HK1210942B - Infection protection agent - Google Patents

Description

Infection defense agents

Technical Field

The present invention relates to an infection phylactic agent (preventive and/or therapeutic agent) that exhibits excellent protection against influenza viruses, is useful for the prevention or treatment of influenza, and has excellent stability and safety. The present invention also relates to an infection phylactic food and drink, an infection phylactic nutritional composition, and an infection phylactic feed, each of which contains the infection phylactic agent.

Background Art

Every year, influenza virus causes human epidemic through airborne transmission. In recent years, health improvements and medical progress have reduced the threat of influenza epidemic, but influenza virus can still cause death. Influenza virus is divided into three types: A, B and C. Among them, type A influenza virus may mutate and cause a pandemic. Vaccine is a common technology for preventing influenza virus infection. Unfortunately, vaccine is not necessarily satisfactory for the prevention of infection, because the infectious influenza virus that may undergo antigenic shift or antigenic drift is often inconsistent with the antigen of the vaccine. Therefore, in Japan, childhood vaccination is not mandatory at present. Influenza therapeutic drugs such as amantadine, oseltamivir and zanamivir need to take into account the appearance of side effects (for example, hallucinations and sleep disorders) and resistant bacteria, so such drugs need to be used with caution. In these cases, there has been a need for the development of food and drink or feed that can be safely taken in daily life and is expected to be useful for the prevention or treatment of influenza.

Traditional food ingredients that have a protective effect against influenza virus infection include sphingomyelin, lactoferrin, and κ-caseinglycomacropeptide, which is produced by the action of rennet or pepsin on κ-casein. It is also known that the exopolysaccharide produced by Lactobacillus bulgaricus 1073R-1, one of the lactobacilli contained in fermented milk, acts on the intestine to increase IgA production and enhance NK activity via Peyer's patches, thereby exhibiting a protective effect against influenza virus infection.

Lactadherin is a glycoprotein present in the milk fat globule membrane and accounts for 10% of the protein contained in the milk fat globule membrane. Lactadherin has a molecular weight of 43kDa to 53kDa, an isoelectric point of 7.0, and includes two epidermal growth factor-like domains. Lactadherin contained in cow's milk is also known as PAS-VI-VII (PAS6/7), and lactadherin contained in mouse milk is also known as MFG-E8. Lactadherin is believed to play a role in regulating the gastrointestinal function of newborns and is incorporated into infant formula for the purpose of preventing newborns from being infected with rotavirus. Unfortunately, it is not yet known that lactadherin and/or its degradation products have an infection protection effect against influenza virus.

Prior art literature

Patent Literature

Patent Document 1: Japanese Patent Application Laid-Open No. 2008-37788

Non-patent literature

Non-patent document 1: Bioscience, Biotechnology and Biochemistry, 57, 1214-1215, 1993

Non-patent literature 2: International Immunopharmacology, 11, 2246-2250, 2011

Summary of the Invention

Problems to be solved by the invention

The present invention aims to provide an infection prevention agent that exhibits infection prevention effects against influenza viruses and is useful for preventing or treating influenza. Another object of the present invention is to provide an infection prevention food and drink, an infection prevention nutritional composition, and an infection prevention feed, each comprising the infection prevention agent.

Solutions for solving problems

The present inventors have conducted extensive studies to solve the above-mentioned problems and, as a result, have found that lactadherin and/or a degradation product of lactadherin have an excellent protective effect against infection with influenza virus.

The present invention includes the following aspects:

Aspect (1). An infection protection agent against influenza virus, comprising lactadhesiven and/or a degradation product of lactadhesiven as an active ingredient.

Aspect (2). The agent for protecting against influenza virus infection according to aspect (1), wherein the lactadhesive and/or the degradation product of the lactadhesive is derived from cow's milk.

Aspect (3). The agent for protecting against influenza virus infection according to aspect (1) or (2), wherein the degradation product of lactadhesin is prepared by decomposing lactadhesin with a protease.

Aspect (4). The infection protection agent against influenza virus according to aspect (3), wherein the protease is at least one selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin and papain.

Aspect (5). A food or drink for infection prevention, a nutritional composition for infection prevention, or a feed for infection prevention, comprising the infection protection agent against influenza virus according to any one of aspects (1) to (4).

Aspect (6). A method for protecting against infection with influenza virus, comprising orally ingesting the infection protection agent according to any one of aspects (1) to (4).

Aspect (7). A method for using an infection protection agent comprising lactadhesin and/or a degradation product of lactadhesin as an active ingredient, the method comprising administering the infection protection agent to a patient infected with influenza virus.

Effects of the Invention

The infection protection agent (preventive agent and/or therapeutic agent) of the present invention exhibits a significant infection protection effect against influenza virus and is useful for the prevention or treatment of influenza.

DETAILED DESCRIPTION

Lactadhesin, which is an active ingredient of an infection protection agent, can be produced by desalting or concentrating component (A), (B), or (C) using an ultrafiltration (UF) membrane, a microfiltration (MF) membrane, a reverse osmosis (RO) membrane, or an ion exchange resin, wherein component (A) is buttermilk, which is an aqueous phase component produced during the production of butter from raw milk; component (B) is an aqueous phase component discharged during the phase inversion of high-fat cream having a fat content of 60% or more obtained by heating or shearing cream having a fat content of 40 to 50% obtained by separatory separation of raw milk; and component (C) is butter serum, which is an aqueous phase component separated from heated and melted butter. Alternatively, lactadherin can be prepared by adding hydrochloric acid to butter whey to a pH of 4.4, adding calcium chloride to the resulting mixture, and maintaining the mixture at 50°C for 30 minutes, followed by separation and dissolving or suspending the resulting precipitate in water. The solution or suspension is then desalted or concentrated using an ultrafiltration (UF) membrane, microfiltration (MF) membrane, reverse osmosis (RO) membrane, or ion exchange resin. Lactadherin degradation products, which are active ingredients in infection prevention agents, can be prepared by degrading lactadherin with a protease. Examples of proteases include commercially available food and industrial protease products such as Protease A "Amano" SD (trade name), Thermoase PC10F (trade name), and Protin SD-AY10 (trade name), trypsin, pancreatin, chymotrypsin, pepsin, and papain. These proteases can be used in combination. The lactadherin and/or lactadherin degradation products prepared above can be freeze-dried or spray-dried.

Lactadherin and/or its degradation products can be prepared from the milk of mammals such as humans, cows, buffaloes, goats, or sheep, produced through genetic engineering techniques, or purified from the blood or organs of such mammals. Lactadherin and/or its degradation products can be produced from such materials through simple processes at low cost, and the resulting products can be safely consumed daily. In particular, lactadherin and/or its degradation products are preferably derived from bovine milk. Lactadherin and/or its degradation products can be commercially available purified reagents.

Lactadherin and/or its degradation products can be used as infection protection agents without any treatment, or can be optionally prepared into powders, granules, tablets, capsules, or drinks by conventional processes. Freeze-dried or spray-dried lactadherin and/or its degradation products can be used as infection protection agents without any treatment, or can be prepared into any product by conventional processes.

The resulting lactadherin product can be incorporated into nutritional supplements, food and beverages (eg, yogurt, beverages, and wafers), nutritional compositions, or feed.

In addition to lactadherin and/or its degradation products, the infection prevention food, nutritional composition, or feed of the present invention may contain any material commonly contained in any other food, beverage, or feed. Examples of such materials include stabilizers, carbohydrates, lipids, spices, vitamins, minerals, flavonoids, and polyphenols. The infection prevention food, nutritional composition, or feed may contain any component that exhibits an infection prevention effect in combination with lactadherin and/or its degradation products as an active ingredient, such as sphingomyelin or lactoferrin.

Infection prevention foods, beverages, nutritional compositions, or feeds may contain any amount of lactadhesive and/or lactadhesive degradation products. To achieve an infection prevention effect against influenza virus, the amount of lactadhesive and/or lactadhesive degradation products incorporated into the drug, food, or feed is preferably adjusted so that the daily oral dose of lactadhesive and/or lactadhesive degradation products per adult is 0.1 mg or more.

By adding appropriate adjuvants to the active ingredient lactadhesin and/or lactadhesin decomposition product, the infection protection agent can be prepared as any pharmaceutical product. The preparation of the pharmaceutical product may involve the use of common excipients or diluents, such as fillers, extenders, binders, disintegrants, surfactants or lubricants. Examples of excipients include sucrose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicic acid, magnesium aluminate, synthetic aluminum silicate, magnesium metasilicate aluminate, calcium carbonate, sodium bicarbonate, calcium hydrogen phosphate and carboxymethylcellulose calcium. These excipients are used alone or in combination.

Although the present invention is described with reference to the embodiment, the embodiment constitutes a part of the present disclosure and should not be interpreted as limiting the present invention. Various alternative embodiments, examples and operating techniques will be apparent to those skilled in the art from the present disclosure.

For example, methods for using an infection protection agent are provided, wherein the infection protection agent, comprising the lactadherin and/or lactadherin degradation products described in the embodiments as an active ingredient, is administered to a patient infected with the influenza virus. The lactadherin and/or lactadherin degradation products used in this method can be isolated or purified from the raw materials described in the following examples. The infection protection agent can also be used in non-human mammals, including livestock such as dogs, monkeys, cats, cows, horses, pigs, chickens, and sheep.

Example

The present invention is described in detail through Examples and Test Examples, which should not be construed as limiting the present invention.

Example 1

Acetone (100 kg) was added to freeze-dried unsterilized buttermilk powder (10 kg), and the mixture was treated with a quark separator to completely remove the precipitate. Acetone was removed from the resulting supernatant with an evaporator, and the residue was dissolved in deionized water. Subsequently, the solution was treated with an ultrafiltration membrane with a 60 kDa molecular weight cut-off to recover the filtrate. The filtrate was then treated with an ultrafiltration membrane with a 30 kDa molecular weight cut-off to remove impurities, followed by desalination and concentration. Thereafter, the resulting product was freeze-dried to prepare lactadhesive powder (Example Sample 1) (68 g). The lactadhesive powder had a lactadhesive content of 78%. The lactadhesive thus prepared can be used as an infection defense agent without any treatment.

Example 2

The lactadhesin powder (500 mg) prepared in Example 1 was dissolved in purified water (100 mL), and the pH of the solution was adjusted to 8 with sodium bicarbonate. Thereafter, trypsin (manufactured by Sigma) (i.e., protease) was added to the solution to give a final lactadhesin concentration of 0.01%, and the mixture was treated with the enzyme at 37°C for one hour. The mixture was then heated at 85°C for 10 minutes to inactivate the enzyme. The resulting mixture was freeze-dried to prepare a lactadhesin degradation product powder (Example Sample 2) (463 mg). The lactadhesin degradation product thus prepared has a molecular weight of 5 kDa or less. The lactadhesin degradation product can be used as an infection defense agent without any treatment.

Example 3

The lactadherin powder (10 g) prepared in Example 1 was dissolved in purified water (200 mL), and the solution was then maintained at 45°C. Protease A "Amano" SD (manufactured by Amano Enzyme Inc.) (2 g) was added to the solution, and the mixture was treated with the enzyme for two hours. The mixture was then heated at 80°C for 10 minutes to inactivate the enzyme. The resulting mixture was freeze-dried to prepare a lactadherin degradation product powder (Example Sample 3) (8.2 g). The lactadherin degradation product thus prepared has a molecular weight of 5 kDa or less. The lactadherin degradation product can be used as an infection defense agent without any treatment.

[Test Example 1]

(Confirmation of infection prevention effects against influenza viruses)

Mice (Balb/c, male, 6 years old) were infected nasally with A/Guinzhou virus (i.e., influenza A virus) or B/Ibaraki virus (i.e., influenza B virus). A solution of Example Sample 1 (lactamase content: 100 μg/mL) and a solution of Example Sample 2 (lactamase decomposition compound content: 100 μg/mL) were prepared. Each solution was administered nasally at a dose of 1 μL/nasal cavity (dose: 0.1 μg), and the infection prevention effect against influenza virus was determined by the virus titer in the nasal wash. For comparison, a group of mice infected with each influenza virus were provided as a control. The plaque method of MDCK cells was used to determine the preventive effect. Table 1 shows the results.

[Table 1]

Values: mean ± standard deviation (n = 8).

*: significantly lower than the corresponding control (p<0.05).

Referring to Table 1, nasal administration of lactadherin or a lactadherin-degrading compound showed an infection-preventing effect against influenza A and B viruses, particularly against influenza A virus.

[Test Example 2]

(Confirmation of the preventive effect against influenza virus infection by oral administration)

Mice (Balb/c, male, 6 years old) were infected with influenza virus PR8(H1N1) (1×10 ³ pfu). Prior to viral infection, the mice were divided into two groups: one receiving oral administration of lactadherin (Example Sample 1) or a lactadherin degradation product (Example Sample 3) (dose: 0.1 mg/kg body weight) and one receiving oral administration of lactadherin (Example Sample 1) or a lactadherin degradation product (Example Sample 3) (dose: 10 mg/kg body weight). The protective effect against influenza virus infection was measured by viral titer in nasal wash fluid three days after viral infection.

For comparison, a group of mice that were orally administered lactoferrin (10 mg/kg body weight) before virus infection were provided as a positive control, and a group of mice that were orally administered a solvent (water only) before virus infection were provided as a control. The plaque method using MDCK cells was used to determine the preventive effect. Table 2 shows the results.

[Table 2]

Value: mean ± standard deviation (n = 10)

*1: Significantly lower than the control (p<0.05)

*2: Significantly lower than lactoferrin 10mg (p<0.05)

Referring to Table 2, oral administration of lactadherin or lactadherin degradation products significantly reduced viral titers in the nasal cavity compared to the control. The preventive effect of lactadherin or lactadherin degradation products was significantly greater than that of oral administration of lactoferrin. These results indicate that lactadherin or lactadherin degradation products exhibit excellent preventive effects against influenza virus infection. The results also demonstrate that this effect is achieved by oral administration of infection protection agents at doses of 0.1 mg/kg mouse body weight or greater.

Example 4

(Preparation of capsules for preventing influenza virus infection)

The raw materials were mixed in the ratio shown in Table 3, and the mixture was granulated and then encapsulated by a conventional process to prepare capsules for preventing influenza virus infection.

[Table 3]

Example 5

(Preparation of Tablets for Preventing Influenza Virus Infection)

The raw materials were mixed in the ratio shown in Table 4, and the mixture was formed into a compact (1 g) by a conventional process, followed by tableting, thereby preparing a tablet for preventing influenza virus infection.

[Table 4]

Example 6

(Preparation of liquid nutritional composition for preventing influenza virus infection)

Example Sample 1 (50 g) was dissolved in deionized water (4,950 g), and the solution was heated to 50° C. Thereafter, the solution was stirred at 6,000 rpm for 30 minutes using a TK homogenizer (TK ROBO MICS, manufactured by PRIMIX Corporation) to prepare a lactadherin solution having a lactadherin content of 39 g/5 kg. The lactadherin solution (5.0 kg) was mixed with casein (5.0 kg), soy protein (5.0 kg), fish oil (1.0 kg), perilla seed oil (3.0 kg), dextrin (17.0 kg), a mineral mixture (6.0 kg), a vitamin mixture (1.95 kg), an emulsifier (2.0 kg), a stabilizer (4.0 kg), and a fragrance (0.05 kg). The mixture was put into 200-mL retort pouches and sterilized at 121° C. for 20 minutes using an autoclave (Type 1 pressure vessel, model: RCS-4CRTGN, manufactured by Hisaka Works, Ltd.) to prepare a liquid nutrient composition for preventing influenza virus infection (50 kg).

Example 7

(Preparation of a beverage for preventing influenza virus infection)

Skimmed milk powder (300g) is dissolved in deionized water (409g), and example sample 2 (1g) is dissolved in the solution. The solution is heated to 50 ℃ and stirred 30 minutes at 9,500rpm with an ultra-disperser (ULTRA-TURRAX T-25, manufactured by IKA Japan). The resulting mixture is mixed with maltitol (100g), acidulant (2g), reduced starch syrup (20g), spices (2g) and deionized water (166g). Thereafter, the mixture is put into a 100-mL glass bottle and then sealed at 95 ℃ of sterilizations for 15 seconds, thereby preparing 10 bottles of influenza virus infection prevention beverages (each 100mL).

Example 8

(Preparation of feed for preventing influenza virus infection in dogs)

Example 1 (2 kg) was dissolved in deionized water (98 kg), and the solution was heated to 50° C. Thereafter, the solution was stirred at 3,600 rpm for 40 minutes using a TK homogenizer (MARK II 160, manufactured by PRIMIX Corporation) to prepare a lactadherin solution having a lactadherin content of 1.56 g/100 g. The lactadherin solution (10 kg) was mixed with soybean lees (12 kg), skim milk powder (14 kg), soybean oil (4 kg), corn oil (2 kg), palm oil (23.2 kg), corn starch (14 kg), wheat flour (9 kg), bran (2 kg), a vitamin mixture (5 kg), cellulose (2.8 kg), and a mineral mixture (2 kg), and the mixture was sterilized at 120° C. for four minutes to prepare a feed for preventing influenza virus infection (100 kg).

Claims (4)

1. Use of refined lactoglossin and/or lactoglossin decomposition products in the preparation of an infection defense agent against influenza virus, said infection defense agent comprising refined lactoglossin and/or lactoglossin decomposition products as active ingredients, said lactoglossin decomposition products being prepared by decomposing lactoglossin with a protease. 2. The use according to claim 1, wherein the refined milk agglutinin and/or the decomposition products of the milk agglutinin are derived from cow's milk. 3. The use according to claim 1 or 2, wherein the protease is at least one selected from the group consisting of trypsin, pancreatic enzyme preparations, chymotrypsin, pepsin and papain. 4. Use of the infection defense agent obtained according to any one of claims 1 to 3 in the preparation of a medicament for an infection defense method against influenza virus, said method comprising oral ingestion of the infection defense agent obtained according to any one of claims 1 to 3.