JP7488573B2 - Macrophage activator - Google Patents

Description

IPOD FERM BP-11439

The present invention relates to a macrophage activator.

Macrophages are cells that contribute to the body's defense against infection by phagocytosis, which directly ingests bacteria and viruses. Macrophage cells also have the ability to produce nitric oxide (NO), and the amount of NO produced is considered an indicator of macrophage activity.

Methods for activating macrophages are being investigated. For example, Patent Document 1 discloses that coffee extract has the activity of promoting the proliferation of macrophages.

JP 2009-49523 A

The object of the present invention is to provide a novel macrophage activator.

The macrophage activator of the present invention contains at least one active ingredient selected from the group consisting of Lactobacillus kunkeii BPS402 (accession number FERM BP-11439), honey, and echinacea and its extracts.

The macrophage activator may be for promoting at least one of macrophage NO production and macrophage phagocytosis.

The macrophage activator may contain at least one active ingredient selected from the group consisting of Lactobacillus kunkii BPS402 and honey.

The macrophage activator may be one that contains echinacea or an extract thereof as an active ingredient and is intended to promote macrophage NO production.

The present invention provides a novel macrophage activator.

Graph showing phagocytosis rate and mean fluorescence intensity in BPS402. Graph showing phagocytosis rate and mean fluorescence intensity in BPS402 homogenates. Graph showing phagocytosis rate and mean fluorescence intensity in multi-flower honey. Graph showing phagocytosis rate and mean fluorescence intensity in Manuka honey. Graph showing phagocytosis rate and mean fluorescence intensity in honeydew honey. Graph showing phagocytosis rate and mean fluorescence intensity in heated acacia honey. Graph showing phagocytosis rate and mean fluorescence intensity in unheated acacia honey.

The following describes in detail preferred embodiments of the present invention. However, the present invention is not limited to the following embodiments.

The macrophage activator according to the present embodiment contains at least one active ingredient selected from the group consisting of Lactobacillus kunkii BPS402, honey, and Echinacea and its extracts. The macrophage activator according to the present embodiment contains at least one active ingredient selected from the group consisting of Lactobacillus kunkii BPS402, honey, and Echinacea and its extracts, and is therefore capable of activating macrophages.

Activation of macrophages includes, for example, actions such as promoting NO production by macrophages and promoting phagocytosis by macrophages. Nitric oxide is involved in the body's defense against microorganisms, viruses, and the like. Furthermore, macrophages directly ingest bacteria or viruses through their phagocytic ability, contributing to the body's defense against infection. The macrophage activator according to this embodiment can also be used for promoting macrophage NO production or promoting macrophage phagocytosis.

The macrophage activator according to this embodiment is particularly effective in activating M1 macrophages. That is, the macrophage activator according to this embodiment may be for activating M1 macrophages. M1 macrophages are activated (classically activated) by Th1 type cytokines such as IFN-γ. M1 macrophages produce inflammatory cytokines such as TNFα, IL-6, IL-12, etc., or reactive oxygen species, and can induce a Th1 type immune response as well as exert antibacterial and antiviral activities and antitumor effects.

In research on the function of macrophages, several studies have been conducted using models such as macrophages with induced inflammation and macrophages sensitized to viruses. On the other hand, the macrophage activator according to the present embodiment has an activating effect on macrophages in a healthy state in which neither inflammation has been induced nor sensitized to viruses. Therefore, the macrophage activator according to the present embodiment is considered to be effective in healthy humans, for example, humans who are not sensitized to viruses and do not have inflammation. The macrophage activator according to the present embodiment can be used, for example, to prevent viral infections and inflammations. The subjects to which the macrophage activator according to the present embodiment is administered may be, for example, healthy individuals, more specifically, for example, those who are not sensitized to viruses and/or do not have inflammation. The subjects to which the macrophage activator according to the present embodiment is administered may be those at risk of viral infection or those at risk of inflammation.

[BPS402]
The macrophage activator according to this embodiment may contain Lactobacillus kunkeei BPS402 (hereinafter, sometimes simply referred to as "BPS402") as an active ingredient. BPS402 was deposited on October 3, 2011 at the National Institute of Advanced Industrial Science and Technology (IPOD, address: 1-1-1 Central 6, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan (postal code 305-8566)) under the accession number FERM P-22177 and is available. IPOD has been moved to Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (postal code 292-0818). The strain has now been transferred to an international depository, and the accession number is FERM BP-11439.

BPS402 as an active ingredient may be the bacterial cells themselves or may be a processed bacterial cell product. The bacterial cells may be live or dead bacterial cells. The processed bacterial cell product may be a product of processing the bacteria, such as heating, pasting, drying (freeze drying, vacuum drying, spray drying, etc.), freezing, lysis, crushing, extraction, etc. Crushing may be performed, for example, by ultrasonic waves. A combination of multiple types of processing may be performed. The processed bacterial cell product may be a supernatant obtained after deproteinization of the processed bacterial cell product, or a supernatant obtained by removing solids from a bacterial cell culture and fermentation product. BPS402 may be an isolated bacterial cell, or a fermentation product or culture of bacterial cells.

BPS402 can be cultured according to conventional methods. There are no particular limitations on the medium as long as it can culture the bacterium, and natural, synthetic, or semi-synthetic media can be used. The medium can contain a nitrogen source and a carbon source. Nitrogen sources include meat extract, peptone, casein, yeast extract, gluten, soy flour, soy hydrolysate, and amino acids, while carbon sources include glucose, lactose, fructose, inositol, sorbitol, starch syrup, starch, koji soup, bran, bagasse, and molasses. In addition, inorganic substances (e.g., ammonium sulfate, potassium phosphate, magnesium chloride, salt, calcium carbonate, iron, manganese, and molybdenum), various vitamins, and the like can be added.

The culture temperature may be, for example, 4 to 45°C, or may be 25 to 40°C, 28 to 37°C, or 30 to 33°C. The culture may be performed by shaking with aeration or stirring with aeration. The pH of the medium may be, for example, 4.0 to 9.0, and is preferably 6.0 to 8.0. As a culture method, for example, the cells are inoculated into MRS medium and cultured at 30°C for 48 hours.

When the macrophage activator contains BPS402 as an active ingredient, the macrophage activator may be, for example, a fermented product containing BPS402, a lactic acid bacteria drink, a drink containing lactic acid bacteria, etc.

The macrophage activator according to this embodiment can be used at a dose of 1 mg to 50 mg per day, preferably 3 mg to 30 mg, and more preferably 5 mg to 15 mg, in terms of the solid content of BPS402, for an adult weighing 60 kg. The dose can be set appropriately within the above range depending on factors such as the health condition of the person taking it, the method of administration, and the combination with other agents.

The content of BPS402 in the macrophage activator may be 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.02% by mass or more, 0.03% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 1% by mass or more, or 10% by mass or more, based on the total amount of the agent. The content of BPS402 in the macrophage activator may be 10% by mass or less, 5% by mass or less, 1% by mass or less, 0.1% by mass or less, 0.05% by mass or less, 0.03% by mass or less, or 0.01% by mass or less, based on the total amount of the agent.

[honey]
The macrophage activator according to the present embodiment may contain honey as an active ingredient. Honey is produced by bees using nectar collected from plant nectar, sap, and secretions of insects parasitizing plants as the main ingredient. For example, manuka honey, acacia honey, honeydew honey, flower honey, clover honey, orange honey, astragalus honey, rosemary honey, sunflower honey, rapeseed honey, coffee honey, etc. may be used as the honey. One type of honey may be used alone, or multiple types may be used in combination. The type of bee used to collect honey and the place of origin of honey are not particularly limited, but for example, flower honey may be produced in Japan.

Honey may be processed by removing dirt, beehive residue, etc., filtering, extracting with an organic solvent, refining by fractionation, concentrating, etc.

The honey may be heat-treated or may not be heat-treated. When the honey is heat-treated, the heating temperature may be, for example, greater than 45°C, greater than or equal to 50°C, greater than or equal to 60°C, and less than or equal to 70°C, less than or equal to 65°C, or less than or equal to 60°C.

The macrophage activator according to this embodiment can be used at a dose of 1 g to 50 g per day, preferably 2 g to 40 g, and more preferably 3 g to 35 g, as the solid content of honey for an adult weighing 60 kg. The dose can be set appropriately within the above range depending on factors such as the health condition of the person taking it, the method of administration, and the combination with other agents.

The honey content in the macrophage activator, as solid content of honey, may be, for example, 0.1% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, 25% by mass or more, 30% by mass or more, 40% by mass or more, 45% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, or 100% by mass relative to the total amount of the agent. The honey content in the macrophage activator, as a solid content, may be, for example, 100% by mass or less, 90% by mass or less, 85% by mass or less, 80% by mass or less, 75% by mass or less, 70% by mass or less, 65% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 8% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, or 0.5% by mass or less, based on the total amount of the agent.

(Echinacea)
The macrophage activator according to this embodiment can contain Echinacea or an extract thereof as an active ingredient. Examples of Echinacea include Echinacea purpurea, Echinacea angustifolia, Echinacea pallida, Echinacea tennesseensis, Echinacea paradoxa, Echinacea atrorubens, Echinacea laevigata, Echinacea sanguinea, and Echinacea serotina. serotina, or Echinacea simulata.

Echinacea may be the Echinacea plant itself, or may be a dried product of the plant. The parts of the plant used may be, for example, leaves, flowers, stems, and other above-ground parts, roots, or a mixture of these. The dried product may be, for example, a crushed or pulverized product. The drying method may be natural drying, spray drying, freeze drying, or the like. The active ingredient may be an extract of these Echinacea. The extract may be, for example, a water extract or a hot water extract. The extract is preferably a hot water extract. The dried product of the plant may be ingested, for example, by extracting it with water or hot water at the time of ingestion. The extract may be obtained by further processing the extract liquid, such as concentration, powdering, or granulation.

In the case of a water extract, the extraction temperature may be 0 to 40°C, 10 to 35°C, or 15 to 30°C. In the case of a hot water extract, the extraction temperature may be, for example, 80 to 100°C, or 85 to 95°C. The extraction time may be, for example, 10 minutes to 3 hours, or 30 minutes to 2 hours. The extract may be obtained by further extraction from the residue after extraction. Echinacea and its extract may be commercially available. Specific examples of commercially available echinacea and its extract include echinacea grains (manufactured by Yamada Bee Farm Co., Ltd.), echinacea tea (manufactured by Yamada Bee Farm Co., Ltd.), echinacea (manufactured by DHC Co., Ltd.), etc.

The Echinacea or extract thereof used in the macrophage activator according to this embodiment can be used at a dose of 150 mg to 1000 mg per day for an adult weighing 60 kg, preferably at a dose of 300 mg to 800 mg, and more preferably at a dose of 450 mg to 650 mg. The dose can be set appropriately within the above range depending on factors such as the health condition of the person taking it, the method of administration, and the combination with other agents.

The content of Echinacea or an extract thereof in the macrophage activator may be 60% by mass or more, 70% by mass or more, 80% by mass or more, or 90% by mass or more in terms of solid content relative to the total amount of the agent. The content of Echinacea or an extract thereof in the agent according to this embodiment may be 100% by mass or less, 90% by mass or less, 80% by mass or less, or 70% by mass or less in terms of solid content relative to the total amount of the agent.

The macrophage activator according to this embodiment may contain one active ingredient alone or a combination of multiple active ingredients.

The macrophage activator according to the present embodiment may further contain other ingredients in addition to the above-mentioned active ingredients. Examples of other ingredients include pharma- ceutically acceptable ingredients (e.g., excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, suspending agents, antioxidants), and food acceptable ingredients (e.g., minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavorings, stabilizers, preservatives, sustained-release regulators, surfactants, dissolving agents, and wetting agents). It is preferable that the macrophage activator according to the present embodiment does not contain coffee extract.

The macrophage activator according to this embodiment may be a drug, a quasi-drug, or a food composition. The macrophage activator according to this embodiment can be used as a drug, a quasi-drug, or a food composition itself, or can be used as an ingredient in a drug, a quasi-drug, or a food composition. A drug, a quasi-drug, or a food composition containing a macrophage activator as one ingredient can be produced, for example, by adding the macrophage activator to an intermediate product in the production process of these products.

The macrophage activator according to the present embodiment may be in any form, such as a solid, liquid, or paste, and may be in the form of a tablet (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsule, pill, powder (powder), fine granules, granules, liquid, suspension, emulsion, syrup, paste, or injection (including the case where it is mixed with distilled water or an infusion such as an amino acid infusion or an electrolyte infusion at the time of use to prepare a liquid). These various preparations can be prepared, for example, by mixing the active ingredient with other ingredients as necessary and forming the mixture into the above-mentioned dosage form.

When used as a food composition or as one component of a food composition, the food composition preferably emphasizes the tertiary function of food, i.e., the function of regulating physical condition. Examples of products that emphasize the tertiary function of food include health foods, functional foods, foods with nutritional functions, nutritional supplements, supplements, and foods for specified health uses.

Examples of food compositions include beverages such as soft drinks, such as coffee, juice, and tea drinks, milk drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, and alcoholic beverages, such as sake, Western liquor, fruit wine, and honey wine; spreads, such as custard cream; pastes, such as fruit paste; Western confectioneries, such as chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes, and puddings; Japanese confectioneries, such as daifuku, mochi, manju, castella, anmitsu, and yokan; frozen desserts, such as ice cream, popsicles, and sorbet; cooked foods, such as curry, beef bowl, porridge, miso soup, soup, meat sauce, pasta, pickles, and jam; and seasonings, such as dressings, furikake, umami seasonings, and soup bases.

The macrophage activator according to this embodiment is preferably ingested into the body. The administration mode may be oral administration or parenteral administration. The macrophage activator according to this embodiment may be administered once a day, or may be administered in multiple divided doses, such as twice a day or three times a day.

The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples.

<Test Example 1: Evaluation of macrophage phagocytic activity>
The macrophage activation ability of the test substance was evaluated using phagocytosis as an index. Briefly, macrophages were cultured in a culture medium containing the test substance for 1 hour, and then fluorescently labeled polystyrene beads were added and incubated for 2.5 hours. The cells were collected and subjected to analysis by a flow cytometer. The details are as follows.

The mouse macrophage J774.1 cell line purchased from the JCRB Cell Bank was used in the study. The J774.1 cell line is one of the cell lines commonly used in macrophage phagocytosis studies.

The culture medium used to dissolve the test substance was used as a negative control substance. Lipopolysaccharide (LPSp) purified from Pantoea agglomerans was used as a positive control substance. All dilution procedures were performed aseptically in a clean bench. LPSp was dissolved in water for injection to a concentration of 2.0 mg/mL and stored at 4°C until use. When used, the LPSp solution was heated at 37°C for 5 minutes and then ultrasonicated at 37°C for 1 minute. After ultrasonication, 5 μL of LPSp solution was added to 995 μL of culture medium and mixed well to prepare a 10 μg/mL solution. The solution was further diluted with culture medium and used in the test.

The culture medium used was a mixture of 500 mL of DMEM medium (#044-29765, Fujifilm Wako Pure Chemical Corporation), 55.5 mL of inactivated FBS, and 5.5 mL of penicillin-streptomycin-glutamine (100x). The inactivated FBS was prepared by heating FBS (fetal bovine serum, Cat No. SH3039603, Hyclone) at 56°C for 30 minutes, dispensing it into aliquots, and storing it at -20°C.

[Test substance]
The test substances used were Lactobacillus kunkeii BPS402, crushed BPS402, Echinacea extract, Japanese wild flower honey, Manuka honey, honeydew honey, heated acacia honey, and non-heated acacia honey. The details are as follows.
Lactobacillus kunkeii BPS402: live bacteria heat-treated at 80°C for 1 minute or more, dextrin is added, and the bacteria is freeze-dried and powdered. Lactobacillus kunkeii BPS402 crushed material: the above powdered Lactobacillus kunkeii BPS402 is further crushed. Echinacea extract: dried powder of echinacea extract with hot water at 90°C. Heated acacia honey: acacia honey heat-treated at 60°C for 24 hours. Non-heated acacia honey: acacia honey not heat-treated as above.

The test solutions were prepared aseptically in a clean bench. Honey was dissolved directly in culture fluid and then diluted to the specified concentration. BPS402 and Echinacea were dissolved in water and then diluted to the specified concentration with culture fluid.

[Preculture]
J774.1 cells were subcultured in DMEM medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were cultured in a T25 culture flask, and subcultured at 1 to 2 × 10 ⁵ cells/mL every 3 or 4 days. The cells were cultured in a 5% CO ₂ incubator set at 37°C.

[PE-labeled polystyrene bead suspension]
A 2.5% aqueous suspension (5.68 x 10 ⁹ pieces/mL) of polystyrene beads (Fluoresbrite Polychromatic Red Microspheres 2.0 μm, Polysciences) was prepared. 140.8 μL of the polystyrene bead aqueous suspension was dispensed into a 2.0 mL microtube, centrifuged (2000 x g, 5 minutes), and the supernatant was removed. The beads were then collected as a precipitate. 2000 μL of culture solution was added to the precipitate to prepare a polystyrene bead suspension of 4 x 10 ⁸ pieces/mL (4 x 10 6 pieces/10.0 μL).

[Macrophage phagocytosis test]
The following test procedures 1) to 7) were carried out in a clean bench.
1) J774.1 cells precultured in a T25 culture flask were peeled off from the wall by pipetting, and the resulting cell suspension was transferred to a 15 mL conical tube. The tube was centrifuged at room temperature for 8 minutes at 1000 rpm (190 x g). The supernatant was discarded by decantation, and the cells were collected as a pellet. After loosening the cells by tapping, 10 mL of culture solution was added, and the tube was centrifuged at room temperature for 8 minutes at 1000 rpm (190 x g), the supernatant was discarded by decantation, and the cells were collected as a pellet. 5 mL of culture solution was added to the cell pellet, and the cells were uniformly suspended by pipetting. 100 μL of the cell suspension was transferred to a 1.5 mL microtube, and the cell count and viability were measured.

2) Based on the measured cell count, a cell suspension of 1 x ¹⁰⁶ cells/mL was prepared. 200 μL of the obtained cell suspension was added to each well of a 48-well plate. The cells were cultured overnight in a 37°C, 5% _CO2 incubator.

3) After pre-culture, the plate was removed from the incubator. The culture medium was removed from the wells in which the cells were seeded using a pipette. 200 μL of warmed culture medium was added, and the culture medium was removed. Then, 200 μL of the test liquid was added. The plate was cultured for 1 hour in a 37° C., 5% CO ₂ incubator.

4) 10.0 μL of culture solution containing 4×10 ⁶ PE-labeled polystyrene beads was added, and the cells were cultured in a 37° C., 5% CO ₂ incubator for 2.5 hours.

5) The supernatant was slowly removed with a pipette and 300 μL of warm PBS was added. The PBS was removed with a pipette and 500 μL of warm PBS was added. This procedure was repeated three times.

6) Remove the PBS with a pipette and add 300 μL of flow cytometer buffer (PBS containing 0.5% BSA and 2 mM EDTA).

7) The cells were detached by pipetting and the cell suspension was transferred to a 1.5 mL tube. The tube was stored on ice.

8) The collected cell suspension was subjected to analysis using a flow cytometer (Gallios, Beckman Coulter). 20,000 cells were analyzed, and a histogram of FL2 was created to determine the phagocytosis rate and mean fluorescence intensity (MFI). A two-group t-test (Student's t-test or Welch's t-test) was performed with the negative control (solvent) on the same plate. The phagocytosis rate (proportion of cells that ingested beads) was calculated for each number of beads ingested (1 to 5). The mean fluorescence intensity increases with the number of phagocytosed cells and the number of beads ingested.

Comparison with the negative control solvent group was made with the measurement results of the solvent group on the same plate. When tests were performed using LPSp as a positive control substance, a significantly higher phagocytosis rate and mean fluorescence intensity were observed than in the negative control group (not shown).

The results of the macrophage phagocytosis test are shown in Figures 1 to 7. Figures 1 to 7 (a) are graphs showing the macrophage phagocytosis rate. Figures 1 to 7 (b) are graphs showing the average fluorescence intensity. It was confirmed that the phagocytosis rate and average fluorescence intensity were significantly increased in all test substances compared to the negative control. Furthermore, it was confirmed that the phagocytosis rate and average fluorescence intensity were significantly increased in the Echinacea extract compared to the negative control (not shown). It was confirmed that the test substances used have the effect of enhancing macrophage phagocytosis.

<Test Example 2: Evaluation of NO production ability>
The macrophage activation ability of the test substance was evaluated using the NO production ability as an index. In summary, the amount of NO production of the test substance was measured using the RAW264.7 cell line, which is a mouse macrophage cell line. Specifically, the procedure is as follows.

RAW264.7 mouse macrophage cells were purchased from ATCC. The RAW264.7 cell line is one of the cell lines commonly used to evaluate macrophage activation ability (e.g., NO production test).

The negative and positive control substances were prepared in the same manner as in Test Example 1. The test substances used were Lactobacillus kunkii BPS402, crushed BPS402, Japanese wild flower honey, manuka honey, and echinacea (all prepared in the same manner as in Test Example 1). The culture medium used was a mixture of 500 mL of RPMI1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 55.5 mL of inactivated FBS, and 5.5 mL of penicillin-streptomycin-glutamine (100x).

[Preculture]
RAW264.7 cells were subcultured in RPMI1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were cultured in a T25 culture flask, and subcultured at 0.25×10 ⁵ cells/mL every 3 or 4 days. The cells were cultured in a 5% CO ₂ incubator at 37° C. (hereinafter, incubator).

[Cell Treatment]
All test operations were performed in a clean bench. The cells pre-cultured in 14 T25 culture flasks were detached from the walls by pipetting, and the resulting cell suspension was transferred to a 50 mL conical tube. The tube was centrifuged at room temperature (1000 rpm, 5 minutes), the supernatant was discarded by decantation, and the cells were collected. After loosening the cells by tapping, 5 mL of culture medium was added, and the cells were uniformly suspended by pipetting. 11 μL was transferred to another tube, and 11 μL of 0.5% trypan blue was added, and the cell suspension was transferred to a blood counting chamber to measure the cell count and viability. The viability was 99.6%, exceeding the 90% standard for cells that can be used in the test. The remaining liquid was used for the test.

Based on the measured cell count, the remaining liquid was diluted with culture medium to adjust the cell count to 1.6 x ¹⁰⁶ cells/mL. 100 μL of this cell suspension was added to each well of a 96-well flat-bottom plate. The plate was transferred to an incubator and pre-cultured for 3 hours until the cells attached to the bottom of the well and spread out.

After the 3-hour pre-incubation, the plate was removed and 100 μL of the test solution was added to each well at a double dose. After the addition of each sample, the wells were incubated in an incubator for 24 hours. After incubation, 100 μL of the supernatant was collected in a 96-well flat-bottom plate and used for the NO assay.

NO Assay
Nitrite concentration was measured as an index of NO production ability. For the calibration curve, 200 μM sodium nitrite aqueous solution was serially diluted with culture medium, and 100 μL was transferred to the calibration curve well. 3% sulfanilamide 7.5% phosphoric acid aqueous solution and 0.15% naphthylethylenediamine solution were mixed in a 1:2 ratio to prepare the Griess reagent just before use. 100 μL of Griess reagent was added per well, incubated at room temperature for 10 minutes, and then the absorbance at the main wavelength of 550 nm and the sub-wavelength of 688 nm was measured to measure the nitrite concentration. The results are shown in Table 1.

When the test was performed using a positive control substance, a dose-dependent NO production ability was confirmed. When only a solvent was used as a negative control substance, NO production ability was not confirmed. All of the test substances used in the NO production ability evaluation test were confirmed to have a significant NO production ability compared to the negative control substance.

Claims (2)

A macrophage activator comprising Lactobacillus kunkeii BPS402 (accession number FERM BP-11439 ) as an active ingredient. The macrophage activator according to claim 1 for promoting at least one of macrophage NO production and macrophage phagocytosis.

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