1996). The tampon was infiltrated with Lactobacillus acidophilus and 5% acetic acid for 4 to 8 weeks of vaginal inflammation. The degree of vaginitis relief was 88%, which was much higher than 38% of the acetic acid group (Neri). Et al. , 1993). Capsules containing more than ¹⁰⁹ strains of Lactobacillus rhamnosus and Lactobacillus fermentum strains, once a day for 60 days, had an improvement of vaginitis of 87.5% (Reid et al. , 2004).

However, only a few lactic acid bacteria strains have the properties of acid and bile salt resistance, ability to adsorb mucosal epithelial cells, and ability to survive after passage through the gastrointestinal tract. Therefore, it is necessary to screen for strains that promote health effects and can become functional probiotics. .

In view of the above, finding a suitable Lactobacillus as an anti-inflammatory, particularly anti-vaginal effect, as a functional probiotic strain is currently a goal that requires effort.

An object of the present invention is to provide a food composition and a pharmaceutical composition which can inhibit an inflammatory reaction by inhibiting the production of tumor necrosis factor-α and/or promoting the production of interleukin-10 (IL-10).

Another object of the present invention is to provide a food composition and a pharmaceutical composition which are resistant to acidity and bile salts for use as an oral probiotic.

Another object of the present invention is to provide a food composition and a pharmaceutical composition having the tolerance of a vaginal sputum suppository for use as a treatment for vaginitis.

In one embodiment, a food composition of the invention is for use in inhibiting an inflammatory response comprising a plurality of strains of lactic acid bacteria and a physiologically acceptable excipient or diluent. The lactic acid bacteria strain contains Lactobacillus salivarius AP-32 strain, accession number BCRC 910437, Lactobacillus reuteri TE-33 strain, accession number BCRC 910441, Lactobacillus acidophilus F-1 strain, The accession number BCRC 910469 and the Lactobacillus rhamnosus CT-53 strain, accession number BCRC 910468, wherein the lactic acid bacteria strains have gastric acid, bile salts and the mold inhibitor Clotrimazole tolerance.

In another embodiment, a pharmaceutical composition of the invention is for use in inhibiting an inflammatory response comprising a plurality of strains of lactic acid bacteria and a physiologically acceptable excipient or diluent. The lactic acid bacteria strain contains Lactobacillus salivarius AP-32 strain, accession number BCRC 910437, Lactobacillus reuteri TE-33 strain, accession number BCRC 910441, Lactobacillus acidophilus F-1 strain, The accession number BCRC 910469 and the Lactobacillus rhamnosus CT-53 strain, accession number BCRC 910468, wherein the lactic acid bacteria strains have gastric acid, bile salts and the mold inhibitor Clotrimazole tolerance.

The invention may also broadly include separate or general descriptions of the description of the application. And any and all combinations of any one or more of the components, constituent elements and features, and if the context and the specifics described herein are relevant to the present invention Where known equivalents occur in the art, such known equivalents will be incorporated herein as a separate matter.

The present invention broadly encompasses any of the following biological cultures: Lactobacillus salivarius AP-32 strain, accession number BCRC 910437, Lactobacillus reuteri TE-33 strain (Accession number BCRC 910441) , F-1 strain ( Lactobacillus acidophilus , accession number BCRC 910469), CT-53 strain ( Lactobacillus rhamnosus , accession number BCRC 910468) or a combination thereof, and physiologically acceptable A food composition or pharmaceutical composition comprising an excipient or diluent. The strain of lactic acid bacteria may be an active or inactivated strain.

Wherein, the food composition may include, but is not limited to, fermented milk, yogurt, cheese, dairy drink milk powder, tea, coffee or a combination thereof.

The pharmaceutical composition may comprise an oral dosage form or an external dosage form; for example, an oral dosage form is a tablet, a capsule, a solution, a powder, and the like. The external dosage form is, for example, a cream, a spray, a gel, a powder or a cream.

In addition, the present invention is useful for anti-vaginitis and thus can be used in women's products including, but not limited to, sprays, ointments or sanitary napkins and the like.

Morphology and general properties of the lactic acid bacteria of the present invention

The freeze-dried culture of the strain of the present invention has been deposited at the Taiwan Food Industry Development Research Institute at 331 Food Road, Hsinchu City, Republic of China. The details of the deposit are shown in Table 1: Table 1: Names of lactic acid bacteria and storage materials

The taxonomic characteristics of the strains were confirmed based on the 16S rDNA sequence analysis and the results of the API bacterial identification system analysis. It was found that the strain number AP-32 was Lactobacillus saliva; the strain number TE-33 was Lactobacillus acidophilus; the strain number F-1 was Lactobacillus acidophilus; and the strain number CT-53 was Lactobacillus rhamnosus. The morphological and general characteristics of this strain are detailed in Table 2:

Inhibition of inflammation

It is known that Th17-type helper T cells secrete many cytokines (such as IL-6) under the stimulation of inflammatory hormone tumor necrosis factor-α (TNF-α) in the Th17 inflammatory response. , IL-17) and media to attract the accumulation of inflammatory cells to collectively cause an inflammatory response to infection. Interleukin-10 (IL-10) has been found to have an anti-inflammatory effect and inhibits the production of inflammatory cytokines such as IL-6 and TNF-α. Therefore, by regulating the increase of IL-10 and/or reducing TNF-α, the inflammatory response of Th17 can be inhibited, and the inflammatory phenomenon of Th17 type immune response is improved.

1. Inhibition of TNF-α secretion (using dendritic cells as an in vitro efficacy verification platform)

The inhibitory effect of Th17 type immunity was measured by the co-culture of the lactic acid bacteria to measure the amount of TNF-α secreted. Use the following experimental steps:

1. Draw an appropriate human blood volume of approximately 200 mL each time.

2. Take an equal ratio of Ficoll-paque and blood at 180-20g for 30-40 minutes at 18-20 °C.

3. Take a layer of human peripheral blood mononuclear cell (PBMC) and wash the cells 2 to 3 times with a buffer solution, and then suspend the cells in a suitable medium (for example, RPMI-1640).

4. In the CD14 ⁺ microbeads with a body (microbeads) (MiniMACS system) of the CD14 ⁺ cells were purified using monocytes from PBMC cells.

5. The cells were differentiated into dendritic cells by cytokine IL-4 and growth hormone GM-CSF, and the differentiated dendritic cells were collected at a culture time of 6-7 days.

6. The lactic acid bacteria strain was activated 3 days before co-culture, and then the lactic acid bacteria strain was heat-killed at 100 ° C for 30 minutes.

7. The dendritic cells were co-cultured with the heat-killed lactic acid bacteria strain at a ratio of 1:10 for 48 hours.

8. Collect the supernatant from the cell culture. The content of TNF-α in the supernatant was detected by an enzyme-linked-immunosorbent serologic assay (ELISA).

Statistical analysis of the data is shown in Table 3, expressed as mean ± standard deviation (Mean ± SD). When the mixed culture of 10 ⁶ to 10 ⁸ CFU strains was separately cultured with 10 ⁵ to 10 ⁷ human dendritic cells for 48 hours, the cell supernatant was collected, and TNF-α was detected by enzyme immunoassay. The content in the supernatant. Among them, a mixed culture of commercially available anti-vaginal functional products (Biocan Vagi-guard ^® , whose active ingredient is lactic acid bacteria) was used as a control group, and lipopolysaccharide (LPS) was a positive control group containing only cells (Cell only). The experimental background value was the negative control group, and the TNF-α stimulated concentration was detected. The results showed that the test group of the present invention can significantly inhibit the secretion of TNF-α from human dendritic cells and the positive control group (LPS) is significantly different from the positive control group (LPS) and the control group (Biocan Vagi-guard ^® ). The amount is less than 84 times and 135 times respectively.

Second, promote IL-10 secretion (using dendritic cells as an in vitro efficacy verification platform).

To measure the increase in IL-10 secretion, human dendritic cells were co-cultured with the aforementioned lactic acid bacteria, and the amount of IL-10 secreted was measured to determine the effect of enhancing the immunity against inflammatory cytokines. Use the following experimental steps:

1. Draw an appropriate human blood volume of approximately 200 mL each time.

2. Take an equal ratio of Ficoll-paque and blood at 180-20g for 30-40 minutes at 18-20 °C.

3. Take a layer of human peripheral blood mononuclear cell (PBMC) and wash the cells 2 to 3 times with a buffer solution, and then suspend the cells in a suitable medium (for example, RPMI-1640).

4. Purify the CD14+ mononuclear cells of the cells from PBMC cells with microbeads (MiniMACS system) with CD14+.

5. The cells were differentiated into dendritic cells by cytokine IL-4 and growth hormone GM-CSF, and the differentiated dendritic cells were collected at a culture time of 6-7 days.

6. The lactic acid bacteria strain was activated 3 days before co-culture, and then the lactic acid bacteria strain was heat-killed at 100 ° C for 30 minutes.

7. The dendritic cells were co-cultured with the heat-killed lactic acid bacteria strain at a ratio of 1:10 for 48 hours.

8. Collect the supernatant from the cell culture. The content of IL-10 in the supernatant was detected by ELISA.

Statistical analysis of the data is shown in Table 4, expressed in Mean ± SD. When the mixed culture of 10 ⁶ to 10 ⁸ CFU of heat death was co-cultured with 10 ⁵ to 10 ⁷ human dendritic cells for 48 hours, the cell supernatant was collected, and IL-10 was detected by enzyme immunoassay. The content in the supernatant. Among them, the experimental background value containing only cell (Cell only) was negative control group, the mixed culture of anti-vaginal function product (Biocan Vagi-guard ^® ) was used as the control group, and the phytohemagglutinin (PHA) was positive. The control group was used to detect the IL-10 stimulated concentration. The results showed that the test group of the present invention can significantly stimulate the secretion of IL-10 by human dendritic cells and the negative control group (Cell only) is significantly different, and is 5 times higher than the control group (Biocan Vagi-guard ^® ). . Table 4 shows the secretion of IL-10 by the culture of the heat-killing lactic acid bacteria mixed bacteria and the control group and the control group, respectively.

Test of gastric acid resistant bile salt of lactic acid bacteria of the invention

One of the objects of the present invention is to provide a new choice for the treatment of vaginitis in addition to drugs. Alternatively, the present invention finds lactic acid bacteria that have no side effects and are beneficial to the human body as a new option for the treatment of vaginitis.

Since oral probiotics should exert anti-vaginal effects, in addition to identifying strains with specific functions, it is necessary to confirm that the strains can pass the environment of human gastric acid bile salts, and also have lactic acid bacteria that are resistant to common drugs for vaginitis. The strain, therefore, is required to have the above characteristics, and the lactic acid bacteria of the present invention can provide medical treatment for treating or soothing vaginal symptoms, and reduce the recurrence rate of vaginitis.

In order to examine whether the lactic acid bacteria strain of the present invention has the ability to pass the test of gastric acid bile salt smoothly and exert its anti-inflammatory function in the intestinal tract, the test procedure is as follows:

1. The lactic acid bacteria of the present invention were activated for 3 days.

2. Calculate the original number of bacteria by taking 1 mL of the bacterial solution, and centrifuge the remaining lactic acid bacteria at 500 g for 10 minutes, and add the deionized water to wash the lactic acid bacteria 2 to 3 times.

3. The medium was mixed with hydrochloric acid to a pH of 2.5, and the lactic acid bacteria of the present invention were thoroughly mixed with a medium having a pH of 2.5, and placed in an incubator at 37 °C.

4. After removing 1 mL of the bacterial liquid per hour and washing it with deionized water for 2 to 3 times, the number of viable cells was counted until the incubation time of 3 hours.

5. After the remaining bacterial solution was centrifuged, the precipitate was reconstituted in a medium containing 1.5% (w/V) bovine bile (ox gall, Sigma), thoroughly mixed, and then cultured at 37 °C.

6. After removing 1 mL of the bacterial liquid per hour and washing it with deionized water for 2 to 3 times, the number of viable cells was counted until the incubation time of 4 hours.

7. Record the growth rate of lactic acid bacteria, calculate the tolerance of lactic acid bacteria to gastric acid and bile salts, and compare whether the growth of the lactic acid bacteria of the present invention in the presence of gastric acid and bile salts in the sample is inhibited.

The results and analysis of the ability to resist gastric acid are shown in Table 5 and Figure 1. The results show that the lactic acid bacteria of the present invention treat the strain with an acidic buffer solution and bile salts after the medium is activated. The number of AP-32, TE-33, F-1 and CT-53 bacteria was not affected by gastric acid. The results and analysis of the ability to resist bile salts are shown in Table 6 and Figure 1. In the bile salt part, except for the sensitivity of CT-53, the number of bacteria decreased, AP-32, TE-33 and F-1. The strains are not affected by bile salts. The above proves that the lactic acid bacteria of the present invention can pass the test of the human digestive system environment.

Tolerance test of lactic acid bacteria of the invention for treating vaginitis commonly used as a suppository inhibitor, Clotrimazole

A high-density bacterial count was applied to the plate medium to grow the strain. The fungicide Clotrimazole was placed in the center of the plate and cultured at 37 ° C for 48 hours. The diameter of the inhibition ring was observed to determine the tolerance of the strain. In the tolerance judgment portion, the diameter of the inhibition zone is 4 mm or less for good tolerance, and the tolerance is medium to more than 4 mm and less than or equal to 5 mm, and the tolerance is greater than 5 mm or more. Tolerance results are summarized in Table 7 and Figure 2, which show that strains of AP-32, TE-33, F-1, and CT-53 were all well tolerated.

The lactic acid bacteria combination of the invention has anti-inflammatory reaction and has tolerance to bile salts, acid and mildew inhibitors, and thus is used for oral probiotics and has anti-vaginal ability, including inhibition of vaginal pathogenic flora and inflammation. Symptoms, such as redness, itching, pain, etc., can be used in sprays or sanitary napkins to promote women's health.

The embodiments described above are only intended to illustrate the technical idea and the features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the contents of the present invention and to implement the present invention. That is, the equivalent variations or modifications made by the spirit of the present invention should still be included in the scope of the present invention.

Figure 1 shows that the number of Lactobacillus saliva AP-32 strain, Lactobacillus acidophilus TE-33 strain, Lactobacillus acidophilus F-1 strain and Lactobacillus rhamnosus CT-53 strain is not affected by gastric acid, bile salt In addition to the sensitivity of the Lactobacillus rhamnosus CT-53 strain, Lactobacillus saliva AP-32 strain, Lactobacillus strain L-33 strain, Lactobacillus acidophilus F-1 strain are not affected by bile salts. The number has dropped sharply, which proves that the anti-vaginal lactic acid bacteria of the present invention can pass the test of the strict environment of the human digestive system.

2 shows that the Lactobacillus salivarius AP-32 strain, the Lactobacillus acidophilus TE-33 strain, the Lactobacillus acidophilus F-1 strain, and the Lactobacillus rhamnosus CT-53 strain have diameters of 4 mm or less, and thus A strain that was judged to be well tolerated.

Claims (7)

A food composition for inhibiting an inflammatory reaction, comprising: a plurality of strains of lactic acid bacteria comprising Lactobacillus salivarius AP-32 strain, registered number BCRC 910437, Lactobacillus reuteri TE-33 strain, deposited No. BCRC 910441, Lactobacillus acidophilus F-1 strain, accession number BCRC 910469 and Lactobacillus rhamnosus CT-53 strain, accession number BCRC 910468, wherein the lactic acid bacteria strain has stomach acid and gallbladder Salt and mold inhibitor Clotrimazole tolerance; and a physiologically acceptable excipient or diluent. The food composition of claim 1, wherein the excipient or the diluent is fermented milk, yogurt, cheese, milk powder for dairy drinks, tea, coffee or a combination thereof. The food composition of claim 1, wherein the lactic acid bacteria strain is a strain having activity or deactivation. The food composition according to claim 1 is for use against vaginitis. A pharmaceutical composition for inhibiting an inflammatory reaction, comprising: a plurality of strains of lactic acid bacteria comprising Lactobacillus salivarius AP-32 strain, accession number BCRC 910437, Lactobacillus reuteri TE-33 strain, and storage No. BCRC 910441, Lactobacillus acidophilus F-1 strain, accession number BCRC 910469 and Lactobacillus rhamnosus CT-53 strain, accession number BCRC 910468, wherein the lactic acid bacteria strain has stomach acid and gallbladder Salt and mold inhibitor Clotrimazole tolerance; and a pharmaceutically acceptable excipient or diluent. The pharmaceutical composition according to claim 5, wherein the lactic acid bacteria strain is a strain having activity or deactivation. The pharmaceutical composition according to claim 5 is for use against vaginitis.