WO2001051079A1

WO2001051079A1 - Antigen-specific ige antibody production inhibitors

Info

Publication number: WO2001051079A1
Application number: PCT/JP2001/000161
Authority: WO
Inventors: Takeshi Takahashi; Shinya Nagabuchi; Yoshitaka Nakamura; Takaji Yajima; Tamotsu Kuwata
Original assignee: Meiji Dairies Corporation
Priority date: 2000-01-14
Filing date: 2001-01-12
Publication date: 2001-07-19
Also published as: AU782539B2; CN1205998C; AU2551501A; JP4787445B2; CN1395490A; CN100384472C; CN1626240A; NZ519957A; HK1076735A1

Abstract

Mammalian (including human) IgE antibody production inhibitors and immunomodulatory compositions containing as the active ingredient lactoferrin (LF) or its hydrolyzate; and IFN-η production potentiators containing LF or its hydrolyzate and ceramides.

Description

Description Antigen-specific IgE antibody production inhibitor Technical field

The present invention relates to an IgE antibody production inhibitor that is effective for preventing or improving allergic diseases such as atopic dermatitis. The present invention also relates to a composition that stimulates the immune system in a living body to activate immunity. Skill

Lactoferrin (LF) is an iron-binding glycoprotein found in milk, but also in exocrine fluids such as saliva, tears, bile, and fluid, and is released by activated neutrophils at the site of inflammation You.

LF has a broad spectrum of biological activities. For example, it has an antibacterial effect, an antiviral effect, an antitumor effect, an inhibitory effect on cancer metastasis formation, and the like.

LF also affects cytoin secretion. For example, it promotes the secretion of human neutrophils from inulin-leukin-8 (IL-8), regulates the secretion of IL-1, IL-6, and TNFa, and suppresses the expression of GM-CSF at the gene level. is there.

Selective production of cytokins plays a protective or etiological role in various disease states. For example, most allergies are type I allergies involving IgE antibodies, and this IgE production is induced by a system centered on IL-4, while influenza IFN-r (IFN-r The production is controlled by a system centered on). The production of IFN-α is induced by the higher IL-12 and IL-18.

IFN-α also has antiviral, antimicrobial, and antitumor activities due to various immunomodulatory functions (J. Immunol., 130: 2011-2013, 1983; Proc.

Natl. Acad. Sci. USA 85: 4874-4878, 1988). Nakajima et al. Found that IFN-α production was enhanced when splenocytes of mice after oral administration of P-LF (b LF) were cultured in the presence of mitogen (Con A). 4 production was not enhanced (Biomedical Research, 29 (1): 27-33, 1999).

As described above, LF is involved in a variety of biological activities and regulation of cytodynamic force production. Therefore, an object of the present invention is to find a new biological activity of LF, and to provide a drug or a functional food containing LF as an active ingredient based on the activity. Another object is to find an orally ingestible substance having an effect of enhancing the biological activity of LF when combined with LF. Disclosure of the invention

The present inventors found that immunization of mice that orally ingested LF, heat-treated LF, or LF hydrolyzate with ovalbumin (OVA), a major food allergen, suppressed the production of OVA-specific IgE antibodies. And that the production of antigen-specific IL-4 in the spleen cell culture system was suppressed. In addition, they found that culturing macrophages in the presence of LF enhanced IL-12 production by macrophages. Furthermore, it was found that IFN-r production in spleen cell culture system was markedly enhanced when LF and ceramides were used in combination. Suppression of production of antigen-specific IgE antibodies by oral ingestion of LF is due to suppression of activation of IgE antibody-producing B lymphocytes by suppression of IL-4 production and to Thl dominance of Thl / Th2 balance by enhancement of IL-12 production. Probably due to the shift. In other words, oral ingestion of LF can prevent a decrease in cellular immunity due to a shift toward Th2 immunity, and can prevent or improve allergic diseases and antibody-mediated nephritis. Furthermore, the remarkable enhancement of IFN-production by the combination of LF and ceramides indicates that the combination of LF and ceramides is effective against viral diseases, bacterial infections, or tumors. Understand. That is, the present invention provides an inhibitor of IgE antibody production in mammals, including humans, containing LF or a hydrolyzate thereof as an active ingredient.

The present invention also provides a composition containing LF or a hydrolyzate thereof as an active ingredient, for suppressing IgE antibody production in mammals including humans and regulating immunity.

In addition, the present invention relates to a composition for enhancing IFN-α production of mammals including humans comprising LF or a hydrolyzate thereof and ceramides, an antiviral composition, an antibacterial infection composition, and an antitumor agent. It provides a composition.

The present invention also provides an immunostimulator composition for mammals, including humans, containing LF or a hydrolyzate thereof and ceramides.

The present invention also provides use of LF or a hydrolyzate thereof for producing an IgE antibody production inhibitor in mammals including humans.

The present invention also provides use of LF or a hydrolyzate thereof for producing a composition for suppressing immunity by suppressing the production of IgE antibodies in mammals including humans.

In addition, the present invention relates to a composition for enhancing IFN-τ production in mammals including humans, an antiviral composition, an antibacterial infection composition, and an antitumor composition of LF or a hydrolyzate thereof and ceramides. Provide use for manufacturing.

The present invention also provides use of LF or a hydrolyzate thereof and ceramides for producing an immunostimulant composition for mammals including humans.

The present invention also provides a method for suppressing the production of IgE antibodies in mammals including humans, which comprises administering an effective amount of LF or a hydrolyzate thereof.

The present invention also provides a method for regulating immunity by suppressing the production of IgE antibodies in mammals including humans, which comprises administering an effective amount of LF or a hydrolyzate thereof.

Further, the present invention administers an effective amount of LF or a hydrolyzate thereof and ceramides. It is intended to provide a method for enhancing IFN-τ production in mammals including humans, or a method for treating a viral disease, bacterial infection or tumor.

Further, the present invention provides a method for immunostimulating mammals including humans, which comprises administering an effective amount of LF or a hydrolyzate thereof and ceramides. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the total IgE concentration in the serum after immunization with OVA antigen (average soil SD) in the 1% ゥ LF oral intake group (■: n = 7) and control group (□: n = 8) in mice. * P <0.05).

FIG. 2 is a graph showing the OVA-specific IgE antibody titer in serum (mean value SD; * p <0.05) in the above.

FIG. 3 is a diagram showing the IL-4 producing ability of spleen cells in the same as above (cells were pooled in each group). Hata: ^ ゥシ LF oral intake group, Painting: control group.

Figure 4 shows the total IgE antibody concentration in the serum of mice after the OVA antigen sensitization (3 times) for the LF oral ingestion group (n = 19) and the control group (n = 17). FIG. 0.05)

FIG. 5 is a diagram showing an OVA-specific IgE antibody titer in serum as in the above. (* P less 0.05)

FIG. 6 is a diagram showing 0VA-specific IFN-α production of splenocytes in the above. (ゥ LF oral intake group: n = ll, control group: n = 10)

FIG. 7 is a diagram showing 0VA-specific IL-4 production of splenocytes in the above. Cp <0.05)

Figure 8 shows 0VA-specific IFN of spleen cells in mice after OVA antigen sensitization (4 times) to the LF oral administration group (n = 8) and the control group (n = 7) after heat treatment. It is a figure which shows -a production.

FIG. 9 is a diagram showing OVA-specific IL-4 production of splenocytes in the above. (Control group; 7, 2.2¾bLF; 8) Figure 10 shows the concentration of total IgE antibody in the serum after immunization with OVA antigen in the mice (n = 7) and the control group (n = 8), orally ingesting 1% LF hydrolyzate in mice. FIG. (* p <0.05)

FIG. 11 is a diagram showing the effect on serum OVA-specific IgE antibody titers in the above.

FIG. 12 is a diagram showing OVA-specific IFN-α production of spleen cells in the above. FIG. 13 is a diagram showing OVA-specific IL-4 production of splenocytes in the above. FIG. 14 is a diagram showing IL-12 production in the culture supernatant after culturing mouse peritoneal macrophages in the presence of 100 × g / ml of mouse LF. ("P x 0.01")

Figure 15 shows cultures of splenocytes of mice after intraperitoneal administration of lactosylceramide or splenocytes after non-administration of lactosylceramide in the presence or absence of 100 xg / ml LF. FIG. 4 is a view showing IFN-α production in a supernatant. BEST MODE FOR CARRYING OUT THE INVENTION

It is known that allergen-specific IgE is involved in the pathogenesis of many allergic diseases, and in fact, allergic patients often find serum allergen-specific IgE (see Allergy Clin. I recitation). unol., 16: 161, 1996). Therefore, suppressing IgE production is one of the effective methods for preventing and treating allergies.

Cytokines produced by CD4 + helper T cells (Th cells) play an important role in IgE production. Th cells are roughly divided into two subpopulations, Th1 cells involved in cell-mediated immunity and Th2 cells involved in humoral immunity, depending on the difference in cytodynamic force produced and their function. Involved are Th2 cells (J. Immunol., 136: 2348, 1986).

Th1 cells and Th2 cells are in a relationship of mutually inhibiting their differentiation and function expression by the cytokines produced respectively. That is, Th2 cells have IL-4, IL-5, And produce IL-6 to positively regulate IgE production. Conversely, IFN-r produced by Thl cells suppresses IgE production by suppressing the IgE class switch and Th2 cell response. For this reason, IgE production and allergic diseases are characterized by enhanced Th2 cell responses (Nature, 383: 787, 1996). Therefore, it can be prevented or improved by inducing a Thl cell response to restore the Thl / Th2 balance to normal. Attempts have therefore been made to search for factors that are likely to induce a Thl response from food components. So far, certain types of lactic acid bacteria (Int. Arch. Allergy Immunol., 115: 278, 1998) ゃ nucleotides (Int. Arch. Allergy Immunol., 122: 33, 2000) have been shown to exhibit this effect.

In recent years, host defense effects of oral administration of LF have been reported in various animal models. Therefore, the present inventors first investigated the production of total IgE and antigen-specific IgE in serum when LF was orally ingested freely using mice. As a result, it was revealed that free oral ingestion of LF suppressed the production of whole IgE antibodies and antigen-specific IgE.

It is known that the physiological activity of LF is reduced by heat treatment (J. Pediatr., 90: 29, 1977). Therefore, it is considered that heat treatment of LF may decrease the ability of LF to suppress production of all IgE antibodies and antigen-specific IgE. Therefore, the effects of heat-treated LF on total IgE antibody production and antigen-specific IgE production were examined. Furthermore, spleen cells were cultured in the presence of OVA, and cytokin (IFN-a, IL-4) levels in the culture supernatant were examined. As a result, it became clear that heating LF does not lose its antigen-specific IgE antibody production inhibitory action and IL-4 production inhibitory action.

Oral ingestion of LF and heat-treated LF significantly reduced total IgE antibody production and antigen-specific IgE production relative to the control group. In addition, it was revealed that when spleen cells sensitized with the antigen were cultured in the presence of the antigen, IL-4 production was significantly reduced as compared to the control group. Therefore, the effects of the free oral ingestion of hydrolyzed LF on total IgE antibody production and antigen-specific IgE production were examined. In addition, spleen cells sensitized with the antigen were cultured in the presence of the antigen, and the level of cytodynamic force (IFN-a, IL-4) in the supernatant was examined. As a result, total IgE antibody production and antigen-specific IgE production tended to decrease in the LF hydrolyzate intake group. On the other hand, the production of IFN-α tended to increase in the LF hydrolyzate intake group in the presence of each concentration of OVA. No difference was seen.

IL-12 is a cytokine produced by antigen-presenting cells such as monocytes, macrophages, and dendritic cells, and plays an important role in the production of IFN-α and inducing the differentiation of helper T cells to Thl (Blood , 84: 4008, 1994; J. Leukoc. Biol., 55: 280, 1994). Gazzinelli et al. (J. Immunol., 153: 2533, 1994) reported that when mice were infected with Toxoplasma gondii, IL-12 production in spleen and peritoneal cells was increased, and the mice were further exposed to anti-IL-12 antibodies. It was reported that spleen cell IFN-α production decreased and IL-4 and IL-10 production increased after administration. Also, Nastala et al. (J. Immunol., 153: 1697, 1994) showed that administration of IL-12 to mice transplanted with cancer suppressed cancer growth and increased IFN-a concentration in blood. Heading.

Therefore, mouse peritoneal macrophages induced in the Chodary Collection medium were cultured together with LF to examine IL-12 production. As a result, it became clear that LF induced IL-12 production by macrophages.

By the way, atopic dermatitis, which was previously thought to be mild dermatitis, is now counted as one of the intractable diseases seen from infants to adults, and has become a major social problem. One of the causes is suspected of abnormal metabolism of sphingolipids in the epidermis. In fact, many atopic patients have reduced epidermal ceramide levels. Ceramides have also begun to be used as beauty foods. Therefore, the present inventors considered that oral ingestion of a combination of LF and ceramides would further enhance the effect of LF alone in preventing and improving allergic diseases. Therefore, the effect of the combination of LF and ceramides on IFN-α production was examined. As a result, it was revealed that the combination of LF and ceramides markedly enhanced IFN-α production in splenocytes. In sphingolipids, it is known that lysosphingolipids (sphingosine, sphingomyelin, rozophosphatidylcholine, etc.) in kefir (fermented milk) promote IFN-; 3 production (Biotherapy: 115-123, 1994). ). It has also been reported that administration of sufingosine increased blood IFN-3 and IFN-α in infected mice (Osada, K. et al .: Animal Cell technology: Development towards the 21st

Century 1995, 1067-1071).

Based on these results, suppression of production of antigen-specific IgE antibodies by oral ingestion of LF is due to suppression of activation of IgE antibody-producing B lymphocytes by suppression of IL-4 production and Thl / Th2 balance by enhancement of IL-12 production. Is likely to have shifted to the Thl dominance. Therefore, oral ingestion of LF is effective in preventing and improving allergic inflammatory diseases. Therefore, by adding an effective amount as a nutritional supplement to foods, it is possible to prevent and ameliorate allergic diseases in daily eating habits, and it can also be used as a pharmaceutical for such diseases. Allergic inflammatory diseases include, for example, chronic bronchial asthma, atopic dermatitis, hay fever (allergic rhinitis), allergic vasculitis, allergic conjunctivitis, allergic gastroenteritis, allergic liver disorder, allergic cystitis And allergic purpura. Alternatively, it can be expected to prevent or improve nephritis and the like mediated by antibodies.

In addition, when LF and ceramides are used in combination, IFN-α production is markedly enhanced.Thus, by using LF and ceramides in combination, the Thl / Th2 balance is shifted to the Thl superiority. It can be seen that the production of IgE antibody is suppressed. IFN-a has antiviral, antitumor, cytotoxic T lymphocyte, natural killer cell (NK cell) activity, neutrophil activation, macrophage activation, MHC class II expression promotion effect, IL-2 receptor expression promotion effect, It is known that the expression of Fc receptor is promoted. Also, IFN is a biological substance that originally plays a part in the defense mechanism against foreign substances in living organisms, has extremely high selective toxicity, and is highly safe for living organisms. Thus, when a food or the like containing a composition comprising a combination of LF and ceramides is orally ingested, living cells can be stimulated to promote secretion of IFN-α and to elicit the above-mentioned physiological activity of IFN-α. Therefore, a composition combining LF and ceramides can be an antiviral agent, an antitumor agent, and an antimicrobial agent. Regarding the safety of LF, no abnormalities were observed in the acute toxicity test and the subacute toxicity test of LF in rats using the maximum dose of 2,000 mg / kg / body weight / day. Is not recognized (Milk Science Vol.48, No.3, P227-232, 1999). As for the safety of ceramide, LD _5D Oryza Ceramide to mouse is 5, 000mg / kg or more (FOOD Style Vol.4, No.10: 99-105 , 2000).

The LF that can be used in the present invention includes, for example, commercially available LF, colostrum of mammals (eg, humans, pests, higgs, goats, pests, etc.), transitional milk, normal milk, terminal milk, and the like, or LF separated from these skim milks derived from milk, whey, etc. by conventional methods (for example, ion-exchange chromatography), hydrolysates with acids or enzymes, and apoproteins deferred with hydrochloric acid, citric acid, etc. Includes metal-saturated or partially-saturated LF, which is obtained by chelating LF and apo LF with metals such as iron, copper, zinc, and manganese.

A known hydrolysis means can be used for preparing the LF hydrolyzate of the present invention. When an enzyme is used for hydrolysis, trypsin is used in the present invention as an enzyme, but other enzymes such as pepsin and papain are also used, but are not limited to these enzymes as long as the hydrolyzate has the activity of the present invention. . After deactivating the enzyme by heating, the hydrolyzate may be fractionated by a conventional method such as ultrafiltration and then subjected to a concentration operation. The obtained liquid hydrolyzate may be used as it is or may be lyophilized before use. Recombinant LF includes a polypeptide having substantially the same amino acid sequence as described by Orla III. Conneely et al. (US Patent 5,766,939). Also natural allelic variants that occur in the (native) and LF modified by the insertion, substitution, or deletion of one or more amino acids as compared to the native LF. Recombinant LF also includes recombinant LF expressed in transgenic animals, e.g., pests, wherein the glycosylation pattern may differ from that of native LF obtained from human milk. .

The ceramides used in the present invention contain ceramide-related substances, and for example, sphingo lipids, particularly glycolipids, are preferred. Glycosphingolipids include, for example, the simplest glycosphingolipids are cerebrosides found in milk, brain, and kidneys, as well as sulfatide with a sulfate group, ceramide oligohexoside with a few neutral sugars, and amino. Examples include globosides with sugars and gandariosides with sialic acid. The structure of the glycosphingolipid is different from that of the glycosphingolipid, and the structure of the glycosphingolipid is more than 100 types, but all those having the action of the present invention are included. Preference is given to lactosylceramide, galactosylceramide, darcosylceramide and the like. Also preferred is sufingomyelin, which is a type of phospholipid (contained in milk). In addition, the use of asymmetric synthesis technology enables chemical synthesis of natural ceramides, and optically active ceramides are being developed. Ceramide 2 and Cosmo Farm's Ceramide 3 are such. In the present invention, these ceramides can also be used as long as they have the action of the present invention. In addition, plant-derived ceramides are receiving attention and are starting to be used. For example, rice-based sphingolipids, like animal sphingolipids, have a basic skeleton of hydrophobic ceramide, which is a long-chain base sphingosine with an acid amide bond to a fatty acid. In addition, sphingolipids derived from rice vary in species depending on the difference in carbon number of the long-chain base sphingosine and fatty acids, and the presence or absence of hydroxyl groups and double bonds. According to a report by Prof. Fujino of Obihiro University of Agriculture, there are at least more than 20 species of sphingolipid molecular species. The present invention also includes these ceramides.

In the present invention, LF (all LFs and LFs having the same Of ceramides, preferably 0.00001-0.1% by weight, more preferably 0.00005-0.01% by weight, based on the total weight of the composition. Particularly preferably, 0.0001 to 0.05% by weight, LF is estimated to be preferably 0.005 to 10% by weight, particularly preferably 0.01 to 5% by weight, most preferably 0.01 to 3% by weight, based on the total weight of the composition. However, those skilled in the art will be able to easily determine the optimal amount according to the composition to be used as a control, for example, by experiments described below. Incidentally, the ceramide content in breast milk is known, and it is considered to be a tentative standard in the amount of ceramides to be mixed.

The present invention has revealed that oral ingestion of LF suppresses antigen-specific IgE antibody production. Therefore, a person skilled in the art selects a preferred form of LF and an effective amount applicable to the present invention from the above-mentioned various LFs based on the present invention, for example, using IgE antibody production as an index, and selects clinical data, Alternatively, taking into account the relationship between clinical symptoms and serum IgE in patients, foods, dietary supplements, foods for the sick, infant formulas, health foods, health claims foods, functional foods, foods for specified health use, Alternatively, it is possible to determine the shape and effective dose of the LF when used for pharmaceuticals and the like. Therefore, the type of LF thus determined and its effective dose are encompassed by the present invention. The technology of adding and processing LF to foods and the like is well known and used. When an effective amount of LF is used as a pharmaceutical, it can be processed into various formulations known to those skilled in the art. Example

Hereinafter, the present invention will be described with reference to Test Examples and Examples, but the present invention is not limited to these Test Examples and Examples.

In the following test examples, test examples 1 to 4 and 6 used 3-week-old male BALB / c mice (Japan SLC; Shizuoka, Japan), and test example 5 used 8-week-old female BALB / c mice. (Japan SLC; Shizuoka, Japan) was used. Test for significant difference between control group and experimental group Performed by Student 'st test.

[Test Example 1] Inhibitory effect of LF LF (bLF) on IgE antibody production

During the experimental period, mice were given a diet containing casein as a protein source (in accordance with AIN76) and water containing 1% LF (LF, manufactured by DMV Japan) freely in the LF administration group (n = 7). . The control group (n = 8) had free access to the above diet and water without bLF.

On day 5 and day 19 after the start of the experiment, 10 ^ g of ovalbumin (OVA, manufactured by Seikagaku Corporation) was immunized intraperitoneally with 4 mg of aluminum hydroxide per mouse. On day 26, blood was collected from the orbit, and the serum total IgE antibody level and the OVA-specific IgE antibody level were measured by ELISA. Both serum total IgE antibody level (Figure 1) and OVA-specific IgE antibody level (Figure 2) were significantly (* p <0.05) lower in mice in the bLF group than in the control group.

[Test Example 2] IL-4 production inhibitory effect of bLF

In Test Example 1, the spleen was excised on day 26 to prepare a spleen cell suspension. After hemolysis, splenocytes were cultured for 72 hours in the presence of various concentrations (5, 10, 50, and 100 / g / ml) of OVA. In _c bLF group administered the IL-4 levels in the culture supernatant was measured by ELISA after the culture (n = 7), IL-4 levels in OVA for each concentration, than those in the control group (n = 8) All decreased (Fig. 3).

[Test Example 3] Heat-treated bLF inhibits IgE antibody and cytokine production The diet [bLF (+)] obtained by adding 2.2 bLF to the diet of Test Example 1 was heated at 70 ° C for 1 hour. As a control, a diet without bLF [bLF (-)] was similarly heated at 70 ° C for 1 hour.

Mice were allowed free access to bLF (+) (experimental group) or bLF (—) (control group) during the experimental period. On days 5, 14, 23 and 33 after the start of the experiment, mice were immunized intraperitoneally with 10 g of ovalbumin (0VA, manufactured by Seikagaku Corporation) together with 4 mg of aluminum hydroxide. Day 8 (day 30) after the third intraperitoneal immunization (7 weeks Aged), blood was collected from the tail, and antibodies in the serum were measured by ELISA. Splenocytes were collected individually on day 11 (day 33) after the third peritoneal immunization and day 8 (day 40) after the fourth peritoneal immunization. Splenocytes were treated with 10% FCS, lOOU / ml penicillin G, 100 xg / ml streptomycin and 5X10- 2-mercaptoethanol for 72 hours with various concentrations of OVA (0, 50, 100, 200 and 400 ii / ml). The cells were cultured in RPMI 1640 medium. After culturing, the levels of IFN-α and IL-4 in the culture supernatant were measured by ELISA.

When the immunization was performed three times, the levels of total IgE antibodies and the OVA-specific IgE antibodies in the serum of the experimental group were significantly lower than those of the control group (FIGS. 4 and 5). In addition, IFN-α production of splenocytes at each concentration of OVA (0, 50, 100, and 200 g / ml) showed little difference between the experimental and control groups (Figure 6). . On the other hand, the IL-4 production of splenocytes at 0 VA at each of the above concentrations was generally lower in the experimental group than in the control group, and under the stimulation with 200 ^ g / ml OVA, It was significantly lower than in the control group (Figure 7).

In the case of four immunizations, spleen cells produced IFN-α at each concentration of OVA (0, 50, 100, 200, and 400 ^ g / ml), as in the three immunizations, in both groups. Almost no difference was found between them (Fig. 8). On the other hand, IL-4 production at 0 VA at each of the above concentrations was significantly significantly lower in the experimental group than in the control group (FIG. 9).

From the above results, it was found that heating bLF does not lose its antigen-specific IgE antibody production inhibitory action and IL-4 production inhibitory action.

[Test Example 4] Inhibitory effect of hydrolyzed bLF on IgE antibody and cytokine production

(1) Preparation of hydrolyzed bLF

Trypsin (Wako Pure Chemical Industries, Ltd., 207-09891, for biochemistry, manufactured by pig kidney, 4500 USP trypsin units / mg) was dissolved in sterile PBS to give a trypsin stock solution (X500, 50 mg / ml). 10 g of b LF (Wako Pure Chemical Industries, Ltd., 127-04122, lot.KSG7724) with 25 mM CaCl _2-

It was dissolved in 0.1 M Tris-HCl (pH 8.2) so that This 1L bL F solution After heating to 37 ° C, 2 ml of a trypsin stock solution was added and reacted at 37 ° C for 4 hours. After the reaction, the enzyme was inactivated by heating at 80 ° C for 30 minutes. After centrifugation at 1700Xg for 20 minutes, the supernatant was collected and sterilized by filtration through a 0.45 m filter. The sample was treated with Microacilizer-1 S1 (manufactured by Asahi Kasei Corporation) to remove salts from the sample. After sterilization by filtration through a 0.45 mm filter, the sample was stored at -20 ° C until it was used for the test. b The yield of LF hydrolyzate was almost 100%. Therefore, the bLF hydrolyzate concentration was ^.

Mice were allowed free access to the diet of Test Example 1 and a solution containing the bLF hydrolyzate. The control group had free access to the same chow and water without bLF. On the 5th and 19th days after the start of the experiment, mouse ovalbumin (0VA, manufactured by Seikagaku Corporation) was immunized intraperitoneally with 4 mg of aluminum hydroxide. Blood was collected on day 8 (day 26) after the second immunization, and antibodies in the serum were measured by ELISA. At this time, spleen cells were collected for each individual. Splenocytes were cultured with OVA (0-400 g / ml) for 72 hours, and the levels of IFN-α and IL-4 in the culture supernatant were measured by ELISA. The serum total IgE antibody level and the OVA-specific IgE level in the experimental group tended to be lower than those in the control group (FIGS. 10 and 11).

In the spleen cells, the IFN-τ production at various OVA concentrations tended to be generally higher in the experimental group than in the control group (Fig. 12). On the other hand, there was almost no difference in IL-4 production between the experimental group and the control group (Fig. 13).

[Test Example 5] Effect of LF on IL-12 production

Although IL-12 is a relatively new cytoin, it is known to act on T cells and NK cells to induce the production of IFN-α (J. Exp. Med., 173). :

869-879, 1991; J. Exp. Med., 177: 1199-1204, 1993). To induce IL-12 production

Stimulation of cell components such as LPS is effective, and the cells producing them include a wide variety of cells such as macrophages, B cells, and neutrophils.

The present inventors have studied peritoneal macrophages in a medium supplemented with bLF (manufactured by Wako). After culturing, the level of IL-12 in the culture supernatant was measured by ELISA.

Mice fed a MF diet (Oriental Yeast) freely were intraperitoneally injected with 2.5 ml of Chodari cholate medium (DIFC0), and 4 days after the activation of peritoneal macrophages Dulbecco containing 1% FCS in the peritoneal cavity A phosphate buffer solution (Dulbeccos' PBS, Nissi) was injected to collect peritoneal cells. The cells were washed twice, and suspended in PBS again Dulbecco, it was injected into 96 © El plate (2X 10 ⁵ cells /0.2 ml well), incubated for 1 hour Plate with C0 ₂ incubator. After removing non-adsorbed cells, add 10% FCS, 100 U / ml penicillin G,

RPMI 1640 medium containing streptomycin, 5 × 10 2 -mercaptoethanol, and 100 zg / ml LF (manufactured by Wako) was injected at 200 / i. As a control, the above medium without bLF was used. The plate was 18 hours cultured in a C0 ₂ incubator scratch. After the culture, the IL-12 level in the supernatant was measured by ELISA. The results are shown in FIG. b Addition of LF to the medium markedly enhanced IL-12 production by macrophages.

The above results indicate that lactoferrin induces macrophage IL-12 production.

[Test Example 6] Effect of ceramide on IFN-α production

BALB / c mice (3 weeks old, male) from SLC Japan were used for the experiment. The mice were divided into groups of lactosylceramide-administered group (experimental group) and non-lactosylceramide-administered group control group, with three mice per group so that the average value and the variation in body weight were almost equal. The ceramide used was lactosylceramide (derived from Shibata Milk, Wako Pure Chemical Industries, Ltd., biochemical reagent 126-04491, lot. ELK6191). Lactosylceramide is 0.5%

Disperse in a 0.9 ° NaCl solution containing Tween20 at a concentration of ZOO ^ g / ml until use-

Stored at 20 ° C. Thaw at the time of use, add an equal volume of sterile PBS, mix well, and intraperitoneally administer lactosylceramide solution 200 1 per mouse every day for 14 days (excluding Saturday and Sunday) (20 g / mouse) mouse). Vehicle only (0.5% 0.9% NaCl solution containing Tween 20 and an equal volume of sterile PBS) was administered.

On the 14th day after the administration, 4 × 10 ⁵ splenocytes per mouse were seeded on a 96-well microplate (Falcon, No. 3072). 10% fetal serum for culture

RPMI1640 medium (Gibco, No.11875-093) containing 100 U / ml of penicillin, 100 g / ml of streptomycin, and 2-mercaptoethanol (5X101) (manufactured by Nippon Biotest Co., Ltd., lot.10086-1) Was used. Culture was performed in quadruplicate, and b L F

(Meggle, 0 or 100 g / ml) for 20 hours. After the culture, IFN-α in the culture supernatant was measured using an ELISA kit (END0GEN). Data were analyzed by two-way analysis of variance (2X2, 5% risk factor) due to lactosylceramide administration and bLF stimulation.

The results are shown in FIG. When ceramide was not administered, no IFN-α production was observed regardless of the presence or absence of bLF. When administered ceramide previously, the absence and presence of b LF both IFN- § production and _c has been shown to increase significantly, the interaction is confirmed in the two factors of ceramide and b LF Is

(p = 0.0016) Thus, it was found that administration of lactosylceramide enhances IFN-α production induced by LF.

Example 1

Milk Epitres, a treatment for milk allergy that does not contain whey protein as a raw material, was mixed with 0.01% of LF-LF in milk epitoles (manufactured by Meiji Dairies Co., Ltd.).

Example 2

Hydrolyzed milk containing whey protein as a raw material, Meiji Nobiyaka (Meiji Dairies Co., Ltd.) 0.01% LF

Industrial applicability According to the present invention, it has been clarified that oral ingestion of LF suppresses IgE antibody production in a living body.

Claims

The scope of the claims

1. An antigen-specific IgE antibody production inhibitor for mammals, including humans, containing lactoferrin or a hydrolyzate thereof as an active ingredient.

2. The inhibitor according to claim 1, further comprising a ceramide as an active ingredient.

3. A composition for regulating immunity by suppressing the production of antigen-specific IgE antibodies in mammals, including humans, containing lactoferrin or a hydrolyzate thereof as an active ingredient.

4. The composition according to claim 3, further comprising a ceramide as an active ingredient.

5. A composition for enhancing IFN-alpha production of mammals including humans, comprising lactoferrin or a hydrolyzate thereof and ceramides.

6. An antiviral composition for mammals including humans, comprising lactoferrin or a hydrolyzate thereof and ceramides as active ingredients.

7. An antibacterial infectious agent composition for mammals including humans, comprising lactoferrin or a hydrolyzate thereof and ceramides as active ingredients.

8. An antitumor agent composition for mammals including humans, comprising lactoferrin or a hydrolyzate thereof and ceramides as active ingredients.

9. An immunostimulator composition for mammals including humans, comprising lactoferrin or a hydrolyzate thereof and ceramides.

10. Use of lactoferrin or a hydrolyzate thereof for producing an antigen-specific IgE antibody production inhibitor for mammals including humans.

11. Use according to claim 10, further comprising ceramides.

1 2. Use of lactoferrin or a hydrolyzate thereof for producing a composition for suppressing immunity by suppressing the production of antigen-specific IgE antibodies in mammals including humans.

13. Use according to claim 12, further comprising ceramides.

14. Use of lactoferrin or a hydrolyzate thereof and ceramides for producing an IFN-alpha production enhancer composition for mammals including humans.

1 5. Use of lactoferrin or a hydrolyzate thereof and ceramides for producing an antiviral composition for mammals including humans.

1 6. Use of lactoferrin or a hydrolyzate thereof and ceramides for producing an antibacterial infectious agent composition for mammals including humans.

1 7. Use of lactoferrin or a hydrolyzate thereof and ceramides for producing an antitumor agent composition for mammals including humans.

18. Use of lactoferrin or a hydrolyzate thereof and ceramides for producing an immunostimulant composition for mammals including humans.

1 9. A method for suppressing production of IgE antibodies in mammals including humans, which comprises administering an effective amount of lactoferrin or a hydrolyzate thereof.

20. A method for suppressing the production of IgE antibodies in mammals including humans, which comprises administering an effective amount of lactoferrin or a hydrolyzate thereof and ceramides.

21. A method for treating a viral disease in mammals including humans, which comprises administering an effective amount of lactoferrin or a hydrolyzate thereof and ceramides.

22. A method for treating bacterial infection in mammals including humans, which comprises administering an effective amount of lactoferrin or a hydrolyzate thereof and ceramides.

23. A method for treating tumors in mammals including humans, which comprises administering an effective amount of lactoferrin or a hydrolyzate thereof and ceramides.

24. A method for regulating immunity by suppressing the production of IgE antibodies in mammals including humans, which comprises administering an effective amount of lactoferrin or a hydrolyzate thereof.

25. A method for enhancing IFN-α production in mammals including humans, comprising administering an effective amount of lactoferrin or a hydrolyzate thereof and ceramides.

26. A method for immunostimulating mammals including humans, which comprises administering an effective amount of lactoferrin or a hydrolyzate thereof and ceramides.