JPH0699313B2 - Macrophage and neutrophil activator - Google Patents

Description

TECHNICAL FIELD The present invention relates to a macrophage containing egg white and a neutrophil activator. More specifically, the present invention relates to an egg white-containing immunity-enhancing / infection-preventing agent which is effective in the prevention and treatment of various diseases and various pathogenic microbial infections that are considered to be due to the reduction of the immune capacity of the living body.

[Problems to be Solved by Prior Art and Invention]

Despite the recent development of antibacterial substances, opportunistic infections and cancers, which are considered to be caused by the deterioration of host defense function on the living body side, have become important issues.

Various illnesses that are thought to be due to a decrease in the immune capacity of the living body,
For example, there are various immunopotentiators or cancer immunotherapeutic agents against cancer, various pathogenic microbial infections and the like, but all of them have serious side effects such as fever and there is no definitive immunotherapy at present.

In addition, research on "functional foods" that have a biological regulation function in addition to the functions of nutrition and taste of foods based on a new concept, which has a special purpose to prevent individual diseases and diseases, as people's desire for health increases. Is being promoted and is becoming a major driving force for revitalizing the food industry, which is also an issue for the future.

On the other hand, in the livestock and fisheries industry, livestock, poultry or farmed fish are centralized, and large-scale management is carried out in response to the health consciousness of the people's dietary habits and the consumption is increasing. To meet market demand.

However, opportunistic infections due to stress due to overcrowding of livestock and immunodeficiency during early life are common. In addition, the damage caused by infectious diseases of cultured fish is also increasing.

Under these circumstances, in recent years to protect against immunodeficiency infection,
Increased use of antibiotics. However, administration of a large amount of antibiotics poses a serious social problem such as the problem of residues in livestock and marine products and the increase of resistant bacteria.

Along with this, legal regulations such as the poultry bird inspection system for ensuring the quality of livestock and marine products have become stricter, and there is growing concern that the administration of antibiotics is not appropriate. Therefore, development of a preventive method having an immunodeficiency / infection protective agent instead of an antibiotic is desired.

The immune system is an important mechanism in the body that identifies and eliminates xenogeneic antigens, and protects against invasion of pathogenic substances such as microorganisms into the body or foreign substances generated in the body such as cancer cells. It works as a defense mechanism.

Therefore, highly safe immune enhancers that enhance the host defense originally possessed by the host are expected.

[Means for Solving the Problems]

As a result of intensive studies on the immunopotentiating / infection-preventing agent, the present inventors have found that egg white has a macrophage and neutrophil activating effect, and completed the present invention.

That is, the present invention provides a macrophage and neutrophil activator characterized by containing egg white.

Hereinafter, the present invention will be described in detail.

The present inventors searched for bioactive components of natural substances such as herbal medicines and foods with the aim of activating macrophages that play a major role in defense against infection for the purpose of enhancing the biological defense system.

That is, macrophages have various functions (functions of macrophages and method for measuring functions; edited by the Japanese Society of Bacterial Education, Yakune Shuppan, 1985). Its basic purpose is to phagocytose and process foreign substances, waste tissues, and microorganisms, but it also produces monokines such as cell-enhancing factors to repair tissues and assist in cell division of self, neutrophils, and lymphocytes. It has a function of activating those cells.

Specifically, it has the following actions 1) to 9).

1) Treatment of foreign substances / waste products 2) Poor diet / bactericidal action / virus inactivation 3) Induction of inflammatory reaction 4) Antitumor action 5) Supporting lymphocyte function 6) Immunosuppression 7) Production of bioprotective substances 8) Tissue repair 9) Involvement in lipid metabolism The present inventors examined the macrophage activating action of mouse, guinea pig and pig, and the activating action of porcine neutrophil using egg white, and found that macrophages derived from various animals were activated. It turned out to turn into. In addition, when egg white was orally administered and the infection protective effect against bacterial infection in mice was examined, it was revealed that the infection resistance to various bacterial infections such as Escherichia coli and Staphylococcus aureus was remarkably enhanced.

Therefore, it was found that egg white has a macrophage and neutrophil activating effect, and has an immunopotentiating / infection-protecting effect.

Although the mechanism of action of the present invention is not necessarily clear, it is presumed that this is due to the fact that it is decomposed in the digestive tract by ingestion of egg white to produce some immunopotentiating substance, and this immunostimulation causes widespread bacterial infections including cancer. , It is presumed to be useful for other diseases caused by immune dysfunction (autoimmune diseases).

The egg white used in the present invention is not particularly limited, as long as it contains raw egg white, whole egg powder, egg white powder or a component constituting egg white or a fraction treated with a digestive enzyme. .

The subject to which the macrophage and neutrophil activator of the present invention is administered is not particularly limited. For example, food nutrition
Foods with so-called "functional foods" that have a bioregulatory function for the purpose of preventing individual diseases in addition to the taste function
Can be used as

Furthermore, there is no influence of resistant bacteria found in antibiotics, there is no problem of persistence, and a feed that is safe and has a biological defense control function,
That is, it can be used as a "functional feed" for domestic animals such as pigs, chickens, cows, horses and sheep, fishes, pets (dogs, cats) and the like.

The dose of egg white is 1 day per kg of body weight for egg white powder.
It is at least mg, preferably about 10 mg to 2 g.

Administration is usually taken orally. The administration period is used at the time of prevention and treatment of various pathogenic microbial infections and diseases such as cancer.

When practicing the present invention, the egg white or its fractions / ingredients are used for oral tablets / capsules / powder /
Granules and syrups can also be used.

That is, when preparing a solid preparation for oral administration, an excipient, a binder, a disintegrating agent, a lubricant and the like are added to the main ingredient, and tablets, capsules, powders and granules are prepared by a conventional method.

In addition, when preparing an oral liquid formulation, the main drug is a buffer,
A syrup or the like is prepared by increasing the amount of the corrigent and the stabilizer by a conventional method. When the egg white or its fraction / component and the antibacterial agent or the antineoplastic agent are used in combination, they may be administered separately, or if necessary, they may be combined.

Further, it may be added to foods such as water, milk, artificial milk, functional foods such as health foods, or feed to be administered. In the case of feed, it can also be used as a feed material or premix material.

〔The invention's effect〕

 Hereinafter, the effects of the present invention will be described with reference to experimental examples.

Experimental Example 1 Macrophage activating effect of mouse, guinea pig and pig, and porcine neutrophil activating effect by in vitro addition of chicken egg white. Preparation method of mouse macrophage: Mouse was anesthetized with ether, and blood was removed by carotid transection, and then cooled. Inject 5 ml of Hank's solution intraperitoneally. After massaging the abdominal wall for 30 seconds, Hanks' solution containing resident macrophages was collected. Centrifuge and wash in MEM medium containing 15% mouse serum
Adjusted to 2.5 × 10 ⁶ / ml.

Guinea pig macrophage preparation method: Guinea pigs (Hartley female: body weight 300 g) injected intraperitoneally with 10 ml of 1% casein Na saline 4 days ago were anesthetized with ether, and blood was removed by carotid artery transection. inject. After massaging the abdominal wall for 30 seconds, Hanks' solution containing peritoneal exudate cells is collected. Centrifuge washed, 15
The concentration was adjusted to 2.5 × 10 ⁶ / ml with MEM medium containing% guinea pig serum.

Preparation method of porcine macrophages (peripheral blood monocytes) and neutrophils: 10 ml of peripheral blood (containing 25 units / ml heparin) was collected from the axillary vena cava of pigs, and 6% (W / V) dextran saline (molecular weight 6
6900) is mixed with 3 ml and left at 37 ° C for 30 minutes to separate the white blood cell layer. Gently pour this on top of 10 ml of Ficoll pack
After centrifugation at 1900 rpm for 4 minutes, monocyte and neutrophil fractions were obtained. The monocyte fraction was adjusted to 2.5 × 10 ⁶ ml except for non-adherent cells (lymphocytes) and used as macrophages. For the neutrophil fraction, erythrocytes were disrupted with 0.85% ammonium chloride solution and centrifuged to collect neutrophils. The concentration was adjusted to 2.5 × 10 ⁶ / ml with MEM medium containing 15% pig serum.

Activation measurement of mouse, guinea pig and pig macrophages: When macrophages are activated, glucose is hypermetabolic. Therefore, macrophage activation can be measured by measuring glucose consumption in culture.

Egg white powder was added in vitro to macrophage cultures at a rate of 100, 10, 1 μg / ml and incubated at 37 ° C for 48 hours under 5% CO _{2 and then} the glucose content in the cultures was determined by the glucose measurement reagent (glucose-B -Test Wako: Wako Pure Chemical Industries, Ltd.).

That is, 20 μl of the culture supernatant was placed in a small test tube, 1.5 ml of an enzyme solution for glucose measurement was added thereto, the mixture was incubated at 37 ° C. for 30 minutes, and then colorimetry was performed at 505 nm. Macrophage activation index
(MAI) was calculated by the following formula.

The results are shown in Table 1.

As is clear from Table 1, when egg white powder was added to mice, guinea pigs and pig macrophages at a rate of 100, 10, 1 μg / ml, glucose consumption was markedly enhanced and macrophages derived from various animals were activated.

Pig neutrophil activation assay: Labtech chamber (4 chambers) pig neutrophils
(2.5 x 10⁶/ ml) 0.5 ml of egg white powder to 100 μg / ml
Added in 5% CO₂The cells were cultured at 37 ° C for 3 hours under End of reaction
After that, the opsonized latex particles (0.81 μm) were added to 0.
05 ml was added and it was made to eat for 45 minutes. Discard the supernatant and wash thoroughly.
It was fixed with tanol and stained with Giemsa. Poor diet rate increased on microscopic examination
I examined whether I was doing it.

The results are shown in Table 2.

As is clear from Table 2, egg white powder was added to pig neutrophils at a rate of 100 μg / ml, and as a result, the phagocytic ability of neutrophils to latex particles was remarkably enhanced.

As described above, it was newly discovered here that egg white activates macrophages and neutrophils that play an important role in the biological defense mechanism and has an immunopotentiating effect.

Experimental Example 2 Oral administration of chicken egg white to prevent infection against bacterial infection in mice In vivo, egg white was orally administered to mice to examine the protective effect against various bacterial infections.

slc: ICR male mice (5-6 weeks old, body weight 25-33 g) were orally administered with egg white or egg white fractions or the amounts shown in Table 3 three times a day, and 24 hours later, clinically-derived Escherichia coli (Escherichia co
li) was inoculated intravenously with a bacterial amount of 5.0 × 10 ⁷ / mouse and infected 7
The survival rate was calculated from the number of survivors on the day. For comparison, the survival rate was also obtained in the case of oral administration of soy protein and skim milk. The results are shown in Table 3.

In addition, instead of clinically-derived Escherichia coli, clinically-derived Staphylococcus aureus 3.8 x 1
The survival effect after infection and the survival rate up to 14 days after inoculation were intravenously inoculated at a bacterial load of 0 ⁷ / mouse. The results are shown in Fig. 1.

From the results of Table 3 and FIG. 1, it was revealed that egg white and egg white fractions or components markedly enhance infection resistance against various bacterial infections.

In addition, a combination experiment with the antibiotic oxytetracycline (OTC) was conducted. That is, E. coli was infected one day after the administration of egg white, and at the same time as the infection, the antibiotic oxytetracycline was orally administered and the combined effect was examined. The results are shown in Figure 2.

As shown in FIG. 2, the combined use of egg white and antibiotics showed a marked increase in survival rate even with a small amount of antibiotics, the amount of antibiotics could be reduced, and a synergistic effect was observed.

This is a new discovery like no other.

Experimental Example 3 Macrophage activation by oral administration of chicken egg whites slc: ICR male mice (5-6 weeks old, body weight 25-33g) were orally administered with the amount of egg white powder shown in Table 4 three times a day. After 24 hours, a carbon clearance test was performed to see if macrophages were activated.

The carbon clearance test measures the macrophage function of the liver and spleen using the clearance of colloidal carbon from the blood as an index. Carbon (manufactured by Pelican) was injected into the tail vein at a rate of 160 mg / kg. 3,6 after injection
After 9,12, and 15 minutes, 2 μm of blood was collected from the retroorbital venous plexus and mixed with 2 ml of 0.1% sodium carbonate in a small test tube. 66 this
Absorbance was measured at a wavelength of 0 nm. Time (minutes) on the horizontal axis,
The logarithmic value of the absorbance is taken on the vertical axis, and the value every 3 minutes is plotted. The gradient of this straight line (phagocytosis index K) was determined, and the corrected dietary index (α) was calculated by correcting the weight of the liver and spleen.

The results are shown in Table 4.

As shown in Table 4, in all of the egg white administration groups, the phagocytic index K and the corrected phagocytic index α value were larger than those of the control, and oral phagocytic administration of egg white promoted and activated the phagocytic ability of mouse macrophages. Became clear.

Experimental Example 4 Oral administration of chicken egg whites to activating peripheral blood neutrophils in pigs Egg white powder 500 mg / kg was added to 4 pigs (body weight: about 20 kg) as a feed, and latex particles of peripheral blood neutrophils were obtained one day later. It was examined whether or not the poor phagocytic ability to

Peripheral blood neutrophils were separated by Ficoll-pack specific gravity centrifugation, and the phagocytic ability to latex particles was performed by the method described in Experimental Example 1.

The results are shown in Table 5.

As shown in Table 5, it was clarified that the dietary intake of egg white in pigs markedly enhanced and activated the phagocytic capacity of peripheral blood neutrophils.

From the results of the above experimental examples, various effects and various pathogenic microbial infectious diseases (bacteria, mycoplasma, viruses, fungi, protozoa, etc.) which are considered to be based on the immune enhancement / infection protection effect of chicken egg white, which is considered to be based on the reduction of the immune ability of the living body ), A preventive or therapeutic agent for cancer, etc., or a functional food and a functional feed (feed additive) material.

[Brief description of drawings]

FIG. 1 is a diagram showing the effect of oral administration of egg white against Staphylococcus aureus in mice, and FIG. 2 is a diagram showing the effect of oral administration of egg white against Escherichia coli in mice in combination with the antibiotic oxytetracycline. .

Claims (1)

[Claims] 1. A macrophage and neutrophil activator containing egg white.