Composition for anti-infection

Abstract

To provide a composition that exhibits anti-infective effects. The present invention provides an anti-infection composition characterized by containing a plasmalogen.

Description

Technical Field

[0001] The present invention relates to a composition that activates natural killer cells and enhances immune function.

Background Art

[0002] Plasmalogen is known to have effects such as promoting neurogenesis, suppressing neuroinflammation caused by lipopolysaccharide (LPS), and suppressing the accumulation of amyloid-β (Aβ) protein in the brain, and is said to be effective in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, depression, and schizophrenia. For example, Non-Patent Document 1 reports that the memory function of patients with mild Alzheimer's disease was improved by oral administration of purified plasmalogen derived from scallops.

[0003] On the other hand, for novel viruses such as the novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), the innate immune function is important, and it is necessary to strengthen this innate immune function.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[17] (2017) 199-205

Disclosure of the Invention

Problems to be Solved by the Invention

[0005] As described above, various reports have been made regarding plasmalogens, but the effects of plasmalogens on natural immune functions have not been examined in detail.

[0006] An object of the present invention is to provide a composition that activates natural killer cells and exhibits an excellent immune function enhancing effect.

Means for Solving the Problems

[0007] As a result of intensive studies to solve the above problems, the present inventors have found that plasmalogens can activate natural killer cells, which are a type of lymphocyte that functions as a major factor in innate immunity, and have completed the present invention.

[0008] That is, the present invention is as follows. [1] A composition for enhancing immune function, characterized by containing plasmalogen. [2] A composition for activating natural killer cells, characterized by containing plasmalogen. [3] A composition for anti-infection, characterized by containing plasmalogen. [4] The composition for enhancing immune function according to the above [1], wherein the plasmalogen is a plasmalogen extracted from animal tissues. [5] The composition for activating natural killer cells according to the above [2], wherein the plasmalogen is a plasmalogen extracted from animal tissues. [6] The composition for anti-infection according to the above [3], wherein the plasmalogen is a plasmalogen extracted from animal tissues.

Effects of the Invention

[0009] The composition of the present invention activates natural killer cells and exhibits an excellent immune function enhancing effect.

Brief Description of the Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] The composition of the present invention is characterized by containing plasmalogen. The composition of the present invention activates natural killer cells (NK cells), which are a type of lymphocyte that functions as a major factor in innate immunity, and enhances innate immune function. Therefore, an anti-infection effect against viruses, bacteria, etc. can be expected. That is, the composition of the present invention is useful as a composition for enhancing immune function, a composition for activating natural killer cells, an antibacterial composition (antibacterial agent), an antiviral composition (antiviral agent), and other anti-infection compositions (anti-infective agents). Note that anti-infection in the present invention is a concept that includes prevention of infection by viruses, bacteria, etc. in healthy individuals and alleviation and treatment of infection symptoms in infected individuals.

[0012] In addition, the composition of the present invention can be expected to have an effect of improving symptoms such as fever in patients with rhizomelic chondrodysplasia punctata (RCDP), and can also be used as a composition for improving RCDP symptoms.

[0013] Plasmalogen is a type of phospholipid having an antioxidant action and is one of glycerophospholipids. It is a specific subclass of glycerophospholipids characterized by having a vinyl ether bond at the sn-1 position of the glycerol backbone, and has been confirmed at high concentrations in the cell membranes of many mammalian tissues.

[0014] The plasmalogen used in the present invention is not particularly limited as long as it is generally classified as a plasmalogen. For example, choline-type plasmalogen, ethanolamine-type plasmalogen, inositol-type plasmalogen, and serine-type plasmalogen can be mentioned. Among these, choline-type plasmalogen and ethanolamine-type plasmalogen are preferred, and ethanolamine-type plasmalogen is particularly preferred.

[0015] The plasmaogen of the present invention can be extracted from animal tissues. The animal tissues are not particularly limited as long as they contain plasmaogen, and examples include aquatic animals such as shellfish, sea squirts, sea cucumbers, salmon, saury, and skipjack, and birds. Among these, shellfish, sea squirts, and birds are preferred, and shellfish are particularly preferred. As the part to be used, an edible part is preferred. These animal tissues may be cut pieces, but it is preferable to use pulverized materials because plasmaogen can be extracted more efficiently.

[0016] Examples of shellfish include edible bivalves such as scallops, oysters, and abalones, and whelks, and scallops are particularly preferred. Scallops are edible bivalves belonging to the family Pectinidae, and examples include those belonging to the genus Mizuhopecten and the genus Pecten. Specifically, the Japanese scallop (scientific name: Mizuhopecten yessoensis) collected in Japan, the European scallop (scientific name: Pecten maximus (Linnaeus)) collected in Europe, etc. can be mentioned. Examples of the edible part include scallop adductor muscles and strings.

[0017] Sea squirts are edible chordates belonging to the family Halocynthiidae, and examples include those belonging to the genus Halocynthia and the genus Akahalocynthia. Specifically, Halocynthia roretzi (scientific name) and Akahalocynthia aurantium (scientific name) etc. can be mentioned. Examples of the edible part include the body part (fascia body).

[0018] Birds are not particularly limited as long as they are edible birds, and examples include chickens, silky fowl, ducks, etc. As the edible part, the breast meat rich in plasmaogen is preferred.

[0019] The extraction of plasmaogen can be carried out using water, an organic solvent, or a water-containing organic solvent, and it is preferable to use enzyme treatment in combination. For example, the ethanol extraction method and the hexane extraction method can be mentioned, and the ethanol extraction method is preferred.

[0020] As the ethanol extraction method, there is no particular limitation as long as it is a method of extraction using ethanol (including hydrated ethanol). For example, the methods described in JP-A-2019-140919, JP-A-2018-130130, Re-Published 2012-039472, JP-A-2010-065167, JP-A-2010-063406, etc. can be cited.

[0021] As the hexane extraction method, there is no particular limitation as long as it is a method of extraction using hexane. For example, the methods described in Re-Published 2009-154309, Re-Published 2008-146942, etc. can be cited.

[0022] The composition of the present invention can be used, for example, as so-called health foods such as pharmaceuticals (including quasi-drugs), foods for specified health uses, foods with nutritional functions, and foods with functional claims whose efficacy has been recognized by a specified authority.

[0023] When the composition of the present invention is used as a pharmaceutical or a health food, the composition of the present invention contains plasmalogen and can be distinguished from other products as a product in that it is used for enhancing immune function, activating natural killer cells, and / or anti-infection. There is no particular limitation as long as it can be distinguished from other products as a product. For example, any of the main body, packaging, instruction manual, and promotional materials of the product according to the present invention that display or suggest that it has an immune function enhancing function, a natural killer cell activating function, and / or an anti-infection function is included in the scope of the present invention.

[0024] The composition of the present invention may be for oral use or for parenteral use such as injection or infusion. However, from the viewpoint of easy intake, oral use is preferred. In the case of oral use, examples of its form include tablet form, capsule form, powder form, granule form, liquid form, granule form, bar form, plate form, block form, solid form, round form, paste form, cream form, caplet form, gel form, chewable form, stick form, etc. Among these, the capsule form is preferred.

[0025] The content of plasmalogen in the composition of the present invention may be appropriately contained within the range where the effect is exhibited. Although it depends on the form, for example, when converted to dry mass, plasmalogen is 10 -10 mass% or more of the whole composition of the present invention, preferably 10 -5 mass% or more, more preferably 0.1 mass% or more, and particularly preferably 1.0 mass% or more.

[0026] The intake amount of the oral composition of the present invention is not particularly limited, but from the viewpoint of more significantly exerting the effect of the present invention, the intake amount of plasmalogen is preferably such that it is 10 -6 μg / day or more per adult per day, more preferably 1 μg / day or more, still more preferably 500 μg / day or more, and particularly preferably 1000 μg / day or more. The upper limit is, for example, 20,000 μg / day, preferably 10,000 μg / day.

[0027] The oral composition of the present invention can be contained in one container or, for example, divided into 2 to 3 multiple containers so that the intake amount per day is the above-mentioned intake amount, as one-day supply.

[0028] The composition of the present invention can be produced by a known method by adding other components other than the components of the present invention, if necessary. Examples of other components other than the components of the present invention include vitamins, minerals, proteins, peptides, amino acids, animal oils, and vegetable oils.

[0029] Hereinafter, the present invention will be described in detail based on examples.

Examples

[0030] Using mice, the activity of NK cells (NK activity) by plasmalogen was evaluated.

[0031] [Plasmalogen] The plasmalogen used was a hexane-extracted ethanolamine-type plasmalogen (mainly containing ethanolamine-type plasmalogen and also containing choline-type plasmalogen) prepared from scallops (scientific name: Mizuhopecten yessoensis) by the following method.

[0032] 1. Add and mix kokurase P (manufactured by Mitsubishi Chemical Foods Co., Ltd.) and phospholipase A1 (PLA1) (manufactured by Mitsubishi Chemical Foods Co., Ltd.) to fresh scallop beards. 2. Next, add hexane / isopropanol and aspirate and filter the supernatant. 3. Add an aqueous sodium sulfate solution and mix well. 4. Dry the upper layer to dryness with a rotary evaporator. 5. Add and mix acetone cooled to 4°C. 6. Centrifuge at 3000 rpm, 10 min, 4°C. 7. Discard the supernatant and collect the precipitate. 8. Dry overnight in a desiccator.

[0033] [Administration of plasmalogen to mice] Seven-week-old C57BL / 6J male mice purchased from Oriental Yeast were used. They were housed in a SPF (Specific pathogen free) environment in the university animal experiment facility throughout the entire experimental period.

[0034] Plasmalogen dissolved in corn oil was orally administered to 3 mice in each of 4 groups at 200 μl / mouse (plasmalogen 20, 2, 0.2 mg / kg) for 4 days. The experiment was conducted with n = 2 only for 0.2 mg / kg. As a control, corn oil without plasmalogen was administered.

[0035] [Isolation of monocytes] The spleen and liver were excised from the mice. The spleen and liver were ground on a metal mesh (0.12×80, 0.2 mm mesh opening) of a 10 cm petri dish, and 10 ml of 3% FCS-RPMI was added. This solution was transferred to a 50 ml tube and centrifuged at 1500 rpm for 5 minutes.

[0036] To the pellet of the obtained spleen cells, 1 ml of lysing buffer was added to lyse the red blood cells, then 10 ml of 3% FCS-RPMI was added, and mononuclear cells were obtained by centrifuging at 1500 rpm for 5 minutes.

[0037] On the other hand, the liver was suspended in 33% Percoll solution with the pellet obtained by grinding with a metal mesh, and centrifuged at 1800 rpm for 15 minutes. 1 ml of lysing buffer was added to the obtained pellet to lyse the red blood cells. It was transferred to a 15 ml tube, 10 ml of 3% FCS-RPMI was added, and mononuclear cells of the liver were obtained by centrifuging at 1500 rpm for 5 minutes.

[0038] [Measurement of NK activity] NK cell activity was measured by a 4-hour Cr release assay using the NK-sensitive cell line YAC-1 as the target cell. YAC-1 was cultured in the presence of Na 51 CrO4 for 1 hour, and the cells were 51 labeled with Cr, and then suspended in RPMI1640 medium containing 10% fetal calf serum (FCS) at 1×10 5 cells / ml. Mononuclear cells isolated from the spleen and liver of mice were suspended in the culture medium according to the Effector (mononuclear cell):Target (YAC-1) ratio respectively. 100 μl of each was mixed in a 96-well round-bottomed microtitre plate (NUNC, Roskilde, Denmark), cultured at 37 °C for 4 hours in humidified air in the presence of 5% CO2, the supernatant was collected, and the γ-ray dose of the solution was measured. The percentage of specific lysis was calculated based on the following formula.

[0039] % specific lysis = (experimental release - spontaneous release) / (maximal release - spontaneous release) x100

[0040] Figure 1 shows the results of the cytotoxic activity against target cells of monocytes derived from the spleen and liver. The results are expressed as the mean value ± SD of the values obtained for each mouse in each group.

[0041] As shown in Figure 1, it was confirmed that monocytes from mice administered with plasmalogen had higher cytotoxic activity against the NK-sensitive cell line YAC-1 compared to control monocytes. That is, it became clear that oral administration of plasmalogen activates NK cells. Therefore, it is considered that the administration of plasmalogen enhances the immune function, and an anti-infection effect against viruses, bacteria, etc. can be expected.

Example

[0042] Mice were used to evaluate the increased expression of interferon (IFN) by plasmalogen.

[0043] [Plasmalogen] As the plasmalogen, ethanol-extracted ethanolamine-type plasmalogen (mainly containing ethanolamine-type plasmalogen and also containing choline-type plasmalogen) prepared from scallops (scientific name: Mizuhopecten yessoensis) by the following method was used.

[0044] 1. Add Cochlearase P (manufactured by Mitsubishi Chemical Foods Co., Ltd.) to fresh scallop mantle and mix. 2. Next, add ethanol and perform suction filtration. 3. Dry the filtrate with a rotary evaporator.

[0045] [Administration of plasmalogen to mice] Seven-week-old male C57BL / 6J mice purchased from Oriental Yeast were used. They were housed in a SPF environment at the university animal experiment facility throughout the entire experimental period.

[0046] Plasmalogen dissolved in water (1 mg / kg / day) was orally administered to 5 mice for 6 weeks. For the control group, water was administered.

[0047] [Measurement of the expression level of interferon-γ (IFN-γ)] The brain was removed from the mice, and the expression level of interferon-γ (IFN-γ) in the cerebral cortex was confirmed by real-time PCR.

[0048] The results are shown in Figure 2. As shown in Figure 2, the expression of IFN-γ in the cerebral cortex of the mice administered plasmalogen was significantly increased compared to the control. That is, it was confirmed that the administration of plasmalogen increased the expression of interferon-γ involved in the suppression of virus infection and the like.

Example

[0049] The inhibitory effect of plasmalogen on virus infection was evaluated using mice.

[0050] [Plasmalogen] As in Example 2, ethanolamine-type plasmalogen extracted with ethanol was used as the plasmalogen.

[0051] [Administration of plasmalogen to mice] The mice were bred in the same manner as in Example 2.

[0052] Plasmalogen dissolved in water (0.02, 1, 10 mg / kg / day) was orally administered to mice (n = 4) for 5 weeks. The control mice were administered drinking water without plasmalogen.

[0053] [Evaluation of virus infection in mice] Murid herpesvirus 1 (ATCC(R)VR-1399 TM ) was intraperitoneally injected into mice at a concentration of 1x108 pfu (Plaque-Forming Unit), and the mice were sacrificed 72 hours later. Each tissue of the lung, spleen, and liver was collected.

[0054] Viral DNA was amplified by RT-PCR (reverse transcription Polymerase Chain Reaction) using virus-specific primers for the extracted genomic DNA of each tissue. For the PCR method, an equal amount of genomic DNA (100 ng) was used from each group. Genomic DNA of the Gapdh gene (Glyceraldehyde 3-phosphate dehydrogenase) was amplified by the PCR method and used as an internal control.

[0055] Figure 3 shows the results of RT-PCR of viral DNA in the lung tissue of mice. Figure 4 shows the number of white blood cells in the blood of mice. Figure 5 shows the measurement results of the spleen weight of mice.

[0056] As shown in Figure 3, a significant increase in viral DNA was observed in the lung tissue of the control group without plasmalogen administration. On the other hand, in the lung tissue of mice administered with plasmalogen, a significant decrease in viral DNA was confirmed compared to the control group. Therefore, it became clear that the administration of plasmalogen suppressed viral infection in mice.

[0057] As shown in Figure 4, in the control group without plasmalogen administration, the number of white blood cells in the blood significantly decreased due to viral infection. On the other hand, in mice administered with plasmalogen, the decrease in the number of white blood cells in the blood was significantly suppressed compared to the control group. Therefore, it was suggested that the administration of plasmalogen suppressed viral infection in mice.

[0058] As shown in Fig. 5, in the control group not administered with plasmalogen, a significant increase in the weight of the spleen was observed due to virus infection. On the other hand, in the mice administered with plasmalogen, the increase in spleen weight was significantly suppressed compared to the control group. Therefore, it was suggested that the administration of plasmalogen suppressed virus infection in mice.

[0059] From the above, it is considered that the administration of plasmalogen improves NK activity and exerts an anti-infection effect against viruses and the like.

Example

[0060] The effect of plasmalogen deficiency on bacterial infection was investigated. The resistance to pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus aureus) was compared between wild-type (WT N2) nematodes with normal genes and nematodes lacking the plasmalogen-producing gene (acl-7, ads-1, fard-1).

[0061] Specifically, the following was done. 1. Culture Pseudomonas aeruginosa and Staphylococcus aureus overnight in LB medium. 2. Prepare Nematode Growth Medium (NGM) agar medium, apply a part of the above suspension, and place it at 37 °C for 24 hours and then at 25 °C for 24 hours. 3. Culture adult nematodes in the above medium, confirm life and death, and determine the survival rate.

[0062] Fig. 6 shows the survival rate of nematodes infected with Pseudomonas aeruginosa, and Fig. 7 shows the survival rate of nematodes infected with Staphylococcus aureus. As shown in Figs. 6 and 7, nematodes lacking the plasmalogen-producing gene had a significantly lower survival rate when infected with Pseudomonas aeruginosa and Staphylococcus aureus compared to the wild type. Therefore, it was suggested that plasmalogen exhibits an anti-infection effect against bacteria.

Example

[0063] The NK activity in humans by plasmalogen was evaluated.

[0064] [Plasmalogen] As the plasmalogen, ethanolamine-type plasmalogen extracted in the same manner as in Example 2 was used.

[0065] [Administration of plasmalogen] Four subjects were a 32-year-old male with obesity, a 52-year-old female with anxiety disorder, a 73-year-old male with diabetes, and a 59-year-old male with hyperlipidemia. Hard capsules containing 0.5 mg of plasmalogen were administered to the subjects 2 capsules each in the morning and evening, for a total of 2 mg (2 mg / day) for 4 days.

[0066] [NK activity] The measurement of NK activity was entrusted to BML Co., Ltd. The test method was performed by the 51Cr release method.

[0067] Figures 8 and 9 show the results of NK activity of monocytes derived from human blood. Figure 8 shows the results when the ratio of monocytes:target cells is 10:1, and Figure 9 shows the results when the ratio of monocytes:target cells is 20:1.

[0068] As shown in Figures 8 and 9, it was confirmed that the NK activity of all subjects increased 4 days after plasmalogen intake. That is, it is expected that the administration of plasmalogen to humans activates NK cells and enhances immune function.

Example

[0069] The frequency of colds in humans by plasmalogen was evaluated.

[0070] [Plasmalogen] As the plasmalogen, ethanolamine-type plasmalogen extracted by ethanol extraction in the same manner as in Example 2 was used.

[0071] [Administration of plasmalogen to humans] The subjects were 33 in total, including 10 males and 23 females who visited the clinic. Hard capsules containing 0.5 mg of plasmalogen were administered to the subjects, 1 capsule each in the morning and evening, for a total of 1 mg (1 mg / day) for 12 months.

[0072] [Evaluation of the frequency of colds] An inquiry was made about the number of colds in the subjects in the year before taking plasmalogen and in the 12 months from the start of taking it.

[0073] Figure 10 shows the frequency of colds before taking plasmalogen and in the 12 months from the start of taking it.

[0074] As shown in Figure 10, before taking plasmalogen, the frequency of colds was 4 times / year in 21.2% of the subjects, but 12 months after the start of taking it, the frequency of colds was 0% in the subjects, and a significant decrease in the frequency of colds was confirmed. Also, in the trend test, a statistically significant decrease (Trend P = 0.0001) was observed. Therefore, it is considered that the administration of plasmalogen to humans improves NK activity and exerts an anti-infection effect against viruses and the like.

Example

[0075] Plasmalogen was administered to patients with rhizomelic chondrodysplasia punctata (RCDP) in whom the plasmalogen blood concentration had decreased and who had developed a viral infectious disease, and the effect was evaluated.

[0076] [Plasmalogen] As in Example 2, ethanolamine-type plasmalogen extracted with ethanol was used as plasmalogen.

[0077] [Administration of plasmalogen to RCDP patients] The subject was a 3-year-old girl with RCDP. At the start of taking it, plasmalogen was administered at 0.25 mg / day, and the dosage was gradually increased to a maximum of 6.0 mg / day.

[0078] [Evaluation of Plasma Logen Blood Concentration, CRP Inflammatory Response, and Fever Frequency] Blood samples were collected from the subjects before and after the start of taking plasma logen, and the plasma logen blood concentration and CRP inflammatory response were measured. In addition, the frequency of fever was confirmed as an indicator of viral infection.

[0079] Figure 11 shows the changes in plasma logen blood concentration, CRP inflammatory response, and fever frequency in RCDP patients with the administration of plasma logen.

[0080] As shown in Figure 11, it was confirmed that the blood concentration of plasma logen increased with the increase in the dosage of plasma logen. In addition, the CRP inflammatory response decreased with the duration of continuous administration of plasma logen. Furthermore, regarding the number of fevers, the subject had a fever once a week before taking plasma logen, but the number of fevers was three times during about two months after the start of administration. After further increasing the dosage of plasma logen to 6.0 mg / day, there was no fever. Also, before taking plasma logen, the subject often developed pneumonia presumably caused by viral infection, but no pneumonia occurred after taking plasma logen. That is, in RCDP patients with low plasma logen blood concentration, plasma logen is considered to have an anti-infection effect against viruses and the like.

[0081] From the results of Examples 1 to 7 above, it became clear that the administration of plasma logen can prevent and improve infections caused by viruses and bacteria. As the mechanism of action of this infection prevention and treatment effect, it is considered that the administration of plasma logen activates NK cells and enhances immune function.

Example

[0082] [Formulation Example] Hard capsules were manufactured with the following formulation. Scallop-extracted plasma logen 0.5 mg Cyclodextrin 3.3 mg Amino acid 1.2 mg Pine binder 185.0 mg

Industrial Applicability

[0083] The composition of the present invention is industrially useful because it contributes to enhancing innate immunity.

Claims

**Claim 1** An anti-infective composition containing plasmalogen and used for the prevention or treatment of acrodermatitis chronica atrophicans, or infection by herpes simplex virus type 1, or Pseudomonas aeruginosa or Staphylococcus aureus. **Claim 2** An anti-infective composition containing plasmalogen and used for the prevention of colds in healthy individuals. **Claim 3** The anti-infective composition according to claim 1 or 2, characterized in that the plasmalogen is a plasmalogen extracted from animal tissues.

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