JP6941828B2 - Macrophage activator and food composition for macrophage activation - Google Patents

Description

The present invention relates to a macrophage activator and a food composition for activating macrophages. According to the macrophage activator of the present invention and the food composition for activating macrophages, macrophages can be activated.

Macrophages are leukocytes produced by monocyte differentiation in tissues. Macrophages are responsible for the treatment of waste products in the body and for the defense against pathogens (eg, viruses or microorganisms) and tumor cells. It also functions as an effector of cell-mediated immunity through the presentation of antigens to T cells and the production of interleukin (IL). Therefore, it is important to activate macrophages for the treatment of infectious diseases and tumors.

Various substances that activate macrophages have been conventionally proposed, and in recent years, attempts have been made to activate macrophages with mutant-introduced yeast (Patent Document 1). In addition, it is known that certain polysaccharides have the ability to activate macrophages (Patent Document 2). However, the current situation is that a safe substance that has a sufficient desired macrophage activating function and can be actually used as a drug or food has not yet been developed.

On the other hand, spices are one of the most frequently used foods in our diet, among which Myristica fragrance seeds and arils have a unique aroma and are of Myristica fragrance. Dried seeds and arils are mainly used as spices in meat dishes, dairy products, baked goods and the like.

Japanese Unexamined Patent Publication No. 2010-284085 Japanese Unexamined Patent Publication No. 2001-314172

The present inventors have compared and examined the macrophage activating ability of various spices, and newly found that the extract of myristica fragrance seeds or arils exhibits macrophage activating ability. .. It is not known at all that an extract of myristica fragrance seeds or arils activates macrophages, and the present invention is based on this finding.

Therefore, the present invention
[1] A macrophage activator comprising an extract of myristica fragrance seeds and / or aril as an active ingredient.
[2] The macrophage activator according to [1], wherein the extract is an extract of an alcohol, an aqueous solvent, or a mixture of an alcohol and an aqueous solvent.
[3] The macrophage activator according to [1] or [2], wherein the extract is an extract of nutmeg and / or mace.
[4] The macrophage activation is one or more selected from cytokine production promotion, phagocytosis activity promotion, active oxygen production promotion, nitric oxide production promotion, and antigen presentation function enhancement, [1] to The macrophage activator according to any one of [3].
[5] A food composition for activating macrophages, which comprises an extract of myristica fragrance seeds and / or aril as an active ingredient.
[6] The food composition for macrophage activation according to [5], wherein the extract is an extract of an alcohol, an aqueous solvent, or a mixture of an alcohol and an aqueous solvent.
[7] The food composition for macrophage activation according to [5] or [6], wherein the extract is an extract of nutmeg and / or mace, and [8] the macrophage activation promotes cytokine production. The macrophage activation according to any one of [5] to [7], which is one or more selected from promotion of phagocytic activity, promotion of active oxygen production, promotion of nitric oxide production, and enhancement of antigen presentation function. Regarding food compositions for use.

According to the macrophage activator of the present invention, macrophages can be activated. Furthermore, according to the macrophage activator of the present invention, it is possible to provide foods and drinks having a macrophage activating ability and high safety.

It is a photograph of Raw264.7 cells treated with solvent control (ethanol only). It is a photograph of Raw264.7 cells treated with an ethanol extract of nutmeg. It is a table which shows the experimental result of the experiment which treated Raw264.7 cells with the alcohol extract of nutmeg. It is a photograph of mouse peritoneal macrophages treated with solvent control (ethanol only). It is a photograph of mouse peritoneal macrophages treated with an ethanol extract of nutmeg. It is a table which shows the experimental result of the experiment which treated the mouse peritoneal macrophage with the alcohol extract of nutmeg. 6 is a graph showing the experimental results of an experiment in which Raw264.7 cells treated with an alcohol extract of nutmeg were subjected to flow cytometry. It is a graph which shows the experimental result of the experiment which measured the amount of nitric oxide release of Raw264.7 cells treated with the alcohol extract of nutmeg.

[1] Macrophage activator The macrophage activator of the present invention contains an extract of myristica fragrance seed and / or aril as an active ingredient.

(Millistica Fragrance)
Myristica fragrance is a type of evergreen tree of the Myristicaceae family. Myristica fragrance is an evergreen tree that reaches about 20 meters in height, the bark of the trunk is taupe and has a smooth surface, the leaves are 8 to 15 cm in length, the front side is dark green, and the back side is light green. It is a single leaf. Myristica fragrance bears egg-shaped yellow fruits about 5 cm in length, similar to plums or apricots, with a reticulated red aril and dark brown seeds wrapped in this aril. ..

(seed)
The seeds of Myristica fragrance are covered with arils. In the seeds, there are egg-shaped, taupe-brown seeds with a diameter of about 2.5 cm inside the hard seed coat. The seeds or dried seeds are commonly used as spice nutmeg.

(Aril)
The aril is an accessory that covers the surface of the seed and is also called a seed coat. The aril is a structure in which a flower peduncle or placenta develops and covers the outside of the seed. Dried arils of Myristica fragrance are commonly used as spice maces.

The site of use of myristica fragrance used for extracting the active ingredient of the macrophage activator of the present invention is a seed and / or aril, preferably a seed. Myristica fragrance may be used raw when performing the extraction operation, but a dried one is preferable because the extraction efficiency is better. In addition, it is preferable to process it into a crushed product or powder so that the extraction efficiency is improved.

(nutmeg)
As used herein, nutmeg means the seeds of Myristica fragrance or dried seeds. Nutmeg is commonly used as a spice. Nutmeg has a sweet and pungent aroma and is mainly used to deodorize meat dishes. Nutmeg is also used in vegetable dishes, dairy products, and Western confectionery.
Nutmeg is often sold as a powder and then sealed in a bottle, but because the aroma is easily lost, nutmeg is sometimes sold as seeds. If the seeds are sold as they are, they are used after being finely divided using a nutmeg mill or the like at the time of use.

(mace)
As used herein, mace means a dried aril of Myristica fragrance. Mace is commonly used as a spice. Mace has a sweet and pungent scent. Similar to nutmeg, mace is used in meat dishes, vegetable dishes, dairy-based dishes, and Western confectionery.

The active ingredient of the macrophage activator of the present invention can be obtained by extracting an extract of myristica fragrance seeds and / or arils with a suitable solvent. The dried product of Myristica fragrance seeds is the spice nutmeg, and the dried product of Myristica fragrance aril is the spice mace. Nutmeg and mace have similar flavors, and they also share ingredients such as aroma components. The active ingredient that activates macrophages contained in the extract of the present invention is considered to be contained in the whole plant of Myristica fragrance, but is particularly concentrated in the seed and aril parts and contained in a large amount. It is thought that there is. For example, nutmeg prepared from seeds and mace prepared from arils contain a common component, and are considered to contain a large amount of an active ingredient that activates macrophages.

(Extract)
The extraction solvent for extracting the extract of myristica fragrance seed and / or pseudoseed coat contained in the macrophage activator of the present invention is as long as the active ingredient can be sufficiently extracted in the extract. However, for example, an organic solvent, an aqueous solvent, or a mixture of an organic solvent and an aqueous solvent can be used.

(Organic solvent)
Extracts of myristica fragrance seeds and / or pseudoseed coats contained in the macrophage activator of the present invention include organic solvents such as alcohol, acetone, benzene, esters, ethyl acetate, hexane, chloroform, and diethyl ether. Can be extracted with, but preferably with alcohol. The alcohol is not limited as long as the active ingredient can be sufficiently extracted in the extract, but for example, methanol, ethanol, propyl alcohol, isopropyl alcohol, butyl alcohol, pentanol and the like have 1 to 1 carbon atoms. 5 monohydric alcohols can be used. Polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol, dipropylene glycol, and glycerin can also be used. Preferred alcohols are monohydric alcohols having 1 to 5 carbon atoms, and more preferable alcohols are methanol and ethanol.

(Aqueous solvent)
The extract of myristica fragrance seed and / or aril contained in the macrophage activator of the present invention can be extracted with an aqueous solvent. The aqueous solvent is not limited as long as it contains water, and for example, water, physiological saline, a buffer solution, or the like can be used. Examples of the buffer solution include a phosphate buffer solution, a sodium phosphate buffer solution, a sodium carbonate buffer solution, a citrate buffer solution, a citrate phosphate buffer solution, an acetate buffer solution, and a Tris buffer solution. The pH of the aqueous solvent is not particularly limited, and is, for example, 3 to 10, 5 to 8, or 7 to 8.

(Mixture of organic solvent and aqueous solvent)
The extract of myristica fragrance seed and / or aril contained in the macrophage activator of the present invention can be extracted by a mixture of an organic solvent and an aqueous solvent. The amount of the aqueous solvent contained in the extraction solvent is not limited as long as the active ingredient can be sufficiently extracted in the extract, but is, for example, 50% by mass or less based on the total amount of the extraction solvent. 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, 5.0% by mass or less, 4.0% by mass or less, 3.0% by mass or less, 2.0% by mass % Or less, or 1.0% by mass or less, or 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 85% by mass or more, 90% by mass or more, 95% by mass. % Or more, 96% by mass or more, 97% by mass or more, 98% by mass or more, or 99% by mass or more.

The extraction temperature is not particularly limited as long as the active ingredient can be sufficiently extracted into the extract, but is preferably −50 ° C. to 100 ° C., preferably −25 ° C. to 50 ° C. The temperature is more preferably −20 ° C. to 25 ° C., further preferably −10 ° C. to 20 ° C., and most preferably 0 ° C. to 15 ° C.

Further, it is preferable to carry out the extraction while stirring or shaking so as to improve the extraction efficiency. The extraction time is, for example, the site of myristica fragrance, the state of myristica fragrance (raw or dried, or the processed state when processed into crushed or powdered material), extraction. It can be appropriately determined depending on the temperature of the liquid, the presence or absence of stirring or shaking, or the conditions, but it is usually 1 minute to 72 hours, preferably 1 hour to 48 hours, and 12 hours to 36 hours. Is most preferable.

The active ingredient in the present invention, an extract of Myristica fragrance seeds and / or pseudo-seed coat, is a conventional carrier or dilution that is acceptable alone or preferably pharmacologically or veterinarily. Together with the drug, it can be administered in an effective amount to subjects [animals, preferably mammals (particularly humans)] who require treatment or prevention of various infectious diseases such as tuberculosis and influenza, and cancers such as lung cancer and gastric cancer. It is also possible to feed animals other than humans in the form of food and drink as feed.

The dosage form of the macrophage activator of the present invention is not particularly limited, and is a powder, a fine granule, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, an extract, or a pill. Oral preparations such as, or parenteral preparations can be mentioned.

The oral preparation includes, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannit, carboxymethyl cellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid ester, talc, magnesium stearate. , Polyethylene glycol, magnesium silicate, silicic anhydride, or synthetic aluminum silicate and other excipients, binders, disintegrants, surfactants, lubricants, fluidity enhancers, diluents, preservatives, colorants , Fragrance, flavoring agent, stabilizer, moisturizing agent, preservative, antioxidant, etc., can be produced according to a conventional method.

Examples of parenteral preparations include injections. In the preparation of injections, in addition to the active ingredient, for example, a water-soluble solvent such as physiological saline or Ringer's solution, a water-insoluble solvent such as vegetable oil or fatty acid ester, an isotonic agent such as glucose or sodium chloride, and solubilization assistance. Agents, stabilizers, preservatives, suspending agents, emulsifiers and the like can be optionally used.

The macrophage activator of the present invention is, but is not limited to, an extract of nutmeg, for example, 1 to 1000 μg / mL, preferably 3 to 500 μg / mL, more preferably 5 to 300 μg / mL. , More preferably 10 to 250 μg / mL, still more preferably 10 to 200 μg / mL, and most preferably 15 to 150 μg / mL.

When the macrophage activator of the present invention is used, the dose can be appropriately determined according to the type of disease, the age, sex, body weight, degree of symptoms, administration method, etc. of the patient, but Myristica fragrance. As an extract (dry matter) of, for example, 0.01 mg / kg body weight / day or more, 0.05 mg / kg body weight / day or more, 0.1 mg / kg body weight / day or more, 0.3 mg / kg body weight / day or more. 0.5 mg / kg body weight / day or more, 0.75 mg / kg body weight / day or more, 1 mg / kg body weight / day or more, 2 mg / kg body weight / day or more, 5 mg / kg body weight / day or more, 10 mg / kg body weight / Day or more, 20 mg / kg body weight / day or more, 30 mg / kg body weight / day or more, 50 mg / kg body weight / day or more, or 75 mg / kg body weight / day or more, or 1000 mg / kg body weight / day or less, 750 mg / kg body weight / Day or less, 500 mg / kg body weight / day or less, 250 mg / kg body weight / day or less, 200 mg / kg body weight / day or less, 150 mg / kg body weight / day or less, 125 mg / kg body weight / day or less, or 100 mg / kg body weight / day Can be less than a day. Specific ranges include, for example, 0.01 to 1000 mg / kg body weight / day, 0.05 to 500 mg / kg body weight / day, 0.3 to 250 mg / kg body weight / day, 0.5 to 200 mg / kg body weight. It can be 0.75 to 150 mg / kg bw / day, or 1-100 mg / kg bw / day.

(Macrophage activation)
The specific action of macrophage activation exerted by the macrophage activator of the present invention is selected from, for example, cytokine production promotion, phagocytosis activity promotion, active oxygen production promotion, nitric oxide production promotion, and enhancement of antigen presentation function. One or more.

Examples of cytokines whose production is promoted include interleukins, interferons, chemokines, hematopoietic factors, cell growth factors, and cytotoxic factors. Examples of interleukins include IL1, 6, 10, 12, 15, and 18. Examples of interferon include IFN-α and the like. Examples of chemokines include MIP and CCL. Examples of hematopoietic factors include granulocyte monocytic colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF). Examples of cell growth factors include platelet-derived growth factor (PDGF), epithelial growth factor (EGF), hepatocyte growth factor (HGF), fibroblast growth factor (bFGF), and vascular endothelial cell growth factor (VEGF). Be done. Examples of cytotoxic factors include TNF-α and TNF-β. Cytokines whose secretion is promoted are preferably IL-6 and / or TNF-α. The cytokine whose secretion is promoted may be one of these or two or more of them.

The phagocytosis of macrophages is a function in which macrophages take in foreign substances such as bacteria, viruses, or dead cells and decompose them, and is also called phagocytosis or phagocytosis. The macrophage activator of the present invention can promote the phagocytic action of this macrophage.

Macrophages kill or eliminate foreign substances by producing reactive oxygen species such as superoxide or hydrogen peroxide, or nitric oxide, which are highly toxic to foreign substances such as microorganisms, viruses, and cancer cells. .. The macrophage activator of the present invention can promote the active oxygen production promoting action and / or the nitric oxide production promoting action of the macrophage.

Antigen presentation is a mechanism in which an antigen-presenting cell takes up an antigen such as a bacterium into the cell, decomposes it, and then presents a part of the antigen to the cell surface. Macrophages break up and decompose bacteria by phagocytosis into several fragments, bind them to MHC class II molecules present in macrophages, and present them on the cell surface. The macrophage activator of the present invention can promote the antigen-presenting function of the macrophage.

[2] Food Composition for Macrophage Activation The food composition for macrophage activation of the present invention contains an extract of myristica fragrance seeds and / or aril as an active ingredient. Specifically, a beverage containing a mystica fragrance seed and / or aril extract, or a mystica fragrance seed and / or aril extract is moistened by appropriate means (eg, lyophilization). Beverages or foods, which are contained as a dried product from which the above-mentioned substances have been removed.

Beverages include tea, juice, milk, soy milk, alcoholic beverages, coffee, black tea, sencha, oolong tea, sports beverages, yogurt and the like. Foods include cookies, bread, biscuits, hardtack, cakes, rice crackers, sheep rice crackers, puddings, jellies, ice creams, chewing gum, crackers, chips, sweets such as candy, noodles such as udon or soba, and kamaboko. Examples include fish paste products such as ham or fish sausage, dairy products such as cheese or butter, seasonings such as miso, soy sauce, dressing, mayonnaise or sweets, tofu, or konjac.

Food compositions include functional foods and health foods (beverages). In the present specification, the "health food" means a food that has or can be expected to have some effect on health, and the "functional food" is the above-mentioned "health food". Foods designed and processed to fully express various bioregulatory functions (that is, regulatory functions of physiological systems such as digestive system, circulatory system, endocrine system, immune system, or nervous system). Means.

Hereinafter, the present invention will be specifically described with reference to Examples, but these do not limit the scope of the present invention.

<< Production Example 1: Preparation of Alcoholic Extract of Myristica Fragrance >>
The dried seeds of Myristica fragrance seeds were crushed to prepare nutmeg powder. 6 grams of nutmeg powder was weighed. 35 mL of 100% ethanol or 100% methanol was added to the weighed nutmeg powder. In a low greenhouse (8 ° C.), rotary extraction was performed for 24 hours using a rotator. The supernatant was collected by centrifugation (1400 × g, 20 minutes). It was concentrated using an evaporator and the amount of dry matter was weighed. 100% ethanol or 100% methanol was added to adjust the dry matter concentration to 100 mg / mL, which was used in the experiment.

<< Example 1: Examination of the effect of promoting the phagocytic activity on Raw264.7 cells by an alcohol extract of Myristica fragrance >>
In this example, the effect of promoting the phagocytic activity in Raw264.7 cells by the alcohol extract of Myristica fragrance was examined.

(1. Pre-culture of Raw264.7 cells)
The supernatant was removed from the Raw264.7 cell culture dish, the Raw264.7 cells were washed with 10 mL of PBS, and 2 mL of 1 mM EDTA-PBS was added to the culture dish. After that, the cells were incubated for 3 minutes, and Raw264.7 cells were scraped off using a scraper. The scraped Raw264.7 cells were suspended using a pipette and centrifuged (600 × g, 5 minutes). Using a hemocytometer and counting the number of cells was adjusted to cell number 2 × ¹⁰ 5 cells / mL. To a 24-well plate supplemented with 500 μL of 20% FBS-DMEM, a cell suspension adjusted for the number of cells was added to 500 μL / well, and the mixture was incubated overnight.

(2. Addition of alcohol extract)
Nutmeg ethanol extract (100 mg / mL) and nutmeg methanol extract (100 mg / mL) were diluted 1600-fold with 10% FBS-DMEM medium. The concentration of ethanol or methanol added to 10% FBS-DMEM was 0.0625%. The 1600-fold diluted product was serially diluted 2-fold and 4-fold, respectively. Then, 1 mL / mL of nutmeg ethanol extract was added to each well to which Raw264.7 cells were added as the High group (62.5 μg / mL), Medium group (31.3 μg / mL), and Low group (15.6 μg / mL). Wells were added. Nutmeg methanol extract was added to each well to which Raw264.7 cells were added as the High group (62.5 μg / mL), Medium group (31.3 μg / mL), and Low group (15.6 μg / mL). bottom. At the time of sample addition, 0.5 μL / well of latex beads was added and cultured for 24 hours. 0.0625% ethanol-10% FBS-DMEM medium and 0.0625% methanol-10% FBS-DMEM medium were prepared and used as controls. 10% FBS-DMEM medium was prepared and used as a negative control. The culture supernatant was collected, washed 5 times with 1 mL of PBS, and the cells were fixed with 100% methanol.

(3. Evaluation of phagocytic activity)
The cells were observed using a microscope, and the state of the cells was photographed. The total number of cells in the photograph taken, the number of cells phagocytosed, and the number of beads phagocytosed by the cells were measured. The total number of phagocytosed beads calculated as the ratio of phagocytic cells existing in one visual field (phagocytosis rate) by dividing the number of phagocytic cells by the number of cells existing in one visual field. It was calculated as the ratio of beads phagocytosed by macrophages (phagocytosis) by dividing by.

Photographs of Raw264.7 cells treated with solvent control (ethanol only) and Raw264.7 cells treated with an ethanol extract of nutmeg are shown in FIGS. 1 and 2, respectively. Raw264.7 cells treated with an ethanol extract of nutmeg have a higher ratio of the number of cells phagocytosing latex beads to the total number of cells than Raw264.7 cells treated with solvent control (ethanol only). Can be observed.

In each of the nutmeg ethanol extract and the methanol extract-added groups, an increasing tendency of phagocytosis rate and phagocytosis was observed as compared with the control group (Fig. 3). In the nutmeg ethanol extract, the phagocytosis rate and phagocytosis were statistically significantly increased relative to the control in the Medium group and the High group. In the Low group of nutmeg ethanol extract, the phagocytosis rate and phagocytosis tended to increase. In the nutmeg methanol extract, phagocytosis and phagocytosis were statistically significantly increased relative to control in all groups. From these results, it was found that the nutmeg ethanol extract and the nutmeg methanol extract improve the phagocytic activity of Raw264.7 cells.

<< Example 2: Examination of the effect of promoting the phagocytic activity on mouse peritoneal macrophages by an alcohol extract of Myristica fragrance >>
In this example, the effect of alcohol extract of Myristica fragrance on promoting phagocytosis activity in mouse peritoneal macrophages was investigated.

(1. Preculture of mouse peritoneal macrophages)
BALB / c mice (8-week-old female) were intraperitoneally injected with 3% thioglycolate medium and bred for 3 days. Mice were sacrificed by cervical dislocation. The abdomen of the mice was disinfected with 70% ethanol. A small cut was made in the center of the abdomen, and the skin was torn across both ends to expose the peritoneum. Approximately 5 mL of cooled EDTA-PBS was injected intraperitoneally using a syringe, the mouse paws were grabbed and shaken for 3 minutes to massage the abdomen. PBS was collected from the infused portion of cooled EDTA-PBS and transferred to a centrifuge tube. 50 μL of PBS was taken for cell number measurement, and the rest was centrifuged (1000 rpm, 5 minutes). The number of cells was adjusted to ^{2 × 10 5 cells / mL.} To a 24-well plate supplemented with 500 μL of 20% FBS-DMEM, a cell suspension adjusted for the number of cells was added to 500 μL / well, and the mixture was incubated overnight.

(2. Addition of alcohol extract)
Nutmeg ethanol extract (100 mg / mL) and nutmeg methanol extract (100 mg / mL) were diluted 800-fold with 10% FBS-DMEM medium. The concentration of ethanol or methanol added to 10% FBS-DMEM was 0.125%. The 800-fold dilutions were serially diluted 2-fold and 4-fold, respectively. Then, 1 mL / well of nutmeg ethanol extract was added to each well to which mouse peritoneal macrophages were added as a high group (125 μg / mL), a medium group (62.5 μg / mL), and a low group (31.3 μg / mL). .. Nutmeg methanol extract was added as a High group (125 μg / mL), a Medium group (62.5 μg / mL), and a Low group (31.3 μg / mL) to each well to which mouse peritoneal macrophages were added. At the time of sample addition, 0.5 μL / well of latex beads was added and cultured for 24 hours. LPS was adjusted with 10% FBS-DMEM medium so that the final concentration was 20 ng / mL, and used as positive control. 0.125% methanol-10% FBS-DMEM medium and 0.125% methanol-10% FBS-DMEM medium were prepared and used as controls. 10% FBS-DMEM medium was prepared and used as a negative control. The culture supernatant was collected and washed 5 times with 1 mL of PBS. It was fixed with 100% methanol. LPS was added to the wells other than the positive control group, that is, the wells of the control group, the high group, the medium group, and the low group so that the final concentration of LPS was 1 ng / mL.
In mouse peritoneal macrophages, the response of macrophages was weaker than that of Raw264.7 cells, so the positive control group conducted experiments at an LPS concentration of 20 ng / mL, and the control, high, medium, and low groups Experiments were carried out by adding LPS to each well so that the final concentration of LPS was 1 ng / mL.

(3. Evaluation result of phagocytic activity)
The cells were observed using a microscope, and the state of the cells was photographed. The total number of cells in the photograph taken, the number of cells phagocytosed, and the number of beads phagocytosed by the cells were measured. The total number of phagocytosed beads calculated as the ratio of phagocytic cells existing in one visual field (phagocytosis rate) by dividing the number of phagocytic cells by the number of cells existing in one visual field. It was calculated as the ratio of beads phagocytosed by macrophages (phagocytosis) by dividing by.

Photographs of mouse peritoneal macrophages treated with solvent control (ethanol only) and photographs of mouse peritoneal macrophages treated with an ethanol extract of nutmeg are shown in FIGS. 4 and 5, respectively. Mouse peritoneal macrophages treated with an ethanol extract of nutmeg may have a higher ratio of the number of cells phagocytosing latex beads to the total number of cells than mouse peritoneal macrophages treated with solvent control (ethanol only). Can be observed.

In the nutmeg methanol extract, phagocytosis and phagocytosis were statistically significantly increased in all groups (Fig. 6). Similarly, in the Low group of nutmeg ethanol extract, the phagocytosis rate and phagocytosis tended to increase with respect to the control. From these results, it was found that the nutmeg ethanol extract and the nutmeg methanol extract improve the phagocytic activity of mouse peritoneal macrophages.

<< Example 3: Examination of the effect of promoting phagocytic activity on Raw264.7 cells by an alcohol extract of Myristica fragrance using flow cytometry >>
In this example, flow cytometry was used to examine the effect of the alcohol extract of Myristica fragrance on promoting phagocytosis activity in Raw264.7 cells.

(1. Pre-culture of Raw264.7 cells)
The supernatant was removed from the Raw264.7 cell culture dish, the Raw264.7 cells were washed with 10 mL of PBS, and 2 mL of 1 mM EDTA-PBS was added to the culture dish. After that, the cells were incubated for 3 minutes, and Raw264.7 cells were scraped off using a scraper. The scraped Raw264.7 cells were suspended using a pipette and centrifuged (600 × g, 5 minutes). The number of cells was measured using a hemocytometer, and the number of cells was adjusted to ^{5 × 10 5 cells / mL.} To a 24-well plate supplemented with 500 μL of 20% FBS-DMEM, a cell suspension adjusted for the number of cells was added to 500 μL / well, and the mixture was incubated overnight.

(2. Addition of alcohol extract and flow cytometry)
Nutmeg ethanol extract (100 mg / mL) and nutmeg methanol extract (100 mg / mL) were diluted 800-fold with 10% FBS-DMEM medium. The concentration of ethanol or methanol added to 10% FBS-DMEM was 0.125%. The 800-fold dilutions were serially diluted 2-fold and 4-fold, respectively. Then, 1 mL / well of nutmeg ethanol extract was added to each well to which Raw264.7 cells were added as the High group (125 μg / mL), Medium group (62.5 μg / mL), and Low group (31.3 μg / mL). bottom. Nutmeg methanol extract was added at 1 mL / well to each well to which Raw264.7 cells were added as the High group (125 μg / mL), Medium group (62.5 μg / mL), and Low group (31.3 μg / mL). At the time of sample addition, 0.5 μL / well of Texas Red fluorescent beads was added and cultured for 24 hours. LPS was prepared in 10% FBS-DMEM medium so that the final concentration was 1 ng / mL, and used as a positive control. 0.125% methanol-10% FBS-DMEM medium and 0.125% methanol-10% FBS-DMEM medium were prepared and used as controls. 10% FBS-DMEM medium was prepared and used as a negative control. The sample was incubated overnight. The culture supernatant was removed and washed twice with PBS at room temperature. Ice-cold EDTA-PBS was added at 1 mL / well, suspended, and cells were collected in an Eppen tube. Centrifugation (400 xg, 5 minutes) was performed and the supernatant was removed. The cells were lightly suspended in the supernatant remaining in Eppen. 0.5 μL of an antibody (CD11b: anti-mouse / human CD11b antibody (clone M1 / 70), BioLegend) was added. It was shaded and incubated at 4 ° C. for 30 minutes. PBS at room temperature was added, centrifugation was performed (400 × g, 5 minutes), and the supernatant was removed. 1 mL of FACS Buffer (2% FBS-PBS) was added, and further centrifugation was performed (400 × g, 5 minutes) to remove the supernatant. 1 mL of FACS Buffer (2% FBS-PBS) was added again, suspended, filtered with a 40 μm cell strainer, placed on ice, and measured with a flow cytometer.

(3. Evaluation result of phagocytic activity)
The results of flow cytometry are shown in FIG. The vertical axis shows the ratio of the number of cells that phagocytosed the beads to the total number of cells. As a result of evaluating the ability to promote the phagocytic activity of the nutmeg ethanol extract and the nutmeg methanol extract using a flow cytometer, the fluorescent beads were phagocytosed in each of the nutmeg ethanol extract and the nutmeg methanol extract as compared with the control. The proportion of Raw264.7 cells showed an increasing tendency (Fig. 7). From experiments using a flow cytometer, it was estimated that nutmeg extract may increase the phagocytic activity of Raw264.7 cells and activate innate immunity.

<< Example 4: Examination of nitric oxide production promoting effect in Raw264.7 cells by alcohol extract of Myristica fragrance >>
In this example, the effect of promoting the production of nitric oxide in Raw264.7 cells by the alcohol extract of Myristica fragrance was examined using the grease method.

(1. Pre-culture of Raw264.7 cells)
The supernatant was removed from the Raw264.7 cell culture dish, the Raw264.7 cells were washed with 10 mL of PBS, and 2 mL of 1 mM EDTA-PBS was added to the culture dish. After that, the cells were incubated for 3 minutes, and Raw264.7 cells were scraped off using a scraper. The scraped Raw264.7 cells were suspended using a pipette and centrifuged (600 × g, 5 minutes). The number of cells was measured using a hemocytometer, and the number of cells was adjusted to ^{2 × 10 6 cells / mL.} To a 24-well plate supplemented with 500 μL of 20% FBS-DMEM, a cell suspension having an adjusted cell number was added to 500 μL / well, and the mixture was incubated for 2 hours.

(2. Addition of alcohol extract and grease method)
Nutmeg ethanol extract (100 mg / mL) and nutmeg methanol extract (100 mg / mL) were diluted 800-fold with 10% FBS-DMEM medium. The concentration of ethanol or methanol added to 10% FBS-DMEM was 0.125%. Then, 1 mL / well of each of the prepared 125 μg / mL nutmeg ethanol extract and nutmeg methanol extract was added to each well to which Raw264.7 cells were added. At the time of sample addition, 0.5 μL / well of Texas Red fluorescent beads was added and cultured for 24 hours. 0.125% methanol-10% FBS-DMEM medium and 0.125% methanol-10% FBS-DMEM medium were prepared and used as controls. 10% FBS-DMEM medium was prepared and used as a negative control. The sample was incubated for 24 hours and the culture supernatant was collected. 1 mL of phosphoric acid was dissolved in 39 mL of Milli-Q water to prepare a phosphate buffer, and 400 mg of sulfanylamide and 40 mg of N-1-naphthylethylenediamine dihydrochloride were dissolved in this phosphate buffer to prepare a Griess reagent. 69 mg of sodium nitrite was dissolved in 10 mL of Milli-Q water to prepare a 100 mM aqueous sodium nitrite solution. A 100 mM sodium nitrite aqueous solution is diluted 1000 times with 10% FBS-DMEM medium, and a 100 μM sodium nitrite aqueous solution prepared with a 100 μM sodium nitrite aqueous solution is serially diluted to 50 to 1.56 μM with 10% FBS-DMEM medium. , Standard solution was prepared. The collected culture supernatant and standard solution were added to a 96-well plate at 100 μL / well. 100 μL / well of Griess reagent was added. Incubated for 20 minutes at room temperature. Absorbance was measured at 540 nm. A calibration curve was prepared from the values of the standard solution, and the nitrite concentration in the culture supernatant was calculated.

(3. Evaluation result of nitric oxide production promoting effect)
Using the Griess method, when macrophages phagocytosed bacteria and the like, the concentration of NO released to eliminate foreign substances such as bacteria was measured. As a result, in the treatment group of nutmeg ethanol extract and nutmeg methanol extract, the NO concentration produced by the phagocytosed Raw264.7 cells tended to increase as compared with the control. From this result, it was confirmed that the nutmeg extract promotes phagocytic activity and increases the concentration of nitric oxide associated with the phagocytic reaction.

The macrophage activator of the present invention can activate macrophages. Furthermore, the macrophage activator of the present invention can provide foods and drinks having a macrophage activating action and high safety.

Claims (6)

Mirisutika-fragrance seeds alcohol, activators of macrophages that have not been activated, characterized in that it comprises as an active ingredient an extract with a mixture of an aqueous solvent, or alcoholic and aqueous solvents. Wherein the extract is a nutmeg extract, activators of macrophages that have not been activated according to claim 1. The invention according to claim 1 or 2, wherein the macrophage activation is one or more selected from promotion of cytokine production, promotion of phagocytic activity, promotion of active oxygen production, promotion of nitric oxide production, and enhancement of antigen presentation function. activators of macrophages that have not been activated. A food composition for activating non- activated macrophages , which comprises, as an active ingredient, an alcohol, an aqueous solvent, or an extract of a mixture of an alcohol and an aqueous solvent of Myristica fragrance seeds. Wherein the extract is a nutmeg extract, non-activated activating food composition of macrophages according to claim 4. 4. activating food composition of macrophages which are not activated.

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