Endothelial type nitrogen monoxide synthase (NOS3) production promotor

Abstract

To provide a novel endothelial type nitrogen monoxide synthase (NOS3) production promotor.SOLUTION: An endothelial type nitrogen monoxide synthase production promotor includes, as an active ingredient, n-6DPA (n-6 docosapentaenoic acid) alone or includes n-6DPA and DHA (docosahexaenoic acid).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an agent, food, or pharmaceutical product that contains n-6DPA (n-6 docosapentaenoic acid) and has an excellent effect of promoting the production of endothelial nitric oxide synthase (NOS3). [Background technology]

[0002] With the recent trend toward Westernized lifestyles, lifestyle-related vascular diseases such as hypertension, angina pectoris, myocardial infarction, and cerebral circulatory disorders are on the rise. It has been revealed that factors contributing to these diseases include decreased vascular flexibility, decreased vascular endothelial function, and increased platelet aggregation, resulting in thrombus formation and decreased blood flow, and this vascular endothelial dysfunction is deeply involved in the onset of arteriosclerosis (Non-Patent Documents 1 and 2). Because lifestyle-related diseases are chronic, their prevention and treatment require a long period of time. Therefore, there is a need for food ingredients that can safely and effectively improve vascular function over the long term.

[0003] Nitric oxide (NO) is produced as a by-product of the conversion of L-arginine to L-citrulline by nitric oxide synthase (NOS), and has a vasodilatory effect by relaxing vascular smooth muscle. Three different isoforms of NO are known: neuronal nitric oxide synthase (NOS1, also known as nNOS), inducible nitric oxide synthase (NOS2, also known as iNOS), and endothelial nitric oxide synthase (NOS3, also known as eNOS).

[0004] Of these, NOS3 is localized in vascular endothelial cells and bound to the cell membrane. It has been revealed that NOS3-derived NO has vascular protective effects, such as inhibiting arteriosclerosis. As mentioned above, NO was initially recognized as a vasodilator. However, its endothelial cell protective effects have been reported, including anti-inflammatory and antioxidant effects, inhibition of adhesion molecules, inhibition of smooth muscle cell migration, and inhibition of thrombosis. Piperine is known to promote NOS3 activity (Patent Document 1). Therefore, increased expression of NOS3 is expected to improve vascular flexibility through increased NO production.

[0005] One test method that has recently attracted attention as a method for evaluating vascular endothelial function is flow-mediated vasodilation (FMD). It is a non-invasive method for measuring vascular endothelial function, with higher values ​​indicating greater vascular flexibility. According to the physiological diagnostic criteria for vascular insufficiency published by the Japanese Society of Vascular Insufficiency, an FMD of less than 4% is considered abnormal, 4% to 7% is considered borderline, and 7% or more is considered normal.

[0006] Furthermore, it is known that continuous intake of fish oil can lead to the prevention of thrombosis, arteriosclerosis, hypertension, etc., and it has been suggested that DHA (docosahexaenoic acid) contained in fish oil has an anti-vasoconstrictive effect (Non-Patent Document 3), and EPA (eicosapentaenoic acid) has an anti-platelet aggregation effect (Non-Patent Document 4). It has been suggested that n-6DPA, an (n-6) PUFA (polyunsaturated fatty acid), promotes vascular endothelial cell migration in vitro (10 times more than EPA, Non-Patent Document 5) and has an anti-inflammatory effect (Non-Patent Document 6). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 6969041 [Non-patent literature]

[0008] [Non-Patent Document 1] Journal of Thrombosis and Hemostasis, Japanese Society on Thrombosis and Hemostasis, 2015, Vol. 26, No. 3, pp. 302-309 [Non-patent document 2] Chemistry and Biology, The Agricultural Chemical Society of Japan, 2016, Vol. 54, No. 10, pp. 713-719 [Non-patent document 3] Journal of Pharmacology, Japanese Pharmacological Society, 2016, Vol. 147, pp. 63-65 [Non-patent document 4] Chemistry and Biology, The Agricultural Chemical Society of Japan, 1983, Vol. 21, No. 3, pp. 168-173 [Non-patent document 5] Lipid Nutrition, Society of Lipid Nutrition, 1996, Vol. 5, No. 1, pp. 17-22 [Non-patent document 6] Lipid (2010), 11, pp.1-14 Summary of the Invention [Problem to be solved by the invention]

[0009] As mentioned above, promoting NOS3 expression can contribute to vascular protection. However, existing compositions have not been sufficient in terms of safely and effectively improving vascular function over the long term. Furthermore, there has been no suggestion that n-6DPA has the effect of promoting endothelial nitric oxide synthase (NOS3) production.

[0010] An object of the present invention is to provide a novel promoter of endothelial nitric oxide synthase (NOS3) production. [Means for solving the problem]

[0011] As a result of intensive research to solve the above problems, the present inventors discovered that n-6DPA has an excellent effect of increasing the expression level of the NOS3 gene, and that ingestion of low doses of microalgae containing n-6DPA increases FMD and has an extremely high effect of improving vascular flexibility, leading to the completion of the present invention.

[0012] That is, the first aspect of the present invention is "an endothelial nitric oxide synthase production promoter comprising either a fatty acid composition containing n-6DPA (n-6 docosapentaenoic acid) or n-6DPA." A second aspect of the present invention is a vascular endothelial nitric oxide synthase production promoter, characterized in that the fatty acid composition according to the first aspect of the invention contains DHA (docosahexaenoic acid). The third aspect of the present invention is a vascular endothelial nitric oxide synthase production promoter, characterized in that the mass ratio of DHA (B) to n-6DPA (A) [(B) / (A)] in the fatty acid composition described in the second aspect of the invention is 0.0001 or more and 10,000 or less. A fourth aspect of the present invention is a food composition containing the endothelial nitric oxide synthase production promoter according to any one of the first to third aspects of the present invention. A fifth aspect of the present invention is a pharmaceutical composition comprising the endothelial nitric oxide synthase production promoter according to any one of the first to third aspects of the present invention. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a food composition and a pharmaceutical composition which contain n-6DPA alone or n-6DPA and DHA and have a high effect of promoting the production of endothelial nitric oxide synthase (NOS3). [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a graph showing the results of an example of the present invention. [Figure 2] 1 is a graph showing the results of an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0015] The configuration of the present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0016] Examples of microalgae that can be used to obtain n-6DPA alone or n-6DPA and DHA contained in the agent of the present invention include Chlorella (green algae), Spirulina (cyanophyceae), Euglena (Japanese name: Midorimushi, protozoa / green algae), Dunaliella (green algae), Nannochloropsis (true eyespot algae), and Labyrinthula Thraustochytrids (Labyrinthula is considered a type of microalgae in this specification). Of these, preferred are Spirulina, Euglena, Nannochloropsis, and Labyrinthula Thraustochytrids, which inherently contain PUFAs (polyunsaturated fatty acids), or Chlorella, which can contain PUFAs in its algae by culturing with the addition of fatty acids; more preferred are Nannochloropsis and Thraustochytrids with high PUFA contents; and even more preferred are Labyrinthula Thraustochytrids, which can be cultured in a closed tank and are highly safe. From the viewpoint of production efficiency, n-6DPA alone or n-6DPA and DHA contained in the agent of the present invention are preferably obtained from microalgae, but can also be obtained from any microorganisms, animals, or plants containing n-6DPA alone or n-6DPA and DHA.

[0017] Next, the method for producing the fatty acid composition, n-6DPA alone, or n-6DPA and DHA contained in the agent of the present invention from microalgae will be described. The solid and liquid media used in culturing microalgae can be any known medium base containing natural or artificial seawater, a carbon source, a nitrogen source, inorganic salts, etc. Examples of carbon sources include, but are not limited to, carbohydrates such as glucose, fructose, and galactose, as well as oils and fats such as oleic acid and soybean oil, glycerol, acetic acid, and sodium acetate. The carbon source can be used at a concentration of, for example, 20 to 300 g per liter of medium. In a particularly preferred embodiment, after the initial carbon source has been consumed, the culture can be continued by feeding a carbon source. Cultivation under these conditions allows for the consumption of a larger amount of carbon source, thereby increasing the production of the fatty acid composition obtained from the microalgae. The nitrogen source may be, but is not limited to, organic nitrogen such as yeast extract, corn steep liquor, peptone, polypeptone, sodium glutamate, or urea, or inorganic nitrogen such as ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate, ammonium nitrate, or ammonia. Potassium phosphate and the like may be used in appropriate combination as an inorganic salt.

[0018] The medium containing each of the above-mentioned medium bases is preferably adjusted to a pH of 4.0 to 9.5 with an appropriate acid or base, and then sterilized in an autoclave before use. The pH during cultivation is generally 3.5 to 10.0, preferably 4.0 to 9.5. Liquid cultivation can be carried out for 2 to 10 days under aeration and agitation. The cultivation temperature is generally 10 to 45°C, preferably 15 to 35°C. The cultivation temperature is preferably controlled to a temperature at which a fatty acid composition can be produced. The scale of the liquid cultivation is not particularly limited, and commercially available closed cultivation equipment can be used. The algae can be recovered from the obtained culture solution by known conventional methods, such as centrifugation or filtration.

[0019] A fatty acid composition containing n-6DPA alone or n-6DPA and DHA can be prepared from the algae by known methods. That is, after cultivation, wet or dried algae can be physically disrupted using a mill or ultrasonic waves, or chemically dissolved in a solvent, followed by solvent extraction using chloroform, hexane, methanol, ethanol, or the like to obtain a fatty acid composition containing PUFAs. Furthermore, the obtained fatty acid composition can be chemically or physically modified or processed using any known technique based on the requirements of the composition.

[0020] n-6DPA alone or n-6DPA and DHA can be prepared by known methods, for example, from the above-mentioned fatty acid composition or other fatty acid compositions containing n-6DPA alone or n-6DPA and DHA. That is, they can be separated and purified from mixed fatty acids or fatty acid esters by, for example, the urea addition method, the cooling separation method, or the column chromatography method. They may also be separated and purified by other methods known in the art.

[0021] The fatty acid composition extracted from microalgae, n-6DPA alone or n-6DPA and DHA may be mixed with carbohydrates, emulsifiers, milk proteins, antioxidants, etc. in a conventional manner to prepare an emulsion, which may then be processed into powdered oils and fats by methods including, but not limited to, spray heat drying, spray cooling, pulverization freeze drying, pulverization cooling solidification, coating microencapsulation, coating spray mixing, or similar processes.

[0022] The content of DHA in the composition of the present invention may be suitably contained within the range in which synergistic effect with n-6DPA is recognized.For example, in the examples, in the combination of DHA and n-6DPA in the concentration range of 1 ng / mL to 10 μg / mL in which the effect of each of them alone is confirmed, the mass ratio of DHA (B) to n-6DPA (A) [(B) / (A)] is preferably 0.0001 or more and 10000 or less, more preferably 0.1 or more and 100 or less, and even more preferably 1 or more and 10 or less.

[0023] The food composition of the present invention contains the agent of the present invention and is used to promote NOS3 production. Specifically, it may be a functional food (including functional foods and foods for specified health uses, including food additives) or a nutritional supplement. Its form may be solid, semi-liquid, or liquid.

[0024] Examples of foods include, but are not limited to, agricultural products (bread, noodles, rice, sweets, tofu and processed products thereof, etc.), fermented foods (sake, medicinal liquor, mirin, vinegar, soy sauce, miso, etc.), livestock products (yogurt, ham, bacon, sausage, etc.), seafood products (kamaboko, fried tempura, hanpen, etc.), and beverages (fruit juice drinks, soft drinks, sports drinks, alcoholic drinks, tea, etc.).

[0025] Functional foods and nutritional supplements can be produced as foods and beverages in the form of powders, granules, tablets, capsules, troches, oral liquids, suspensions, emulsions, syrups, drinks, natural liquid diets, semi-digested nutritional foods, elemental nutritional foods, enteral nutrients, etc. In such cases, other nutritional components or functional components may be blended together with the algae of the present invention. Examples of processed forms include pharmaceutical preparations, such as natural liquid diets, semi-digested nutritional foods, elemental nutritional foods, drinks, enteral nutrients, etc., in which the algae of the present invention are blended with proteins, sugars, fats, trace elements, vitamins, emulsifiers, flavorings, etc., but the forms of the above-mentioned foods and beverages are also possible.

[0026] The pharmaceutical composition of the present invention contains the agent of the present invention and is used to promote NOS3 production.

[0027] The pharmaceutical composition can be formulated for oral administration using known pharmaceutical preparation techniques and may be in any form, such as solid, liquid, or semisolid. The n-6DPA and DHA, or a mixture thereof, in the pharmaceutical composition may be in a free form or in the form of a pharmaceutically acceptable salt, such as a sodium salt, potassium salt, lithium salt, or other alkali metal salt, or other metal salt such as zinc salt, calcium salt, or magnesium salt, or in various forms, such as a monoglyceride, diglyceride, triglyceride, ester of a lower alcohol, phospholipid, glycolipid, or amide. Here, "lower alcohol" refers to a monohydric alcohol having 6 or fewer carbon atoms, and examples thereof include methanol, ethanol, propanol, isopropanol, butanol, pentanol, and hexanol. [Example]

[0028] The present invention will be described in detail below with reference to examples. However, the examples are merely illustrative and the present invention is not limited thereto.

[0029] Evaluation of NOS3 gene expression level

[0030] [Test substance] For n-6DPA (all-cis-4,7,10,13,16-docosapentaenoic acid: manufactured by Cayman Chemical Co.), DHA (4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid: manufactured by Cayman Chemical Co.), and EPA (5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acid: manufactured by Cayman Chemical Co.), the ethanol was removed by nitrogen gas distillation, and the solvent was replaced with DMSO.

[0031] n-6DPA, EPA, and DHA were each adjusted to a concentration of 2 mg / mL with DMSO and diluted with Endothelial Cell Growth Medium 2 (EGM-2) medium (manufactured by Lonza) to prepare the test substances of Examples 1 to 6 and Comparative Examples 1 to 8 shown in Tables 1 to 3.

[0032] [Table 1]

[0033] [Table 2]

[0034] [Table 3]

[0035] [Cell culture] Human umbilical vein endothelial cells (HUVECs: manufactured by Lonza) were cultured in EGM-2 medium. 1 × 10 cells were plated onto a collagen-coated 96-well plate. 4 HUVECs were seeded at 100 μL / well and pre-cultured in a 5% CO incubator at 37°C. After 24 hours, the medium was removed from each well, and a test substance listed in Tables 1 to 3 was added at 100 μL / well, followed by 24 hours of culture.

[0036] [Measurement of NOS3 gene expression level] After removing the medium and washing once with PBS, total RNA was purified using an RNeasy Mini Kit (QIAGEN). Real-time PCR was performed on the obtained RNA using the One Step TBGreen PrimeScript RT-PCR Kit II (Takara). Primers (Takara) were used to measure the gene expression levels of NOS3 and GAPDH (endogenous control).

[0037] [Evaluation of NOS3 gene expression] The expression level of the NOS3 gene is shown in FIG. 1, where the expression level in the DHA 10 μg / mL administration group (Comparative Example 8) in Table 3 is set to 1.

[0038] The test substance that showed the highest NOS3 gene expression level was n-6DPA 0.03 μg / mL + DHA 0.1 μg / mL (Example 1), with a value of 1.60. The test substance that showed the highest expression level alone was n-6DPA 0.1 μg / mL (Example 4), with a value of 1.36. n-6DPA increased NOS3 gene expression level more than EPA and DHA, and a synergistic effect was observed when used in combination with DHA.

[0039] Evaluation of the effect of intake of a supplement containing n-6DPA-producing microalgae on vascular endothelial function

[0040] [Production of dried algae] The SPA-214 strain, which was created by UV mutagenesis from Aurantiochytrium sp. NBRC111922 (provided by the National Institute of Technology and Evaluation), was cultured in a 600L culture vessel in liquid medium (4% glycerol, 4% yeast extract, 0.9% salt) for 4 days. The culture medium was centrifuged to obtain approximately 15 kg of wet algae, which was then vacuum freeze-dried to yield approximately 3,200 g of dried algae.

[0041] [Manufacturing supplements containing microalgae] A microalgae-containing supplement was manufactured, with 280 mg of dried algae packed into each capsule. The n-6DPA, DHA, and EPA contents per capsule are shown below. n-6DPA: 14mg DHA: 45mg, EPA: 0.42mg

[0042] [FMD measurement] FMD was calculated by measuring the brachial artery vascular diameter at rest using an ultrasound diagnostic device, then avascularizing for 5 minutes, and measuring the vascular diameter at maximum diastole again after avascularization was released, using the following formula. FMD (%) = maximum vascular dilation (mm) ÷ resting vascular diameter (mm) × 100

[0043] [Evaluation of the effects of taking supplements containing microalgae] Example 7 and Comparative Example 9: A 44-year-old male with an FMD of approximately 6% (borderline) at the time of screening and pre-intake testing and not undergoing any medication for any chronic disease was tested. He took one capsule of a microalgae-containing supplement per day for 12 weeks. His FMD was measured before (Comparative Example 9) and after (Example 7) intake (Figure 2). After 12 weeks of intake of dried algae, FMD increased by 1.4%, from 6.1% to 7.5%, demonstrating an improvement in vascular flexibility.

[0044] Example 8 and Comparative Example 10: A 50-year-old woman with an FMD of approximately 6% (borderline) at the time of screening and pre-intake testing and not undergoing any medication for any chronic disease was the subject. After 12 weeks of taking one capsule of a microalgae-containing supplement per day, her FMD was measured before (Comparative Example 10) and after (Example 8) intake (Figure 2). After 12 weeks of intake of dried algae, FMD increased by 2.6%, from 6.1% to 8.7%, demonstrating an improvement in vascular flexibility.

[0045] Example 9 and Comparative Example 11: A 40-year-old woman with an FMD of approximately 8% (normal range) at the screening and pre-intake test and not undergoing any medication for any chronic disease was tested. She took one capsule of a microalgae-containing supplement per day for 12 weeks. Her FMD was measured before (Comparative Example 11) and after (Example 9) intake (Figure 2). After 12 weeks of intake of dried algae, her FMD increased by 3.8%, from 8.6% to 12.4%, demonstrating an improvement in vascular flexibility.

[0046] In a clinical trial evaluating the effects of a supplement containing n-6DPA-producing microalgae on vascular endothelial function, 12 weeks of 280 mg / day of microalgae containing n-6DPA and DHA increased FMD and improved vascular flexibility. The 280 mg of algae used in the study contained 14 mg of n-6DPA and 45 mg of DHA. Assuming that 100% of the n-6DPA and DHA in the algae ingested daily by a 60 kg subject (estimated blood volume: 4.6 L) is transferred to the blood, the resulting blood concentrations would be 3 μg / mL for n-6DPA and 9.8 μg / mL for DHA. The test substance concentrations used to evaluate NOS3 gene expression were estimated to cover the blood concentrations observed in clinical trials. It is believed that the increased NOS3 gene expression following algae ingestion improved vascular flexibility.

Claims

1. An endothelial nitric oxide synthase production promoter comprising either a fatty acid composition containing n-6DPA (n-6 docosapentaenoic acid) or n-6DPA.

2. 2. A vascular endothelial nitric oxide synthase production promoter, characterized in that the fatty acid composition according to claim 1 contains DHA (docosahexaenoic acid).

3. A vascular endothelial nitric oxide synthase production promoter characterized in that the mass ratio [(B) / (A)] of DHA (B) to n-6DPA (A) in the fatty acid composition according to claim 2 is 0.0001 or more and 10,000 or less.

4. A food composition comprising the endothelial nitric oxide synthase production promoter according to any one of claims 1 to 3.

5. A pharmaceutical composition comprising the endothelial nitric oxide synthase production promoter according to any one of claims 1 to 3.

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