Composition for IAP activation

Abstract

To provide a novel composition for IAP activation.SOLUTION: The present invention provides a composition for IAP activation, a pharmaceutical composition for IAP activation, a food composition for IAP activation, or a feed composition for IAP activation, each containing D-lactic acid as an active ingredient. Further provided are a composition for prevention or improvement of intestinal inflammation, a pharmaceutical composition for prevention or improvement of intestinal inflammation, a food composition for prevention or improvement of intestinal inflammation, or a feed composition for prevention or improvement of intestinal inflammation, each containing the composition for IAP activation.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a component that enhances intestinal alkaline phosphatase (IAP) activity. [Background technology]

[0002] Intestinal alkaline phosphatase (IAP) is a dephosphorylating enzyme expressed on the small intestinal brush border. IAP detoxifies inflammation-inducing substances (e.g., lipopolysaccharides and flagellin) derived from enterobacteria in the intestinal lumen, thereby preventing inflammation in the intestinal tract (Non-Patent Documents 1 and 2).

[0003] Decreased IAP activity has been reported to be involved in various diseases, including diabetes, chronic renal failure, metabolic syndrome, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), necrotizing enterocolitis (NEC), sepsis, and antibiotic-associated diarrhea (Non-Patent Documents 1 and 2). Therefore, activating IAP is expected to have preventive and ameliorative effects on these diseases.

[0004] Various substances have been reported to activate IAP. Examples include hormones such as adrenocorticotropic hormone and thyroid hormone, lipids such as long-chain and medium-chain triacylglycerols, carbohydrates such as starch, cellulose, and oligosaccharides, as well as amino acids, peptides, vitamins, and minerals (Non-Patent Documents 1 and 2). Some of these components have been suggested to have inhibitory effects on the various diseases mentioned above (Non-Patent Documents 1 and 2). Since food components have been reported to have the ability to activate IAP, it is thought that improving the nutritional composition of one's daily diet can maintain high IAP activity and prevent various diseases.

[0005] Short-chain fatty acids are known to be one of the substances that activate IAP. For example, Non-Patent Document 3 reports that propionic acid, butyric acid, and valeric acid enhance IAP gene expression in human colon adenocarcinoma cell lines. It has also been reported that butyric acid activates IAP in mouse intestinal epithelial cell lines and suppresses inflammation induced by lipopolysaccharide (Non-Patent Document 4). However, these documents do not examine the IAP-activating effect of lactic acid. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lalles JP. Recent advances in intestinal alkaline phosphatase, inflammation, and nutrition. Nutr Rev.2019 October;77(10):710-724. [Non-patent document 2] Lalles JP. Intestinal alkaline phosphatase: multiple biological roles in maintenance of intestinal homeostasis and modulation by diet. Nutr Rev. 2010 Jun;68(6):323-32. [Non-patent document 3] Hinnebusch, BF, Meng, S., Wu, JT, Archer, SY, & Hodin, RA The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation. J Nutr. 2002 May;132(5):1012-7. [Non-patent document 4] Bol-Schoenmakers M., Fiechter D., Raaben W., Hassing I., Bleumink R., Kruijswijk D., Maijoor K., Tersteeg-Zijderveld M., Brands R., & Pieters R. Intestinal alkaline phosphatase contributes to the reduction of severe intestinal epithelial damage. Eur J Pharmacol. 2010 May 10;633(1-3):71-7. Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a novel composition for activating IAP. [Means for solving the problem]

[0008] As a result of extensive research to solve the above problems, the inventors discovered that D-lactic acid has a strong IAP activation effect, and that the use of D-lactic acid can produce a composition with high IAP activation ability.

[0009] Specifically, the present invention is as follows: The term "composition" as used herein includes preparations, foods and drinks, feeds and other substances that can be ingested by animals (including humans). <1> A composition for activating IAP, containing D-lactic acid as an active ingredient. <2> The concentration of D-lactic acid is 0.135% by mass or more in terms of free lactic acid. <1> The composition for activating IAP described above. <3> <1> or <2> A composition for preventing or ameliorating intestinal inflammation, comprising the composition for activating IAP described in 1. <4> <1> or <2> A pharmaceutical composition for activating IAP, a food composition for activating IAP, or a feed composition for activating IAP, comprising the composition for activating IAP described above. <5> <1> or <2> A pharmaceutical composition for preventing or improving intestinal inflammation, a food composition for preventing or improving intestinal inflammation, or a feed composition for preventing or improving intestinal inflammation, comprising the IAP activating composition described in the above. [Effects of the Invention]

[0010] According to the present invention, a composition for activating IAP containing D-lactic acid as an active ingredient can be provided. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a graph showing IAP activity (U / mg protein) when 3.75 to 30 mM L-lactic acid or 3.75 to 30 mM D-lactic acid was added to human colon cancer-derived cell line Caco-2, which had been cultured for two weeks, and then cultured for one week. DETAILED DESCRIPTION OF THE INVENTION

[0012] The composition for activating IAP of the present invention is characterized by containing D-lactic acid as an active ingredient. The present invention also relates to pharmaceutical compositions, food compositions, and feed compositions for activating IAP, which contain the composition. Furthermore, the present invention relates to compositions for preventing or ameliorating intestinal inflammation, which contain the composition for activating IAP. The active ingredients and compositions for each purpose are described below.

[0013] (D-lactic acid) The active ingredient of the present invention, D-lactic acid, refers to the D-form of lactic acid (chemical formula CH3-CH(OH)-COOH), an organic acid. Unless otherwise specified, lactic acid in this specification may be free lactic acid or a lactate salt, both of which are included in the lactic acid of the present invention. Lactate salts are not particularly limited, but examples include alkali metal salts such as sodium lactate and potassium lactate, alkaline earth metal salts such as calcium lactate and magnesium lactate, aluminum lactate, zinc lactate, manganese lactate, and iron lactate. In the present invention, the lactate salts may also be hydrates.

[0014] The origin of the D-lactic acid used in the present invention is not particularly limited, and it may be derived from fermentation using microorganisms, synthetic or semi-synthetic products obtained by chemical synthesis, or natural products derived from animals, plants, or microorganisms. The appropriate method for obtaining the D-lactic acid can be selected based on the desired purity of the lactic acid and production costs. Natural materials containing natural products may be used as a source and incorporated directly into the composition of the present invention. Here, "natural products" refers to products extracted from natural materials containing D-lactic acid by known methods, crudely purified products, or products further purified from these. The D-lactic acid used in the present invention may also contain L-lactic acid, regardless of its optical purity. The D-lactic acid used in the present invention may also be commercially available.

[0015] The method for producing the D-lactic acid of the present invention by fermentation using microorganisms such as lactic acid bacteria typically involves saccharifying starch with malt, adding calcium carbonate to produce wort, adding lactic acid bacteria to the wort for fermentation, and then purifying, filtering, concentrating, etc. to obtain an aqueous lactic acid solution. Microorganisms that can be used to produce the D-lactic acid used in the present invention include lactic acid bacteria, yeast, mold, etc., but are not particularly limited as long as they can ferment sugars to produce D-lactic acid.

[0016] Methods for chemically synthesizing D-lactic acid for use in the present invention include, but are not limited to, synthesizing lactic acid by reacting acetaldehyde with hydrogen cyanide and hydrolyzing the resulting cyanohydrin, or synthesizing lactic acid by reacting acetaldehyde with carbon monoxide under high pressure. Lactic acid produced by these synthetic methods is a racemate with an L-lactic acid:D-lactic acid ratio of 1:1, but there is no limitation on the D / L lactic acid ratio, and any product containing D-lactic acid can be used in the present invention.

[0017] The composition for activating IAP of the present invention, which contains D-lactic acid as an active ingredient, preferably contains 0.135% by mass or more of D-lactic acid, calculated as free lactic acid, and more preferably 0.27% by mass or more. The upper limit of the content is not particularly limited, but examples include 10% by mass or less and 5% by mass or less. The content ranges from 0.1 to 10% by mass, with 2 to 5% by mass being preferred.

[0018] (How to use) The composition for activating IAP of the present invention can contain D-lactic acid derived from microbial fermentation, synthetic, semi-synthetic, or natural products as its active ingredient, and therefore can be widely used as a raw material for pharmaceuticals, foods, beverages, and feed.

[0019] (Pharmaceutical composition for IAP activation) When the IAP activating composition of the present invention is formulated as a pharmaceutical, it can be appropriately mixed with pharmaceutically acceptable excipients, stabilizers, flavoring agents, etc., and then concentrated or lyophilized. These dried, concentrated, and paste-like forms are also included. Furthermore, the composition can be formulated by mixing excipients, binders, disintegrants, lubricants, flavoring agents, suspending agents, coating agents, and any other optional agents within the scope of not interfering with the IAP activating activity of the composition. Possible dosage forms include tablets, capsules, granules, powders, dusting agents, and syrups, and these are preferably administered orally. They can also be administered parenterally in parenteral forms such as gastrostomy tubes, injections, infusions, and patches.

[0020] (Food composition for IAP activation) The IAP-activating composition of the present invention may be incorporated into any food or beverage, or may be added to ingredients during the manufacturing process of the food or beverage. Examples of foods and beverages include, but are not limited to, dairy products such as cheese, fermented milk, dairy lactic acid bacteria drinks, lactic acid bacteria drinks, butter, and margarine; beverages such as milk drinks, fruit juice drinks, and soft drinks; egg products such as jelly, candy, pudding, and mayonnaise; confectioneries and breads such as butter cake; various types of powdered milk; infant foods; and nutritional compositions. The D-lactic acid to be incorporated into a food composition may be present in any amount that does not impair the flavor of the food and is expected to have an IAP-activating effect. For example, the amount may be 0.1 to 10% by mass, with 2 to 5% by mass being a preferred range.

[0021] The classification of the above-mentioned food and drink is not particularly limited, and it can be used as a general food, a health functional food, a food for special uses, etc. Furthermore, as a health functional food, it can be used as a food for specified health uses, a food with nutrient function claims, a food with functional claims, etc.

[0022] When indicating the uses of the IAP activating composition of the present invention, examples include indications stipulated by various laws, enforcement regulations, guidelines, etc. Indications of use include indications on packaging such as wrapping and containers, as well as indications on advertising media such as pamphlets.

[0023] (Feed composition for IAP activation) The IAP activating composition of the present invention can be incorporated into feed. It may be incorporated into any feed, similar to the aforementioned foods and beverages, or may be added to the raw materials during the feed production process. The D-lactic acid to be incorporated into the feed composition may be present in any amount that does not impair the flavor of the feed and is expected to have an IAP activating effect, for example, in an amount of 0.1 to 10% by mass, with 2 to 5% by mass being a preferred range.

[0024] (Target of administration) The recipient of the IAP activating composition of the present invention is not particularly limited, and may be a human or a non-human animal (e.g., a dog, a cat, a horse, or a rabbit). When the recipient is a human, the composition may be administered to a minor under 20 years of age, an adult, or an elderly person 65 years of age or older. The recipient of the IAP activating composition of the present invention may be a healthy subject or a subject with a condition requiring IAP activation. Specific examples of subjects with a condition requiring IAP activation include subjects diagnosed with diabetes, chronic renal failure, metabolic syndrome, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), necrotizing enterocolitis (NEC), sepsis, antibiotic-associated diarrhea, etc. Further examples include subjects who do not have the above symptoms and for whom prevention of intestinal inflammation is desired.

[0025] (Dosage / Intake) When D-lactic acid, the active ingredient of the present invention, is incorporated into a composition for activating IAP, a food or beverage composition for activating IAP, a feed composition for activating IAP, or a pharmaceutical composition for activating IAP, the dosage can be determined taking into consideration the symptoms, age, etc. of the recipient.

[0026] (Method for assessing IAP activity) The evaluation can be performed by the method described in the Examples, that is, the method using the human colon cancer-derived cell line Caco-2. Specifically, the method is as follows. (1) Caco-2 cells are cultured on collagen-coated (Nitta Gelatin) 48-well plates. The culture medium is high-glucose DMEM (Nacalai Tesque) containing 10% FBS (Gibco), 1% NEAA (Nacalai Tesque), and 1% penicillin-streptomycin (Gibco). The cells are cultured at 37°C and 5% CO2. (2) 0.5 × 10 Caco-2 cells were plated on a collagen-coated 48-well plate. 5 The cells are seeded at 1000 cells / well, and the day they become confluent is considered to be day 0 of culture. Culture is continued for 2 weeks with medium changes every 2-3 days. (3) After two weeks, the medium is replaced with a medium containing 2 mM sodium butyrate or samples of various concentrations dissolved therein, and the cells are cultured for seven days, with the medium being replaced every two to three days. (4) After adding the sample, the cells were cultured for 7 days and washed three times with physiological saline. 200 μl / well of 1% Triton-X, 1 mM PMSF, and 10 mM TBS (pH 7.3) was added to lyse the cells. The entire cell lysate was collected in a 1.5 ml tube and sonicated on ice (on time 15 seconds, 30% amp). The cells were centrifuged at 4,800 × g for 5 minutes at 4°C, and the supernatant was collected. The collected supernatant was used as the crude enzyme extract and stored at -80°C until use in the assay. (5) IAP activity of crude enzyme extracts was determined by Pierce TM Measurements were performed using the PNPP Substrate Kit (37620, Thermo Fisher Scientific). Bacterial alkaline phosphatase (BAP, Takara) serially diluted with 1% Triton-X, 1 mM PMSF, and 10 mM TBS (pH 7.3) for the calibration curve, or a 10-fold diluted sample, was added to a 96-well plate at 10 μl / well. PNPP substrate solution was added at 90 μl / well, the plate was shaken on a plate shaker, and the plate was left to stand at room temperature in the dark for 30 minutes. After standing, the reaction was stopped by adding 50 μl / well of 2N NaOH, and the absorbance at 405 nm was measured using a Varioskan Flash (Thermo Fisher Scientific). The calibration curve generated from the BAP measurement results was used to calculate IAP activity (U / ml). (6) The protein concentration of the crude enzyme extract was measured using Pierce TM Measurement was performed using a BCA Protein Assay Kit (23225, Thermo Fisher Scientific). IAP activity (U / ml) was corrected for protein concentration (mg / ml), and the corrected value was used as IAP activity (U / mg protein). If the IAP activity (U / mg protein) calculated in this manner is found to increase when the component to be evaluated is contained compared to when it is not contained, it can be determined that the component to be evaluated has an IAP activating effect.

[0027] (Composition for preventing or improving intestinal inflammation) The composition for preventing or ameliorating intestinal inflammation of the present invention is a composition containing the above-mentioned composition for activating IAP, and by ingesting an effective amount of D-lactic acid as an active ingredient, it has the effect of preventing or ameliorating intestinal inflammation via IAP activation. [Example]

[0028] The present invention will be described in more detail below based on test examples and examples, but the present invention should not be construed as being limited to these examples, etc. Unless otherwise specified, % indicates % by mass.

[0029] [Test Example] IAP activation ability evaluation test of L-lactic acid and D-lactic acid To verify the IAP activating effects of L-lactic acid and D-lactic acid, the following evaluation was performed. 0.5 × 10 Caco-2 cells were cultured on a collagen-coated 48-well plate. 5 The cells were seeded at 1000 cells / well, and the day they became confluent was designated as day 0 of culture. The medium was changed every 2 to 3 days and the cells were cultured for 2 weeks.

[0030] After 2 weeks of culture, the medium was replaced with a medium containing 3.75 to 30 mM L-lactic acid (71718, Sigma Aldrich) or 3.75 to 30 mM D-lactic acid (71716, Sigma Aldrich), and the cells were cultured for 7 days, with the medium replaced every 2 to 3 days.

[0031] After adding the sample, the cells were cultured for 7 days and washed three times with physiological saline. 200 μl / well of 1% Triton-X, 1 mM PMSF, and 10 mM TBS (pH 7.3) was added to lyse the cells. The entire cell lysate was collected in a 1.5 ml tube and sonicated on ice (on time 15 seconds, 30% amp). The cells were centrifuged at 4,800 × g for 5 minutes at 4°C, and the supernatant was collected. The collected supernatant was used as a crude enzyme extract and stored at -80°C until use in the assay.

[0032] IAP activity of crude enzyme extracts was determined using Pierce TM Measurements were performed using the PNPP Substrate Kit (37620, Thermo Fisher Scientific). Bacterial alkaline phosphatase (BAP, Takara) serially diluted in 1% Triton-X, 1 mM PMSF, and 10 mM TBS (pH 7.3) for the calibration curve, or a 10-fold diluted sample, was added to a 96-well plate at 10 μl / well. PNPP substrate solution was added at 90 μl / well, the plate was shaken on a plate shaker, and then allowed to stand at room temperature in the dark for 30 minutes. The reaction was then stopped by adding 50 μl / well of 2N NaOH, and the absorbance at 405 nm was measured using a Varioskan Flash (Thermo Fisher Scientific). IAP activity (U / ml) was calculated using a calibration curve constructed from the BAP assay results.

[0033] The protein concentration of the crude enzyme extract was determined by Pierce TM Measurement was performed using a BCA Protein Assay Kit (23225, Thermo Fisher Scientific). IAP activity (U / ml) was corrected by protein concentration (mg / ml), and the corrected value was used as IAP activity (U / mg protein).

[0034] Comparison between the control group and the control group was performed using Dunnett's test. The significance level was set at P = 0.05 ( * P<0.05, ** P<0.01).

[0035] The IAP activation ability of L-lactate and D-lactate was evaluated. The results are shown in Figure 1. As shown in this figure, for cells cultured in a medium containing L-lactate, no increase in IAP activity was observed at any concentration between 3.75 and 30 mM. On the other hand, a significant increase in IAP activity was observed at 15 mM (0.135% by mass in terms of free lactate) and 30 mM (0.27% by mass in terms of free lactate). This demonstrates that D-lactate has the ability to activate IAP.

[0036] [Example 1] Production of supplements 10 g of D-sodium lactate powder was mixed with 40 g of an equal mixture of vitamin C and citric acid, 100 g of granulated sugar, and 60 g of an equal mixture of cornstarch and lactose. The mixture was packed into a stick-shaped bag to produce the IAP activating supplement of the present invention.

[0037] [Example Product 2] The ingredients were mixed according to the formulation shown in Table 1, filled into a container, and then heat sterilized to produce the IAP activating beverage of the present invention.

[0038] [Table 1] [Industrial Applicability]

[0039] According to the present invention, a composition for activating IAP containing D-lactic acid as an active ingredient can be provided.

Claims

1. A composition for activating IAP, which contains D-lactic acid as an active ingredient.

2. 2. The composition for activating IAP according to claim 1, wherein the concentration of D-lactic acid is 0.135% by mass or more in terms of free lactic acid.

3. A composition for preventing or ameliorating intestinal inflammation, comprising the composition for activating IAP according to claim 1 or 2.

4. A pharmaceutical composition for activating IAP, a food composition for activating IAP, or a feed composition for activating IAP, comprising the composition for activating IAP according to claim 1 or 2.

5. A pharmaceutical composition for preventing or improving intestinal inflammation, a food composition for preventing or improving intestinal inflammation, or a feed composition for preventing or improving intestinal inflammation, comprising the IAP activating composition according to claim 1 or 2.

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