A blood nitric oxide concentration increasing agent that improves breathing ease during exercise.

Abstract

To provide an agent or a food having the action of increasing a nitric oxide (NO) concentration within blood vessels.SOLUTION: It is found out that the ingestion of tablets, concentrated extracts, beverages, etc., containing genus Bidens, particularly Bidens Pilosa, or their components causes the increase in the NO concentration within blood vessels and affects breathing during exercise.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to an agent that regulates blood nitric oxide concentration and can improve ease of breathing during exercise. [Background Art]

[0002] Nitric oxide (NO) is a nitrogen oxide synthesized from arginine by nitric oxide synthase (NOS) in vivo and exhibits various functions. NO produced from vascular endothelial cells has a vasodilatory effect (hypotensive effect) and is called endothelium-derived relaxing factor (EDRF). In addition, NO is known to have various other physiological activities including an inhibitory effect on platelet aggregation (anti-atherosclerotic effect), an inhibitory effect on proliferation of vascular smooth muscle cells, an information transmission effect in neurotransmission and other processes, and a bactericidal effect. It is known that when NO is released into blood vessels, it has an effect of improving blood flow through vasodilation. When blood flow is improved, ease of breathing during exercise is enhanced, and as a result, exercise performance is expected to be improved. [Prior Art Literature] [Non-Patent Literature]

[0003] [Non-Patent Literature 1] Hypertens Res 31: 2105-2113, 2008. [Non-Patent Literature 2] Hypertension 63: 376-382, 2014. [Summary of the Invention] [Problem to be Solved by the Invention]

[0004] An object of the present invention is to provide an agent or food having an effect of increasing blood nitric oxide (NO) concentration. [Means for Solving the Problem]

[0005] We have completed the present invention by confirming that administering a composition containing components extracted from plants of the genus Bidens, particularly Bidens pilosa, increases the concentration of NO in the blood. Furthermore, it has been found that ingesting the blood nitric oxide concentration-increasing agent of the present invention also affects the ease of breathing during exercise. In other words, the present invention provides the following: (1) A blood nitric oxide concentration increasing agent characterized by containing plant components of the genus Bidens. (2) The blood nitric oxide concentration increasing agent according to (1), characterized in that the plant component of the Bidens genus is an extract of the above-ground part of a Bidens genus plant. (3) A blood nitric oxide concentration increasing agent according to (1) or (2), wherein the plant of the genus Bidens is Bidens pilosa. (4) A blood nitric oxide concentration increasing agent described in any one of items (1) to (3), which increases the blood nitric oxide concentration compared to when the blood nitric oxide concentration increasing agent is not taken. (5) A blood nitric oxide concentration increasing agent described in any one of items (1) to (4), which reduces the ease of breathing during exercise compared to when the blood nitric oxide concentration increasing agent is not taken. [Effects of the Invention]

[0006] Administration of the blood nitric oxide concentration-increasing agent of the present invention resulted in a significant increase in blood nitric oxide concentration. Furthermore, it was found that continued administration of the blood nitric oxide concentration-increasing agent of the present invention further enhanced the effect of increasing blood nitric oxide concentration, reducing shortness of breath during exercise and improving ease of breathing. [Brief explanation of the drawing]

[0007] [Figure 1] This graph shows the results of measuring the increase in blood NO due to cold exposure one day after ingestion of the blood nitric oxide concentration increasing agent of the present invention (BP tablets, BP tea, and BP extract powder). [Figure 2]This graph shows the results of measuring the increase in blood NO after 7 consecutive days of intake of the blood nitric oxide concentration increasing agent of the present invention (BP tablets, BP tea, and BP extract powder) due to cold exposure. [Figure 3] This graph compares the amount of NO in the blood before and after 7 consecutive days of intake of the blood nitric oxide concentration increasing agent (BP tablets, BP tea, and BP extract powder) of the present invention. [Figure 4] This graph shows the effect of administering the blood nitric oxide concentration-increasing agent of the present invention on exercise (running). [Figure 5] This graph shows the percentage of subjects who felt that they could breathe more easily during exercise (running) after being administered the blood nitric oxide concentration increasing agent of the present invention. [Modes for carrying out the invention]

[0008] In the present invention, "blood nitric oxide concentration" refers to the concentration of nitric oxide (NO) in the blood of a subject. Blood nitric oxide concentration can be measured by any method, but in this document, the value measured using the "Total NO / Nitrite / Nitrate Assay Kit" (R&D KGE001) (assay principle: nitrates are converted to nitrites by an enzyme, nitrites are converted to azo dye products, and the light absorption of the resulting azo derivative at a wavelength of 540 nm is measured). There are several reports on the resting blood nitric oxide concentration in healthy adult males, some reporting values ​​of 0.1 μM (0.1 μmol / L) or less (J Int Soc Sports Nutr. 2016; 13: 6), but it is generally considered to be at most 2000 nM (2 μmol / L) or less (European Journal of Applied Physiology volume 110, pages 341-351 (2010)). In the experiments conducted by the present inventors, the value at rest was ~0.5 μmol / L. The increase in blood nitric oxide concentration induced by the administration of the blood nitric oxide concentration-increasing agent of the present invention can be detected by a single or continuous administration of the blood nitric oxide concentration-increasing agent. Furthermore, since the increase in nitric oxide concentration as a vasodilator becomes more pronounced by performing, for example, a cold stress test, it is also possible to confirm the effect by performing such a stress test; however, such a stress test is not an essential condition for increasing blood nitric oxide concentration. An example of a cold load test is performed as follows: The skin temperature of the index, middle, and ring fingers of each subject's hands was measured using thermography. After waiting for 10 minutes in a constant temperature and humidity environment room at 25±1°C and 50±5% humidity, a blood nitric oxide concentration increasing agent was administered, and after waiting for another 20 minutes, the body surface temperature was measured immediately before the cold load. Subsequently, a cold stimulus test was performed under the condition that both hands were immersed in 15°C water up to the wrists for 1 minute.

[0009] The present invention's blood nitric oxide concentration increasing agent contains components from plants of the genus Bidens. The Bidens genus plants used in this invention belong to a group of plants scientifically known as the Bidens genus, as described in Japanese Patent Publication No. 2001-178390 and Japanese Patent Publication No. 2001-233727. There are many different species, and they hybridize with each other, resulting in many varieties. This has led to botanical confusion, and the correspondence between scientific names, Japanese names, and Chinese names is also mixed, making identification extremely difficult. However, the Bidens genus plants used in this invention include those listed below.

[0010] Bidens pilosa L. (Bidens pilosa, White Bidens, Ham Pung Grass) Bidens pilosa L. var. minor (Blume)Sherff (White-flowered Bidens, White Bidens, Small White Bidens, Small Bidens, Ham Peng Grass) Bidens pilosa L. var. bisetosa Ohtani et S.Suzuki (Awayukisendangusa) Bidens pilosa L. f. decumbens Scherff (Hayawuyukisendangusa) Bidens pilosa L. var. radiata Scherff (may also include Tachiawuyukisendangusa and Hayawuyukisendangusa) Bidens pilosa L. var. radiata Schultz Bipontinus (Shironosendangusa, Oobananosendangusa) Bidens biternata Lour. Merrill et Sherff (Sendangusa) Bidens bipinnata L. (Kobanosendangusa, Sendangusa) Bidens cernua L. (Yanagitaukogi) Bidens frondosa L. (Amerikasendangusa, Seitakataukogi) Bidens maximowicziana Oett (Bidens maximowicziana) Bidens parviflora Willd (Hosobanosendangusa) Bidens radiata Thuill. var. pinnatifida (Turcz.)Kitamura (Ezonotaukogi) Bidens tripartita L. (Taukogi)

[0011] Among the above Bidens plants, Bidens pilosa species are particularly preferred from the viewpoint of effect.

[0012] As for the part to be used of the above Bidens plant, any part of the root, the aerial part (stem, leaf, flower, etc.) or the whole plant may be used. In particular, use of aerial parts such as leaves and stems is preferable from the viewpoint of efficacy. The above plant of the genus *Bidens* may be used raw, or may be used in the form of a dried product or a processed dried product. Usually, a product obtained by drying a fresh plant by sun drying or hot air drying (for example, at 70 to 80°C), or a product obtained by steaming with steam for, for example, about 1 hour to 1 and a half hours and then drying is used. Alternatively, a product processed and dried by the method described in Japanese Patent Application Laid-Open No. 2001-178390 may be used.

[0013] Furthermore, an extract obtained by adding water or a water-containing solvent or the like and performing extraction at normal temperature or under heating may also be used. As for the extraction method, for example, an extract can also be obtained by immersion and standing, or by using an extraction apparatus such as a Soxhlet extractor.

[0014] In the blood nitric oxide concentration increasing agent of the present invention, other plants may be further added for purposes such as flavor improvement in addition to the above-mentioned plant of the genus *Bidens*. Any other plant may be used as long as it does not inhibit the body temperature maintenance effect of the present invention. Specifically, the blood nitric oxide concentration increasing agent of the present invention may further contain barley and / or turmeric in addition to the above plant of the genus *Bidens*.

[0015] Barley Barley can be used particularly for the purpose of improving flavor, and in that case, it is more preferable to use roasted barley. Roasted barley can be further ground before use. When barley is mixed with a plant of the genus *Bidens*, the mixing ratio is preferably 10 to 400 parts by mass of barley based on dry weight, more preferably 100 to 200 parts by mass, relative to 100 parts by mass of dry weight of the genus *Bidens* plant.

[0016] Turmeric Turmeric may be one used for edible, medicinal and other purposes. A commercially available dried or ground product may be used, or a product obtained by drying and grinding a cultivated plant, or steaming it followed by drying and grinding may be used. The site used is a portion containing at least the root. Only the root portion may be used, or the root and other portions such as stems and leaves may be used. The root portion is particularly preferable from the perspective of the effect of the present invention. When adding turmeric to plants of the genus Bidens, the mixing ratio is preferably 0.1 to 30 parts by weight of turmeric, and more preferably 1 to 10 parts by weight, per 100 parts by weight of the dry weight of the Bidens plants.

[0017] When using dried leaves and stems of plants of the genus Bidens (preferably Bidens pilosa), it is preferable to add 1g to 50g (preferably 10g to 40g) of the dried material to about 1 liter of water and heat the solution at a temperature of 90°C or higher (preferably 95 to 100°C) for 10 minutes or more (preferably 30 to 60 minutes), then let it cool to around body temperature before use. When using dried Bidens plants in combination with dried barley and / or turmeric, it is preferable to add 1g to 50g (preferably 10g to 40g) of the total amount of these to about 1 liter of water, heat the solution at a temperature of 90°C or higher (preferably 95 to 100°C) for 10 minutes or more (preferably 30 to 60 minutes), and then let it cool to around body temperature before use. When consuming the above-mentioned extract solution, it is desirable from the viewpoint of effectiveness to take 10 to 2000 ml per day, preferably 100 to 1500 ml, and more preferably 1000 to 1500 ml. From the viewpoint of the effects of the present invention, it is preferable to ingest 1 g or more per day in terms of the dry weight of plants of the genus Bidens (preferably Bidens pilosa), more preferably 3 g or more, even more preferably 5 g or more, even more preferably 8 g or more, and still more preferably 10 g or more. Furthermore, from the viewpoint of the effects of the present invention, it is preferable to ingest plants of the genus Bidens (preferably Bidens pilosa) for two consecutive days or more, more preferably for four days or more, and even more preferably for six days or more.

[0018] Extract powder Examples of solvents used for extraction to produce the extract powder include water, methanol, ethanol, propanol, butanol, ethylene glycol, propanediol, butanediol, glycerin, and other alcohols, as well as their aqueous solutions, acetone, ethyl methyl ketone, chloroform, methylene chloride and ethyl acetate, dimethyl sulfoxide, dimethylformamide, and their aqueous solutions. Water or hot water is particularly preferred from the viewpoint of ease of post-extraction processing and good extraction rate. A mixture containing two or more of the above solvents may also be used. The amount of solvent added can be, for example, about 1 L to 100 L per 1 kg of the total dry weight of the plants used. From the viewpoint of the effects of the present invention, water extraction or hot water extraction is particularly preferred. The extract may be concentrated by distillation to remove the solvent as needed, yielding a concentrate or solid (dried) product. The extract may be subjected to treatments such as activated carbon treatment, resin treatment such as HP20, low-temperature standing, or filtration to remove decolorization and unwanted substances before use. Furthermore, the extract may be fractionated to obtain a highly active fraction using appropriate separation methods, such as gel filtration, silica gel column chromatography, or reverse-phase or normal-phase high-performance liquid chromatography. In this invention, the Bidens genus plant extract includes such fractions. The obtained extract may be used as is, or as a concentrate or solid (e.g., powder). Other components may also be mixed in depending on the intended use.

[0019] Tablets, capsules, etc. The dried extract of the Bidens genus may be administered as is, or with the addition of excipients such as dextrin, in the form of powder, tablet granules, hard capsules, etc. When forming into tablets, conventionally known carriers, dilution agents, disintegrants, lubricants, etc., can be used.

[0020] Drinkable medicine A drink can be prepared by adding water and other additives to an extract of the Bidens genus plant. The additives are not particularly limited and can be any that are commonly used in general beverages. Examples include sweeteners, acidulants, minerals, colorants, flavorings, emulsifiers, thickeners and stabilizers, antioxidants, preservatives, antifungal agents, vitamins, dietary fiber, and carbon dioxide. Examples of sweeteners include sugars (glucose, fructose, liquid sugar, sucrose, lactose, maltose, oligosaccharides, etc.), honey, dextrins, sugar alcohols (oligosaccharides, erythritol, maltitol, etc.), and artificial sweeteners (stevia, aspartame, acesulfame K, sucralose, etc.). Examples of acidulants include organic acids, preferably citric acid, malic acid, fumaric acid, acetic acid, lactic acid, and tartaric acid. Examples of minerals include potassium, calcium, and magnesium. Examples of emulsifiers include sucrose fatty acid esters and lecithin. Examples of thickening and stabilizing agents include soybean polysaccharides, pectin, carrageenan, gellan gum, xanthan gum, and guar gum. Examples of antioxidants include tocopherol and cysteine ​​hydrochloride. Examples of colorants include carotenoid pigments, anthocyanin pigments, caramel colorants, and various synthetic colorants. Examples of vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, and vitamin E.

[0021] The composition of the present invention can also be used in the form of food. The food may be in the form of a powder, a solid such as a tablet, or a beverage such as a liquid. Examples of food products include confectionery such as cookies and candies, luxury goods such as alcoholic beverages, juices and soft drinks, seasonings such as miso and soy sauce, instant foods, and so-called health supplements. [Examples]

[0022] <Manufacturing example> (1) Method for treating plants of the genus Bidens The above-ground parts of Bidens pilosa, a plant belonging to the Bidens genus, were harvested, washed under running water, cut, steamed, then mixed in a kneading machine to make the steamed material uniform, and finally dried with hot air.

[0023] (2) Method for producing extract powder containing plants of the genus Bidens Approximately 20 times the amount of hot water is added to the dried Bidens pilosa processed in (1) to extract the product. Modified starch is added to the concentrated product, and it is dried into a powder using a spray-drying process (also called Bidens pilosa extract powder or BP extract powder). 1g of Bidens pilosa extract powder is equivalent to 1g of dried Bidens pilosa.

[0024] (3) Method for producing tablets containing plants of the genus Bidens (2) The extract powder prepared in (2) was mixed with an excipient and compressed into tablets (also called Bidens pilosa tablets or BP tablets). The amount of Bidens pilosa contained in one Bidens pilosa tablet is 0.9 g in terms of dry Bidens pilosa weight.

[0025] (4) Method for producing tea containing plants of the genus Bidens The dried Bidens pilosa prepared in (1) was further chopped and packaged in tea bags of 3g each, using roasted barley and turmeric. The proportions were such that, based on dry weight, the roasted barley was in the range of 100-200 parts by mass and the turmeric was in the range of 1-10 parts by mass per 100 parts by mass of Bidens pilosa. The amount of Bidens pilosa contained in one tea bag is 1.26g in terms of dry weight of Bidens pilosa. We used tea (also known as Bidens pilosa tea or BP tea) made by simmering three 3g tea bags in 250mL to 500mL of hot water for 10 minutes.

[0026] (5) Method for producing a drink containing plants of the genus Bidens Using the extract powder prepared in (2), a liquid preparation (also called Bidens pilosa drink or BP drink) (containing honey, erythritol, and citric acid, 29 kcal / 50 ml) was obtained. The amount of Bidens pilosa contained in one bottle (50 ml) of Bidens pilosa drink is 5 g in terms of dry Bidens pilosa weight.

[0027] [Example 1] The following tests were conducted to confirm the effect of improving NO concentration. • Test subjects The study involved 40 healthy men and women aged 20 to 64 (who gave written consent to participate in the study). Measurements were taken at five time points: before cold exposure, immediately after exposure (0 minutes), 10 minutes, 20 minutes, and 30 minutes. Participants were placed in a constant temperature and humidity chamber (25±1℃, 50±5% humidity) for a specified period of time, and then subjected to cold exposure by immersing both wrists in 15℃ water for 1 minute. Measurements were taken every 10 minutes after the cold exposure, up to 30 minutes, and the skin surface temperature of the index, middle, and ring fingers of both hands was measured at each time point. A recovery rate of 60% or higher, as expressed by the following formula, is considered a success. Recovery rate (%) = (Temperature after 20 minutes of cold load - Temperature immediately after cold load) / (Temperature before cold load - Temperature immediately after cold load) × 100 In the above experiment, subjects whose skin temperature recovery rate after 10 minutes of cold exposure was less than 90% were selected, and the effect of administering the blood nitric oxide concentration increasing agent of the present invention was measured. Subjects with a skin temperature recovery rate of less than 90% were selected as test subjects for the following reason: In other words, healthy humans have a mechanism to return to their original skin temperature after a temporary cold exposure. However, some individuals have a poor recovery rate. There are various reasons for this, but poor blood flow is considered to be the main cause. Subjects with a skin temperature recovery rate of less than 90% indicate poor blood flow recovery, and were selected as test subjects because it was thought that they would be more likely to reflect the effect of the blood nitric oxide concentration increasing agent, which has been shown to improve blood flow.

[0028] • Test product consumed and number of test subjects for each test. Participants were randomly divided into four groups: a water intake group (control), a Bidens pilosa tea intake group (BP tea), a Bidens pilosa tablet intake group (BP tablets), and a Bidens pilosa extract powder intake group (BP extract powder). The number of participants in each group was 7 for the water intake group, 9 for the Bidens pilosa tea intake group, 8 for the Bidens pilosa tablet intake group, and 9 for the Bidens pilosa powder intake group. The water intake group consumed 250 mL of water per day. The Bidens pilosa tea intake group consumed three 3 g / packet tea bags brewed in 250 mL of hot water for 10 minutes (equivalent to 3.78 g of dried Bidens pilosa). The Bidens pilosa tablet intake group consumed 9 tablets per day (equivalent to 8.1 g of dried Bidens pilosa). The Bidens pilosa extract powder intake group consumed 15 g per day (equivalent to 15 g of dried Bidens pilosa).

[0029] • Test conditions Each test subject ingested the daily amount of water, Bidens pilosa tea, Bidens pilosa tablets, or Bidens pilosa extract powder as described above for seven consecutive days. Blood samples were taken on each test day, day 1 and day 7, before ingestion of the test product and 30 minutes after the cold loading test (conditions of Example 1), and the blood NO concentration (μmol / L) was measured (Day 1: Figure 1, Day 7: Figure 2).

[0030] ·Blood NO concentration measurement method Using the measurement kit "Total NO / Nitrite / Nitrate Assay Kit" (R&D Co., Ltd. KGE001), blood nitric oxide levels were measured according to the following procedure. (1) The collected plasma (collected using an EDTA2NA blood collection tube) was stored at -80°C until measurement. (2) The plasma was thawed during measurement and diluted 2-fold with the reaction diluent included in the kit. (3) 50 μL of the reaction diluent was added to each well of a 96-well microplate. (4) 50 μL each of the sample or calibration standard solution was added. (5) Add 50 μL of the reaction diluent to each well. (6) 50 μL of Griess Reagent II reagent was added to each well. (7) After standing at room temperature for 10 minutes, the absorbance of each well was measured at a wavelength of 540 nm.

[0031] • Test results Participants in the analysis: Eligibility checks were performed on the clinical trial participants (water: 8 people, BP tea: 9 people, BP tablets: 9 people, BP extract powder: 9 people). One participant from the water intake group (control) and one participant from the Bidens pilosa tablet intake group who were found to have consumed foods related to blood flow were excluded, and the following number of participants were included in the analysis. Wednesday: 7 people BP tea: 9 people BP tablets: 8 people BP extract powder: 9 people The values ​​obtained from the measurements are shown as mean ± standard deviation, and an independent t-test was performed with a significance level of 5% (two-sided) (Table 1, Figures 1-3).

[0032] Table 1 TIFF0007923517000001.tif57144

[0033] In all test products, blood NO concentration increased 7 days after ingestion (Figure 3). Significant differences compared to water were observed in Bidens pilosa tablets and Bidens pilosa extract powder. This is thought to be due to differences in the concentration of Bidens pilosa extract. The effect of the present invention's blood nitric oxide concentration-increasing agent became clearer after continuous intake for 7 days than after a single dose. Continuous intake resulted in a statistically significant improvement in blood NO production (Figure 3).

[0034] [Example 2] (Reference test example) Nitric oxide is a substance released from endothelial cells of blood vessels and is known to dilate blood vessels and improve blood flow. Improved blood flow can lead to easier breathing during exercise, which in turn can improve athletic performance. Therefore, to confirm this effect, we conducted a test to see whether running improved athletic performance.

[0035] • Test method Study participants: Five healthy men and women aged 30 to 60 who regularly run were divided into two groups to ensure a similar gender ratio and average age. Running distance: 3km Exam Schedule: Each examinee took the exam twice, with a one-month interval between each test. Method of Drink Consumption: Participants were randomly assigned either the Bidens pilosa drink (50ml) manufactured according to manufacturing example (5) or the reference drink (Chocola BB Drink Fe Charge (manufactured by Eisai)) (50ml) (5kcal / 50ml), and 50ml was consumed before running. For the same test subject, if they consumed the Bidens pilosa drink the first time, they consumed the reference drink one month later; if they consumed the reference drink the first time, they consumed the Bidens pilosa drink the second time. The test drink groups were determined using a blinding method, so that test subjects did not know whether they were receiving the test drink or the reference drink.

[0036] • Evaluation method Running condition (post-run impressions): Based on the following indicators, scores were assigned, and the average values ​​were calculated for each group of reference drinks and Bidens pilosa drinks (Figure 4). 5 points - I was in excellent form. 4 points - I was in pretty good form. 3 points remain unchanged Two points: Slightly off my game. 1. I was in very poor condition. Ease of breathing: This shows the percentage of people who "felt" they could breathe more easily after running, in each group (reference drink and Bidens pilosa drink).

[0037] It is presumed that the ingestion of the present invention's blood nitric oxide concentration-increasing agent leads to an increase in nitric oxide concentration in the blood vessels, improving blood flow and making breathing easier during running, resulting in improved athletic performance. This point is supported by the fact that, although subjective, it is in good agreement with post-run impressions and evaluations of breathing ease.

Claims

1. A blood nitric oxide concentration increasing agent characterized by containing components of Bidens pilosa, wherein the blood nitric oxide concentration in healthy individuals is increased compared to when the blood nitric oxide concentration increasing agent is not taken.

2. The blood nitric oxide concentration increasing agent according to claim 1, characterized in that the component of Bidens pilosa is an extract of the above-ground part of Bidens pilosa.

3. A blood nitric oxide concentration increasing agent according to claim 1 or 2, for improving the ease of breathing during exercise compared to when the blood nitric oxide concentration increasing agent is not taken.

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