Immunostimulant composition and expression induction composition

Abstract

To provide a novel immunostimulatory composition.SOLUTION: An immunostimulatory composition contains microalgae-derived extracellular membrane vesicles as an active ingredient.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an immunostimulating composition and an expression-inducing composition.

Background Art

[0002] In vivo, nitric oxide is synthesized from arginine and oxygen by nitric oxide synthase. Three isoforms of nitric oxide synthase are known: inducible nitric oxide synthase, neuronal nitric oxide synthase, and endothelial nitric oxide synthase. The expression of inducible nitric oxide synthase is induced at the transcriptional level by interferon-γ and interleukin-1 induced by infection or the like. Inducible nitric oxide synthase synthesizes nitric oxide, which plays an important role in the inflammatory response, and activates a series of biological defense mechanisms.

[0003] Extracellular vesicles are particulate membrane vesicles secreted from cells. Extracellular vesicles contain various biomolecules such as nucleic acids and proteins inside them. These biomolecules function as information transmitters. In recent years, research has been conducted on regulating biological functions using extracellular vesicles. For example, Patent Document 1 discloses a technique for suppressing apoptosis using extracellular vesicles derived from bovine milk. Patent Document 2 also discloses a technique for protecting the skin from external stimuli such as ultraviolet rays and heat using extracellular vesicles derived from yeast.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] <0OO0033>As a result of their diligent research, these researchers discovered that administering extracellular membrane vesicles derived from microalgae to cells induces the expression of inducible nitric oxide synthase and the production of nitric oxide. [Means for solving the problem]

[0006] An immunostimulatory composition that solves the above problems contains extracellular membrane vesicles derived from microalgae as an active ingredient. In one embodiment of the above-described immunostimulatory composition, the particle size of the extracellular membrane vesicles is 450 nm or less.

[0007] In one embodiment of the above-described immunostimulatory composition, the average particle size of the extracellular membrane vesicles is 150 nm or more and 250 nm or less. In one embodiment of the above-described immunostimulatory composition, the microalgae is at least one selected from the thalli of the order Volvocales of the class Chlorophyceae, the thalli of the order Pinnate of the class Diatoms, the thalli of the order Oscillariaes of the class Cyanobacteriaceae, the thalli of the order Nostocales of the class Cyanobacteriaceae, and the thalli of the order Coccolithophores of the class Haptophyceae.

[0008] The expression-inducing composition that solves the above problems contains extracellular membrane vesicles derived from microalgae as an active ingredient. [Effects of the Invention]

[0009] The present invention provides a novel immunostimulatory composition. Furthermore, the present invention provides a novel expression-promoting composition that induces the expression of inducible nitric oxide synthase. [Brief explanation of the drawing]

[0010] [Figure 1] This graph shows the relationship between the administration of extracellular membrane vesicles derived from microalgae and NO production. [Figure 2] This is a Western blot analysis showing the relationship between the administration of extracellular membrane vesicles derived from microalgae and the expression level of iNOS. [Modes for carrying out the invention]

[0011] The following describes one embodiment of the present invention. The composition of this embodiment (hereinafter referred to as "this composition") contains extracellular membrane vesicles derived from microalgae as an active ingredient.

[0012] [Microalgae] First, we will describe the microalgae that secrete extracellular membrane vesicles contained in this composition. Microalgae are algae with a microscopic size of approximately 1 μm to 1 mm. Specific examples of microalgae include thalli of the Volvoxales order of Chlorophyceae, thalli of the Pinnate order of Diatoms, thalli of the Oscillariales order of Cyanobacteriaceae, thalli of the Nostocales order of Cyanobacteriaceae, and thalli of the Coccolithophores order of Haptophyceae. However, the term microalgae is not limited to these thalli.

[0013] Examples of thalli belonging to the order Volvocales of the class Chlorophyceae include thalli belonging to the genus Dunaliella, such as Dunaliella salina, Dunaliella tertiolecta, and Dunaliella bardawil, and thalli belonging to the genus Chlorogonium, such as Chlorogonium sp., Chlorogonium capillatum, Chlorogonium euchlorum, Chlorogonium elegans, Chlorogonium elongatum, Chlorogonium fusiforme, Chlorogonium kasakii, and Chlorogonium neglectum.

[0014] Examples of diatoms belonging to the pinnate order include thallus of the genus Nitzchia in the family Nitzchiaceae, such as Nitzschia acicularis, Bacillaria paxillifer, Cylindrotheca Closterium, Neodenticula seminae, Nitzchia levidensis, Nitzchia martiana, Nitzchia pungens, Nitzchia tenuiarcuata, Pseudo-nitzschia multistriata, and Pseudoeunotia doliolus, as well as thallus of the genus Phaeodactylum in the family Nitzchiaceae, such as Phaeodactylum tricornutum.

[0015] Examples of thalli belonging to the order Oscillarianes of the class Cyanobacteria include those belonging to the genus Arthrospira, such as Arthrospira (Spirulina) platensis and Arthrospira (Spirulina) subsalsa. Examples of thalli belonging to the Nostocales order of the class Cyanobacteria include thalli belonging to the genus Nostoc, such as Nostoc commune, Nostoc flagelliforme, Nostoc sphaericum, Nostoc verrucosum, Nostoc linckia, Nostoc muscorum, and Nostoc punctiforme, and thalli belonging to the genus Aphanizomenon, such as Aphanizomenon flosaquae.

[0016] Examples of thalli in the order Coccolithophytes of the class Haptophyceae include thalli belonging to the genus Pleurochrysis, such as Pleurochrysis carterae and Pleurochrysis roscoffensis. Microalgae may be naturally occurring algal bodies or artificially cultured algal bodies. However, using artificially cultured algal bodies is industrially preferable due to the ability to ensure a stable supply and ease of maintaining quality.

[0017] [Extracellular membrane vesicles] Next, the extracellular vesicles contained in this composition will be described. Extracellular vesicles are particulate substances secreted from the above-mentioned microalgae, and are membrane vesicles covered with a membrane mainly composed of phospholipids. Extracellular vesicles contain biomolecules such as nucleic acids and proteins inside. Examples of the above nucleic acids include miRNA, mRNA, and DNA.

[0018] The size of the extracellular vesicles is not particularly limited. The extracellular vesicles include sEVs (small Extracellular Vesicles) with a particle diameter of 200 nm or less and m / l EVs (medium / large Extracellular Vesicles) with a particle diameter of 200 nm or more.

[0019] The particle diameter of the extracellular vesicles is, for example, 450 nm or less, preferably 220 nm or less. In this specification, the fact that the particle diameter of the extracellular vesicles is below a specific value means that it is of a size that can pass through a membrane filter with a pore diameter of the specific value.

[0020] The average particle diameter of the extracellular vesicles is, for example, 150 nm or more, preferably 170 nm or more. Also, the average particle diameter of the extracellular vesicles is, for example, 250 nm or less, preferably 210 nm or less. The average particle diameter of the extracellular vesicles can be measured by nanoparticle tracking analysis (NTA) which measures based on both the characteristics of scattered light and Brownian motion.

[0021] [Method for preparing extracellular vesicles] Next, the method for preparing extracellular vesicles will be described. Note that the method for preparing extracellular vesicles is not limited to the method described below.

[0022] An example of the method for preparing extracellular vesicles includes a culturing step of culturing microalgae to obtain a culture solution, a separating step of removing the microalgae from the culture solution to separate the culture supernatant, and a purification step of purifying the extracellular vesicles from the culture supernatant.

[0023] The method for culturing microalgae in the cultivation process is not particularly limited, and known cultivation methods appropriate to the target microalgae can be applied. Examples of culture media used in the cultivation process include water and aqueous culture media containing water.

[0024] The separation method in the separation process is not particularly limited, and known solid-liquid separation methods can be applied. Known solid-liquid separation methods include, for example, filtration, centrifugation, ultracentrifugation, fractional centrifugation, equilibrium density gradient centrifugation, density gradient, dialysis, free-flow electrophoresis, and combinations thereof.

[0025] An example of a purification process using ultracentrifugation and size exclusion filtration is described below. First, the culture medium obtained in the culture process is subjected to ultracentrifugation to settle the extracellular membrane vesicles contained in the culture medium. Ultracentrifugation is performed, for example, at 80,000 to 100,000 rpm for 2 to 4 hours. Next, the precipitate containing the extracellular membrane vesicles is collected and dissolved in a lysis solution such as phosphate buffer. Then, the resulting lysis solution is filtered using a filter to remove unwanted materials larger than the extracellular membrane vesicles. An example of a filter used for filtration is a membrane filter with a pore size of 220 to 450 nm.

[0026] The temperature during the separation and purification steps is not particularly limited, but it is preferable to carry them out at a temperature of 4°C to 10°C, for example. [This composition] This composition contains extracellular membrane vesicles derived from microalgae as an active ingredient. The extracellular membrane vesicles contained in this composition may be of only one type, or a combination of two or more types from different origins. For example, this composition may contain only one type of extracellular membrane vesicle derived from thalli of the order Volvoxales of the class Chlorophyceae, extracellular membrane vesicles derived from thalli of the order Pinnate of the class Diatoms, extracellular membrane vesicles derived from thalli of the order Oscillariae of the class Cyanobacteriaceae, extracellular membrane vesicles derived from thalli of the order Nostocales of the class Cyanobacteriaceae, and extracellular membrane vesicles derived from thalli of the order Coccolithophores of the class Haptophyceae, or it may contain two or more types.

[0027] Administering this composition to living organisms such as cells induces the expression of inducible nitric oxide synthase. Therefore, this composition can be used as an expression-promoting composition that induces the expression of inducible nitric oxide synthase. Furthermore, administering this composition to living organisms increases the production of nitric oxide. In living organisms, small amounts of nitric oxide play a role in the body's defense against pathogens and other pathogens. Therefore, this composition can be used as an immunostimulatory composition that exerts an immune-boosting effect based on the production of nitric oxide.

[0028] Each of the immunostimulatory composition and expression-inducing composition comprising this composition can be applied to various fields such as food and beverages, pharmaceuticals, quasi-drugs, and cosmetics. Examples of food and beverages include various types of beverages (fruit or vegetable juice drinks, soft drinks, mineral drinks, sports drinks, tea drinks, coffee, carbonated drinks, dairy products such as milk and yogurt, etc.), jelly-like foods (jelly, agar, jelly-like drinks, etc.), capsules (soft capsules, hard capsules), and various types of confectionery. Food and beverages may appropriately contain gelling agents such as pectin and carrageenan, sugars and sweeteners such as glucose, sucrose, fructose, lactose, stevia, aspartame, and sugar alcohols, food additives such as flavorings, and oils such as vegetable oils and animal oils. Furthermore, there are no particular limitations on the use of food and beverages, and they can be applied as so-called general foods, health foods, functional foods, nutritional supplements, supplements, foods for specified health uses, foods with functional claims, and foods for sick people.

[0029] The method of administration when used as a pharmaceutical, quasi-drug, or cosmetic is not particularly limited. Specific methods of administration include, for example, oral administration, intravascular administration, enteral administration, transdermal administration, and intraperitoneal administration. Furthermore, the dosage form when used as a pharmaceutical, quasi-drug, or cosmetic is not particularly limited. Specific dosage forms include, for example, powders, granules, tablets, capsules, pills, suppositories, liquids, and injections. In addition, the product may contain excipients, bases, emulsifiers, solvents, stabilizers, etc.

[0030] Next, the effects of this embodiment will be described. (1) The immunostimulatory composition contains extracellular membrane vesicles derived from microalgae as an active ingredient. Administration of the immunostimulatory composition having the above configuration increases the amount of nitric oxide produced in the body. Therefore, the immunostimulatory composition having the above configuration exerts an immune-activating effect based on the production of nitric oxide. Furthermore, the increase in nitric oxide production based on the immunostimulatory composition having the above configuration is milder compared to the case when interferon-γ, a known substance that has a similar effect, is administered. Therefore, the immunostimulatory composition having the above configuration is suitable for applications that slightly increase or gradually increase the amount of nitric oxide produced in the body.

[0031] (2) The expression-inducing composition contains extracellular membrane vesicles derived from microalgae as an active ingredient. By administering the expression-inducing composition with the above configuration, the expression of inducible nitric oxide synthase can be induced in vivo. Furthermore, the increase in the expression level of inducible nitric oxide synthase based on the expression-inducing composition with the above configuration is milder compared to the case when interferon-γ, a known substance that produces a similar effect, is administered. For this reason, the expression-inducing composition with the above configuration is suitable for applications that slightly increase or gradually increase the expression level of inducible nitric oxide synthase in vivo.

[0032] This embodiment can be implemented with the following modifications. This embodiment and the following modifications can be combined with each other to the extent that they do not contradict each other technically. Each of the immunostimulatory composition and the expression-inducing composition may contain other components other than extracellular membrane vesicles, to the extent that they do not impair the intended effect of each.

[0033] The content of extracellular membrane vesicles in each of the immunostimulatory composition and the expression induction composition is not particularly limited. The intake amounts and durations of the immunostimulant composition and the expression-inducing composition are not particularly limited and should be determined as appropriate, taking into consideration the physical condition, age, sex, and other conditions of the person taking the supplement.

[0034] Each of the immunostimulatory compositions and expression-inducing compositions can be applied not only to humans, but also to domesticated animals such as livestock. Next, the technical concepts that can be understood from the above embodiments and modified examples are described below.

[0035] [Note 1] An expression-inducing composition for inducing the expression of inducible nitric oxide synthase, comprising extracellular membrane vesicles derived from microalgae as an active ingredient.

[0036] [Note 2] The expression induction composition according to [Appendix 1], wherein the particle size of the extracellular membrane vesicles is 450 nm or less.

[0037] [Note 3] The expression-inducing composition described in [Note 2], wherein the average particle size of the extracellular membrane vesicles is 150 nm or more and 250 nm or less.

[0038] [Note 4] The expression-inducing composition according to any one of [Appendix 1] to [Appendix 3], wherein the microalgae is at least one selected from the thalli of the order Volvocales of the class Chlorophyceae, the thalli of the order Pinnate of the class Diatoms, the thalli of the order Oscillariaes of the class Cyanobacteriaceae, the thalli of the order Nostocales of the class Cyanobacteriaceae, and the thalli of the order Coccolithophores of the class Haptophyceae.

[0039] [Note 5] An immunostimulatory composition containing the expression-inducing composition described in any one of the following [Appendix 1] to [Appendix 4]. [Examples]

[0040] The above embodiments will be further described with reference to test examples below. However, the present invention is not limited to these examples. [Preparation of extracellular membrane vesicle solution] (Test Example 1) The culture medium of Dunaliella Salina (TZ) was centrifuged (14,000 × g, 4°C, 20 minutes), and the supernatant was collected. The collected supernatant was ultracentrifuged (290,000 × g, 4°C, 60 minutes), and the precipitate was collected. The collected precipitate was then dissolved in 200 μL of phosphate buffer (PBS). The solution was filtered using a membrane filter (0.45 μm DISMIC Filter, Advantec Toyo Co., Ltd.). The filtrate was collected and used as the extracellular membrane vesicle solution for Test Example 1.

[0041] A diluted solution of the obtained extracellular membrane vesicle solution was prepared, and the Brownian motion of the extracellular membrane vesicles contained in the diluted solution was recorded. The average particle size of the extracellular membrane vesicles contained in the diluted solution was calculated by analyzing the obtained video using nanoparticle tracking analysis. The results are shown in Table 1.

[0042] Furthermore, the protein concentration of the extracellular membrane vesicle solution from Test Example 1 was measured. The protein concentration was measured using the DC Protein Assay Kit (Bio-Rad), with bovine serum albumin as the standard substance.

[0043] (Test examples 2-8) The extracellular membrane vesicle solutions for Test Examples 2-8 were obtained using the same method as in Test Example 1, except that the culture medium for Dunaliella Salina (TZ) was replaced with the culture medium for the microalgae listed in Table 1 below. The average particle size of the extracellular membrane vesicles was calculated using the same method as in Test Example 1. These results are shown in Table 1. The protein concentration of the extracellular membrane vesicle solutions for Test Examples 2-8 was also measured using the same method as in Test Example 1.

[0044] [Table 1] [Evaluation of immune-boosting effects] The immunostimulatory effects of Test Examples 1-8 were evaluated based on the measurement of nitric oxide production (NO production) in cells administered with Test Examples 1-8.

[0045] Mouse macrophage RAW264.7 cells (2 × 10⁻⁶ cells) 5 500 μL of cells (cells / mL) were seeded into 24-well multiplates and pre-cultured for 12 hours. Then, each test sample was added to the culture medium to a final concentration (protein concentration) of 1 μg / mL and treated for 24 hours. As a control sample (untreated), PBS was added instead of the test sample and treated for 24 hours. As a control sample (positive control), lipopolysaccharide (LPS) and interferon-γ (IFNγ) were added and treated for 24 hours. LPS was added to a final concentration of 200 ng / mL. IFNγ was added to a final concentration of 25 ng / mL.

[0046] After 24 hours of treatment, RAW264.7 cells produced nitrite (NO2), a breakdown product of nitric oxide. - The sample was colored using Griess reagent, and the absorbance at 540 nm was measured using a plate reader. Based on the measured absorbance, the amount of NO produced was indirectly determined. NO production was measured using a sample size of 9, and the average value and standard error of NO production after each treatment were calculated. The results are shown in Figure 1.

[0047] As shown in Figure 1, NO production in cells stimulated with LPS and IFNγ (positive control) was approximately 8.4 times higher than in untreated cells. Furthermore, NO production in cells treated with each test sample was approximately 3.3 times higher than in untreated cells. These results indicate that administering extracellular membrane vesicles derived from microalgae to cells resulted in a moderate increase in nitric oxide production compared to stimulation with LPS and IFNγ. Therefore, it is suggested that extracellular membrane vesicles derived from microalgae exert an immune-stimulating effect based on a moderate induction of nitric oxide production.

[0048] As shown in Figure 1, the amount of NO produced was similar regardless of whether Test Examples 1 to 8 were used. Although detailed test results are omitted here, the same tests were also conducted on extracellular membrane vesicles derived from Dunaliella Salina (SL), a different species from Dunaliella Salina (TZ) used in Test Example 1. The results showed that the average particle size of the extracellular membrane vesicles derived from Dunaliella Salina (SL) was 186 nm. The amount of NO produced using these extracellular membrane vesicles was similar to that produced using extracellular membrane vesicles derived from Dunaliella Salina (TZ). These results suggest that the effect of extracellular membrane vesicles derived from microalgae in inducing nitric oxide production is a general effect of extracellular membrane vesicles derived from microalgae, regardless of the taxonomic genus or species of the microalgae from which they originate.

[0049] [Evaluation of induction of inducible nitric oxide synthase (iNOS) expression] (Preparation of sample solution) RAW264.7 cells (2 x 10 4 Two mL of the sample (cells / mL) was seeded into each 6-well multiplate and pre-cultured for 12 hours. Then, each test sample was added to the culture medium to a final concentration (protein concentration) of 5 μg / mL and treated for 24 hours. As a control sample (untreated), PBS was added instead of the test sample and treated for 24 hours. As a control sample (positive control), LPS and IFNγ were added and treated for 24 hours. LPS was added to a final concentration of 200 ng / mL. IFNγ was added to a final concentration of 25 ng / mL.

[0050] After 24 hours of treatment, cells were lysed with RIPA buffer containing a phosphatase inhibitor (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) to obtain a cell lysate. The cell lysate was then subjected to ultrasonic disruption (10 seconds x 3 times) and centrifugation (13500 x g, 4°C, 15 minutes), after which the supernatant was collected.

[0051] Next, the protein concentration in the recovered supernatant was measured, and then the supernatant was diluted to achieve a specific protein concentration. The protein concentration was measured using a DC Protein Assay Kit (Bio-Rad) with bovine serum albumin as the standard substance. After adding a sample buffer containing 10% mercaptoethanol to the diluted supernatant solution, the solution was heated at 98°C for 5 minutes in a heat block to obtain the sample solution.

[0052] (Western blot analysis) Proteins contained in each sample solution were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF (polyvinylidene fluoride) membrane. The transferred PVDF membrane was then immersed in a 1% skim milk solution dissolved with 1×T-TBS for 1 hour of blocking treatment. After blocking treatment, the PVDF membrane was subjected to primary antibody treatment by shaking wash with 1×T-TBS (5 minutes x 3 times) and then immersion in primary antibody solution and shaking overnight at 4°C. The primary antibody solution used was a 1000-fold dilution of iNOS and β-actin (β-Actin) with antibody diluent (bovine serum albumin: 2%, sodium azide: 0.05%).

[0053] The PVDF membrane, after primary antibody treatment, was subjected to a series of subsequent treatments: shaking wash with 1×T-TBS (5 minutes x 3 times), followed by secondary antibody treatment by immersion in a secondary antibody solution and shaking for 1 hour. The secondary antibody solution used was a horseradish peroxidase-labeled secondary antibody diluted 10,000 times with a 1% skim milk powder-containing T-TBS solution. Next, the PVDF membrane, after secondary antibody treatment, was washed with 1×T-TBS (5 minutes x 3 times). Subsequently, the PVDF membrane was treated with a chemiluminescence detection reagent (GE Healthcare) to induce chemiluminescence in the antigen-antibody complex on the PVDF membrane, and this chemiluminescence was detected using a chemiluminescence detector. The results are shown in Figure 2.

[0054] As shown in Figure 2, iNOS could not be detected in untreated cells. On the other hand, iNOS was detected in cells stimulated with LPS and IFNγ (positive control) and in cells treated with each test sample. Furthermore, the iNOS expression level in cells treated with each test sample was lower compared to the positive control. These results indicate that administering extracellular membrane vesicles derived from microalgae to cells moderately increases iNOS expression compared to stimulation with LPS and IFNγ. Moreover, the trend of increasing iNOS expression in the above results is consistent with the trend of increasing NO production in the immunostimulatory effect evaluation test results described above. Therefore, it is considered that NO production increases in cells treated with extracellular membrane vesicles derived from microalgae based on an increase in iNOS expression. Although detailed test results are omitted, similar tests were also performed on extracellular membrane vesicles derived from Dunaliella Salina (SL). As a result, the expression level of iNOS when using the extracellular membrane vesicles in question was about the same as when using extracellular membrane vesicles derived from Dunaliella Salina (TZ).

Claims

1. An immunostimulatory composition containing, as an active ingredient, extracellular membrane vesicles derived from at least one microalga selected from the thallus of the diatom class pinnate order, the thallus of the cyanobacteria class nostocales, and the thallus of the haptophyceae class coccolithophores.

2. The immunostimulatory composition according to claim 1, wherein the particle size of the extracellular membrane vesicles is 450 nm or less.

3. The immunostimulatory composition according to claim 2, wherein the average particle size of the extracellular membrane vesicles is 150 nm or more and 250 nm or less.

4. A composition for inducing the expression of inducible nitric oxide synthase, An expression-inducing composition containing, as an active ingredient, extracellular membrane vesicles derived from at least one microalga selected from the thallus of the diatom class (pennate order), the thallus of the cyanobacteria class (Nostocales order), and the thallus of the haptophyceae class (coccolithophores order).

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