JP7004774B2 - Uses of Lactobacillus reuteri strain GKR1 for the preparation of compositions that reduce uric acid - Google Patents

Description

CGMCC CGMCC15201

The present invention relates to the use of a single Lactobacillus strain for the preparation of a composition that lowers uric acid, and in particular to the use of the Lactobacillus creuteri strain GKR1 for the preparation of a composition that lowers uric acid.

Purins in the human body are mainly excreted by the kidneys after the liver synthesizes uric acid. In recent years, the proportion of patients with hyperuricemia has been on the rise due to changes in dietary patterns. Serum uric acid when exposed to high pudding foods and drinks (eg, seafood, animal guts, broth, beer, etc.), high-fat diets (eg, fried foods), stress, overwork, sugar-containing beverages and / or strenuous exercise in life May improve concentration. When the serum uric acid concentration deviates from the normal value, it is called hyperuricemia, which is a highly dangerous factor that causes gouty arthritis, nephropathy, vascular heart damage and the like. In general, it is considered that hyperuricemia can be determined when the serum uric acid concentration in women is higher than 6 mg / dL or the serum uric acid concentration in men is higher than 7 mg / dL.

Currently, as drugs that lower serum uric acid concentration, drugs that inhibit uric acid synthesis [for example, trade names: allopurinol tablets and Febuxostat], and drugs that promote the excretion of uric acid [for example, trade names]. : Probenecid tablets (Probenecid) and Benzbromarone tablets (Benzbromarone)] are roughly classified into two types. However, when the above drug is taken for a long period of time, the following side effects are likely to occur. For example, long-term use of allopurinol tablets can cause Stevens-Johnson syndrome or toxic epidermal necrolysis, with a case fatality rate of 20% to 25%. high. Long-term administration of Febuxostat is likely to cause cardiovascular-related symptoms such as heart disease or stroke. Long-term use of probenecid tablets and benzbromarone tablets is prone to urinary stones and is unsuitable for patients with renal insufficiency.

According to previous studies on probiotics, complex strains [eg, Lactobacillus acidophylus and Lactobacillus rhamnosus] can be used to reliably reduce hyperuricemia. However, the interaction or antagonism between complex strains is very complex, and complex strains composed of different strains have not been experimentally proven, and any combination of complex strains is effective for a specific indication. I can't predict. Little research has been done on the treatment of hyperuricemia with a single strain of probiotics.

In view of this, in order to solve the above problems, it is desired to provide a single probiotic strain for reducing serum uric acid.

Therefore, one aspect of the present invention provides the use of the Lactobacillus reuteri strain GKR1 for the preparation of a composition that lowers uric acid.

According to the above aspect of the present invention, in the use of the Lactobacillus reuteri strain GKR1 for the preparation of a composition that lowers uric acid, the composition is a Chinese common microorganism with deposit number CGMCC15201 on July 10, 2019. Lactobacillus re. Submit the use.

According to the above examples of the present invention, the composition comprises a culture containing Lactobacillus reuteri strain GKR1, a liquid containing the culture, a liquid concentrate and / or a dried product.

According to the above examples of the present invention, the composition is an oral composition.

According to the above examples of the present invention, the composition is a food composition or a pharmaceutical composition.

According to the above examples of the invention, the composition further comprises a food or pharmaceutically acceptable carrier, excipient, diluent, auxiliary agent and / or additive.

According to the above examples of the present invention, the dosage form of the composition is an aqueous solution, a suspension, a dispersion, an emulsion, a hydrocolloid, a gel, a solid lipid nanoparticle, a tablet, a granule, a powder and / or It is a capsule.

According to the above examples of the present invention, when the composition is administered to mice, the effective amount of Lactobacillus reuteri strain GKR1 in the composition is at least 0.2 g / Kg body weight / day.

According to the above examples of the present invention, when the composition is administered to an adult, the effective amount of Lactobacillus reuteri strain GKR1 in the composition is at least 0.016 g / Kg body weight / day.

When the composition containing the Lactobacillus reuteri strain GKR1 of the present invention is applied and administered to a subject, serum uric acid can be lowered, and hyperuricemia can be effectively reduced and / or prevented.

The detailed description of the accompanying drawings below is intended to make the above and other objects, features, merits and embodiments of the present invention easier to understand.
It is a column graph which shows the viability of a strain in the culture medium of a different pH value by one Example of this invention. 3 is a columnar graph showing the survival rate of the strain in the culture solution of 0.3% by weight of bile acid according to one embodiment of the present invention. It is a columnar graph which shows the serum uric acid concentration by one Example of this invention.

Based on the above, the present invention relates to the use of the Lactobacillus reuteri strain GKR1 for the preparation of a composition that lowers uric acid, wherein the composition is commonly used in China on July 10, 2019 with a deposit number CGMCC15201. Lactobacillus reuteri (Lactobacillus lactobacillus) for the preparation of a composition containing an effective amount of Lactobacillus reuteri strain GKR1 deposited at the China General Microbiological Culture Center (CGMCC). Provide uses.

In the examples, the composition may be a culture containing Lactobacillus reuteri strain GKR1, a liquid containing the culture, a liquid concentrate and / or a dry product. The above-mentioned culture is a product obtained after the Lactobacillus reuteri strain GKR1 has been subjected to a culture step. The culture form is not particularly limited and may be a conventional solid culture or a liquid culture. The liquid may be a liquid obtained by adding a nutrient solution or a similar substance to the culture after the culture step. The concentrate is a product obtained after the culture and / or the liquid has been subjected to a conventional concentration treatment method (for example, an evaporation concentration method or a membrane concentration method). The dried product is a product obtained by subjecting the culture and / or the liquid to a conventional water removal treatment method (for example, freeze-drying method, vacuum drying method or spray drying method).

The above composition may contain an oral composition, and the type of the oral composition is not particularly limited, and may contain Lactobacillus reuteri strain GKR1.

In the examples, the composition may be, for example, a food composition or a pharmaceutical composition. In the examples, the composition is, for example, a solvent, an emulsifier, a suspending agent, a disintegrant, an adhesive, a stabilizer, a chelating agent, a diluent, a gelling agent, a preservative, a lubricant and / or an absorption retardant. , Food or pharmaceutically acceptable carriers, excipients, diluents, auxiliaries and / or additives may be selectively included.

The dosage form of the composition of the present invention is not particularly limited. In the examples, the dosage forms of the compositions include aqueous solutions, suspensions, dispersions, emulsions (single-phase or polyphase dispersions, single-layer or multi-layered liposomes), hydrocolloids, gels, solid lipid nanos. It may be particles, tablets, granules, powders and / or capsules and the like.

Examples of the food composition include grain products, fruit products, vegetable products, meat products, fish products, egg products, dairy products, beverages, health foods, health foods, functional foods, and nutritional supplements. Alternatively, it may be a special nutrition food, but is not limited thereto.

When the above composition is applied, the administration route thereof is not particularly limited, and may be, for example, oral administration, which may be adjusted according to actual requirements. The dose and frequency of administration of the above composition may also be flexibly adjusted as required. In the examples, when the composition is administered to mice, the effective amount of Lactobacillus reuteri strain GKR1 in the composition is at least 0.2 g / Kg body weight / day. When the composition is administered to an adult, the effective amount of Lactobacillus reuteri strain GKR1 in the composition is at least 0.016 g / Kg body weight / day. [Estimating the maximum safe starting dose in clinical trials for therapeutics in adult body weight per kilogram per person (reference volume per kilogram per kilogram), published by the U.S. Food and Drug Administration in 2005. Convert 12.3 times / kg b.w./d) as 1 times the dose of mice]. The number of administrations may be once a day or divided into several doses.

The composition containing the Lactobacillus reuteri strain GKR1 of the present invention can reduce serum uric acid and can effectively reduce and / or prevent hyperuricemia when administered to a subject. Confirmed experimentally.

Hereinafter, the application of the present invention will be described with reference to Examples, but this is not intended to limit the present invention, and those skilled in the art can make various changes and modifications as long as they do not deviate from the spirit and scope of the present invention. Can be added.

1. 1. Properties of Lactobacillus reuteri strain (1) Preparation of Lactobacillus reuteri strain GKR1

The Lactobacillus reuteri strain GKR1 was deposited at the China Common Microbial Species Conservation Management Center (CGMCC) on July 10, 2019 under the deposit number CGMCC15201.
(2) Evaluation of acid resistance of Lactobacillus reuteri strain GKR1

Acid resistance was tested against Lactobacillus reuteri strain GKR1 and commercially available Lactobacillus reuteri strains (BCRC80379, BCRC80063, BCRC17477, BCRC16091 and BCRC14625). First, the pH values of the cultures were adjusted to pH 6.5 (group without HCl), pH 3.2, with or without addition of HCl to the MRS cultures (De Man, Rogosa & Sharpe broth, MRS broth), respectively. The pH was 2.4 and 2.0. Next, after activating the strain, it was added to a culture solution having a different pH value, and after culturing at 37 ° C. for 3 hours, the number of colonies was calculated. The survival rate of the strains at pH 6.5 (group without addition of HCl) was set to 100%, and the survival rates of the strains at different pH values in the culture medium were converted.

Please refer to FIG. 1, which is a columnar graph showing the viability of the strain in the culture medium having different pH values according to one embodiment of the present invention. The X-axis is the pH values of the culture solutions of pH 6.5, pH 3.2, pH 2.4 and pH 2.0 from left to right, the Y-axis is the cell viability (%), and the vertical bar 101 is Lactobacillus reuteri. The vertical bar 103 represents the survival rate of Lactobacillus reuteri strain BCRC80379, the vertical bar 105 represents the survival rate of Lactobacillus reuteri strain BCRC80063, and the vertical bar 107 represents the survival rate of Lactobacillus reuteri strain BCRC17477. The vertical bar 109 represented the viability of Lactobacillus reuteri strain BCRC16091, and the vertical bar 111 represented the viability of Lactobacillus reuteri strain BCRC14625.

According to the results of FIG. 1, when the pH values of the culture solution are pH 3.2, pH 2.4 and pH 2.0, the Lactobacillus reuteri strain GKR1 (vertical bar 101) of the present invention has a cell viability of 90%. It was higher than 67% and 8%, and the survival rate of both was higher than that of commercially available Lactobacillus reuteri.
(3) Evaluation of lactobacillus reuteri strain GKR1 for bile acid resistance

Lactobacillus reuteri strain GKR1 and commercially available Lactobacillus reuteri strains (BCRC80379, BCRC80063, BCRC17477, BCRC16091 and BCRC14625) were tested for biliary acid resistance. First, the number of colonies was calculated after soaking at 37 ° C. for half an hour with or without adding 0.3% by weight bile acid salt to MRS (De Man, Rogosa & Sharpe broth, MRS) culture medium. did. The survival rate of the strain in the culture solution containing 0.3% by weight of bile acid was converted, assuming that the survival rate of the strain in the culture solution to which no bile acid was added was 100%.

Please refer to FIG. 2, which is a columnar graph showing the viability of the strain in the culture solution of 0.3% by weight of bile salt according to one embodiment of the present invention, and the X axis is added with bile salt from left to right, respectively. The Y-axis is the cell viability (%), the vertical bar 201 represents the viability of the Limosilactobacillus reuteri strain GKR1, and the vertical bar 203 is the Limosilactobacillus. The vertical bar 205 represents the survival rate of the Limosilactobacillus strain BCRC80379, the vertical bar 207 represents the survival rate of the Limosilactobacillus strain BCRC17477, and the vertical bar 209 represents the survival rate of the Limosilactobacillus strain BCRC16091. The vertical bar 211 represented the rate and the viability of the Limosilactobacillus reuteri strain BCRC14625.

According to the results of FIG. 2, the Lactobacillus reuteri strain GKR1 of the present invention has a commercially available cell viability (for example, as shown in vertical bar 201) in a culture medium supplemented with 0.3% by weight of bile acid. It was found to be significantly higher than Lactobacillus reuteri.

2. 2. Creation of animal mode for hyperuricemia mice (1) Induction of hyperuricemia mice

The mouse is an ICR male mouse purchased from the Philadelphia Cancer Institute (Institute of Cancer research) of BioLASCO Taiwan Co., Ltd., Taiwan, and is 5 weeks old and has a weight. Was 27.5 g to 32.5 g. Mice were bred at a temperature of 22 ± 3 ° C., a humidity of 40% to 70%, 12 hours of light and 12 hours of dark circulating light irradiation, and were given sufficient feed and sterile reverse osmosis water to allow the mice to freely ingest. rice field. After acclimatization and adaptation for one week, animal experiments were performed. The experimental process was carried out in accordance with the Agricultural Agency's laboratory animal protection and usage guidance and the relevant provisions of the Taiwan Animal Protection Law.

Prior to the animal experiment, the mice were randomly divided into 4 groups of 8 mice per group, 4 animals per cage, and 2 cages per group. The control group was fed a normal diet and ingested. Negative control groups were fed daily with a diet containing 2.5% by weight of potassium oxonate and 1% by weight of RNA to induce hyperuricemia, but with any drug. I didn't give it. Hyperuricemia was induced in the positive control group by the same method as in the negative control group, and 5 mg / kg b. Daily. w. Allopurinol was orally administered. In the Lactobacillus reuteri (GKR1) group, hyperuricemia was induced by the same method as in the negative control group, and 0.205 g / kg b. Daily. w. Lactobacillus reuteri (GKR1) was orally administered.

This experiment was carried out for a total of 7 days, and after the experiment was completed, 0.5 mL to 0.8 mL of blood was collected from the orbit and the serum uric acid concentration was analyzed.
(2) Evaluation of each basic physiological parameter of mouse

During the experiment, changes in mouse weight and intake were recorded daily and changes in each basic physiological parameter of the mice were observed. Table 1 shows the weight changes of mice during the experiment, in grams (g), the data are statistic by the Duncan test, and there are significant differences between groups when different lowercase letters are compared (). It was shown that p <0.05), and that the same lowercase alphabetic markings did not make a significant difference between the groups (p> 0.05). According to Table 1, it was found that there was no significant difference in the body weight of the mice between the groups on the 0th and 7th days.

Table 2 shows the changes in body weight of the mice on the 7th day of the experiment, and there was no significant difference in the intake of the mice in each group.

According to each of the above physiological parameters, it was explained that the intake of Lactobacillus reuteri strain GKR1 did not affect the growth state of mice.

3. 3. Lowering effect of Lactobacillus reuteri strain GKR1 on serum uric acid

Serum uric acid concentration was detected with a commercially available uric acid quantification kit (QuantiChrom Uric Acid Assay Kit, BioAssay Systems; USA), and the serum uric acid (UA) concentration in mice was evaluated.

Please refer to FIG. 3, which is a columnar graph showing the serum uric acid concentration according to one embodiment of the present invention. The X-axis is from left to right the control group, the negative control group, the positive control group and the Lactobacillus reuteri (GKR1) group. The Y-axis is the serum uric acid concentration, and the unit is mg / dL.

According to the results of FIG. 3, it was found that the serum uric acid concentration of the Lactobacillus reuteri (GKR1) group was clearly lower than that of the negative control group.

As can be seen from the above examples, the use of the Lactobacillus reuteri strain GKR1 for preparing the composition for reducing uric acid of the present invention is to contain the single strain Lactobacillus reuteri strain GKR1. When the composition is administered to a subject, it has an advantage that serum uric acid can be lowered and hyperuricemia can be effectively reduced and / or prevented.

It should be understood that although the present invention created an animal mode of hyperuricemia in male mice, it should not be used to limit the sex of the subject of Lactobacillus reuteri strain GKR1. Those skilled in the art should understand that Lactobacillus reuteri strain GKR1 has the same or similar effect on female mice, as the lowering effect of Lactobacillus reuteri strain GKR1 on serum uric acid has been demonstrated in male mice. Is.

What should be supplemented is that the present invention illustrates the use of the Lactobacillus reuteri strain GKR1 for the preparation of a composition that reduces uric acid of the present invention, exemplifying a specific process and / or a specific analytical method. However, if you are a person skilled in the art, the present invention is not limited to this, and the composition containing the Lactobacillus reuteri strain GKR1 of the present invention can be used in other processes without departing from the spirit and scope of the present invention. It should be understood that other analytical methods or other instruments may be used.

Although the present invention has been described above by a plurality of examples, it is not intended to limit the present invention, and those skilled in the art will have various variations and modifications as long as they do not depart from the spirit and domain of the present invention. Therefore, the scope of protection of the present invention is based on the contents specified in the claims.

101, 103, 105, 107, 109, 111, 201, 203, 205, 207, 209, 211: Vertical bar

Claims (8)

A composition comprising the Lactobacillus reuteri strain GKR1 for reducing uric acid.
The composition comprises an effective amount of Lactobacillus reuteri strain GKR1 deposited with the China Common Microbial Species Conservation Control Center (CGMCC) under Deposit No. CGMCC15201 on July 10, 2019 . The Lactobacillus reuteri strain for reducing uric acid according to claim 1 , which comprises a culture containing the Lactobacillus reuteri strain GKR1, a liquid containing the culture, a concentrate of the liquid and / or a dried product. A composition comprising GKR1 . The composition according to claim 1 , which is an oral composition, comprising the Lactobacillus reuteri strain GKR1 for reducing uric acid . The composition according to claim 1 , which is a food composition or a pharmaceutical composition , comprising Lactobacillus reuteri strain GKR1 for reducing uric acid . The composition comprising the Lactobacillus reuteri strain GKR1 for reducing uric acid according to claim 4 , further comprising a food or pharmaceutically acceptable carrier, excipient, diluent, auxiliary agent and / or additive. thing. The uric acid according to claim 1 , wherein the dosage form is an aqueous solution, a suspension, a dispersion, an emulsion, a hydrocolloid, a gel, solid lipid nanoparticles, tablets, granules, powders and / or capsules. A composition comprising the lactobacillus reuteri strain GKR1 for reducing . The reduced uric acid according to claim 1, wherein when the composition is administered to mice, the effective amount of the Lactobacillus reuteri strain GKR1 in the composition is at least 0.2 g / Kg body weight / day. A composition comprising the Lactobacillus reuteri strain GKR1 for the purpose . The reduced uric acid according to claim 1 , wherein when the composition is administered to an adult, the effective amount of the Lactobacillus reuteri strain GKR1 in the composition is at least 0.016 g / Kg body weight / day. A composition comprising the Lactobacillus reuteri strain GKR1 for the purpose .

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