Method for producing neutral fat suppressing composition using Ryukyu pine needles

Abstract

To provide a neutral fat suppression composition using luchu pine needle which can secure stability and safety property and can be utilized for a food product, by adopting an extract composition in which a plurality of active ingredients efficiently exhibiting a neutral fat suppression action included in luchu pine are found out, a synergic effect thereof can be exhibited, a neutral fat suppression effect can be enhanced as a whole, and ingredient balance in which concentration of some of ingredients does not become high is achieved.SOLUTION: There is provided a neutral fat suppression composition using luchu pine needle which is an extract composition in which the luchu pine needle is subjected to coarse crushing processing, and then to freezing and drying processing, and after the crushing processing, an organic solvent is extracted.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing a neutral fat-lowering composition using an extract composition obtained by solvent extraction of Ryukyu pine needles. [Background technology]

[0002] Ryukyu pine (Pinus luchuensis) is a plant of the pine family and is endemic to the Ryukyu Islands, and is found in abundance throughout Okinawa Prefecture. Ryukyu pine is used for pulp and woodworking in Okinawa Prefecture, but in Hokkaido Since leaves that fall on roads and the ground are unused, new ways of using Ryukyu pine needles are being sought.

[0003] It has been reported that the Pinaceae family contains active ingredients such as procyanidin, which has antioxidant and lipid metabolism-promoting properties.

[0004] Obesity is a condition in which more fat than normal accumulates in adipose tissue, and is a risk factor for diseases such as diabetes, hypertension, and hyperlipidemia.

[0005] Lipids (oils) are essential to the animal body, but if left as is, they do not mix well with the water in the blood, making it difficult for them to be transported through the blood and exchanged between different parts of the body.

[0006] Lipids exist in the body as substances such as triglycerides (TG) and cholesterol. Furthermore, when triglycerides and cholesterol are combined and bound to proteins to make them more water-soluble, they form substances called LDL and HDL.

[0007] Dietary lipids are absorbed into the intestines and initially become substances rich in triglycerides, which then enter the bloodstream. Once they circulate through the bloodstream and reach fat cells, the triglycerides are stored in the fat cells.

[0008] Adipose tissue acts as subcutaneous fat to maintain body temperature and protect internal organs from shock. Triglycerides are also taken up by liver cells, where they are used as an energy source and processed into other lipids.

[0009] Although triglycerides and cholesterol are essential for the body, if there is too much of them, the walls of blood vessels become narrow and fragile, causing various problems throughout the body. It is also said that if triglycerides are too high, they can damage the pancreas.

[0010] A patent has been disclosed that uses extracts from biological materials such as Ryukyu pine as an anti-obesity agent.

[0011] Japanese Patent Application Laid-Open No. 2010-280734 discloses a lipase inhibitor containing an extract obtained from at least one plant selected from the group consisting of Polygonum knotweed, Heritiera japonica, Eucalyptus tschonoskii, Phaseolus vulgaris, Alpinia zerumbet, Nandina, Safflower, Prunus tinctorius, Kalanchoe kaempferi, Gajumaru, Prunus momotsumana, Mulberry serrata, Butterfly leek, and Ryukyu pine, with the addition of diatomaceous earth and 50% ethanol at an extraction temperature of 70 to 90°C.

[0012] The use of biological extracts provides lipase inhibitory activity or antioxidant components. Furthermore, by defining drying and extraction conditions that allow both activities to function simultaneously, the aim is to provide safe formulations and cosmetics that not only prevent obesity and acne, but also reduce oxidative stress in the body, thereby reducing the risk of skin blemishes, freckles, and even malignant neoplasms (cancer). The following test results have been disclosed.

[0013] After cutting each plant to an appropriate size, it was either hot-air dried at 65°C for 12 hours or freeze-dried, crushed, and passed through a 0.5mm mesh. Each biological material was then subjected to high-pressure extraction. 0.5g to 5g of dried herb powder (dry weight) was added to an extraction cell along with 5g of diatomaceous earth. The extraction procedure was carried out under the following conditions: extraction solvent: 50% ethanol, solvent volume: 25ml, extraction temperature: 82°C, extraction pressure: 13.8MPa, extraction time: 10 minutes, extraction cycle: 2. The extract was then filtered through a 0.45μm membrane filter.

[0014] Lipase inhibition rate (%) Antioxidant activity (dilution ratio) Japanese knotweed whole plant 98.18 1,000 Kidney bean seed coat 58.10 1,111 Ryukyu pine leaves 65.78 175

[0015] Procyanidin, one of the active ingredients contained in Ryukyu pine, belongs to the flavan-3-ol flavonoid family and is a secondary metabolite of plants that exists as an oligomer or polymer (2-15 mer) formed by condensation of epicatechin or catechin.

[0016] It is found in large amounts in foods such as cocoa, black soybeans, cinnamon, nuts, apples, and grape seeds, and is expected to have high antioxidant properties.

[0017] The main physiological functions of procyanidins are as follows:

[0018] 1) Antioxidant effect 2) Drug metabolism promoting effect It has been reported that it suppresses cell damage and inflammation by promoting drug metabolism. 3) Inhibition of liver damage 4) Glucose metabolism promotion effect Procyanidins and foods containing large amounts of them have been reported to promote glucose metabolism and have effects on maintaining and improving health, including preventing and improving hyperglycemia. 5) Lipid metabolism promotion effect Although various reports have been published on the effects of procyanidins in promoting lipid metabolism and suppressing obesity and fat accumulation, these experiments have all been conducted using compositions with a high procyanidin content, and the detailed mechanism of action has not yet been fully elucidated. It has been shown that procyanidin-rich compositions derived from cocoa and black soybeans contribute to the prevention of fat accumulation caused by high-fat diet intake. 6) Effect on digestive enzymes Regarding the inhibition of lipase activity involved in lipid metabolism, the inhibitory effect of proanthocyanidins has been reported. For example, it has been shown that procyanidins exert lipase inhibitory effects in both in vitro and in vivo tests, and that the inhibitory activity increases depending on the degree of polymerization from dimers to pentamers. Furthermore, in animal experiments, when lipid loading tests were performed, it was confirmed that the amount of triglyceride absorption was suppressed by one hour of pretreatment with procyanidins.

[0019] It is known that procyanidin is effective in very small amounts, so it is necessary to thoroughly verify its effectiveness in the body at more physiological concentrations and evaluate its safety.It is expected that it will be put to practical use in foods with functional claims, which will also contribute to society.

[0020] The present invention also focuses on quercetin, one of the active ingredients contained in Ryukyu pine.

[0021] Quercetin is a flavonoid found widely in vegetables, fruits, tea, etc. It is known to have a variety of physiological functions, including potent antioxidant activity, inhibition of platelet aggregation and adhesion, vasodilatory properties, and anti-cancer properties.

[0022] Several prospective cohort studies have been conducted in Europe and the United States to date to investigate the relationship between flavonoid intake and lifestyle-related diseases. These cohort studies used food frequency questionnaires and estimated flavonoid intake from a database of the flavonoid content of each food, and examined the relationship between flavonoid intake and the risk of cardiovascular disease, etc., and reported that people with high intakes of flavonols, including quercetin and kaempferol, had a lower rate of death from coronary heart disease (Non-Patent Document 1).

[0023] In a Finnish study of approximately 10,000 people, Knekt et al. reported that people with high quercetin intake had lower mortality rates from ischemic heart disease and lower incidence of asthma, and that men also had lower incidence of lung cancer (Non-Patent Document 1).

[0024] In terms of foods, it has been reported that asparagus, red lettuce, onion, bell pepper, and romaine lettuce, which are rich in rutin (quercetin-3-rutinoside), are high in quercetin. Onions are known to have a particularly high quercetin content.

[0025] In Japan, the main source of quercetin is green tea, but it is also consumed in large amounts from onions, asparagus, tomatoes, etc. The quercetin content of Japanese onions is said to be approximately 10-50 mg / 100 mg.

[0026] In mouse studies, Quercetin's diabetes-improving effects By feeding diets containing 0.5% quercetin for two weeks, elevated blood glucose levels due to diabetes were reduced, and the decreased insulin levels were increased, improving the symptoms of diabetes (Non-Patent Document 1).

[0027] The effects of quercetin on improving obesity and metabolic syndrome When quercetin was added to a Western-style diet at a rate of 0.05% and given to mice, after 20 weeks, the increase in body weight and fat mass induced by the Western-style diet was suppressed, and hyperglycemia and increases in blood insulin and cholesterol levels were ameliorated (Non-Patent Document 1).

[0028] In human trials, Egert et al. reported that taking capsules containing 150 mg of quercetin for six weeks reduced systolic blood pressure and oxidized LDL levels in overweight people with a BMI of 25 or more, and that taking onion skin extract containing 162 mg of quercetin for six weeks reduced systolic blood pressure in overweight to obese (BMI of 30 or more) hypertensive patients (Non-Patent Document 1).

[0029] Thus, there is a need to clarify the effectiveness of quercetin ingested from food, as well as effective intake methods and mechanisms of action.

[0030] Patents have been disclosed regarding the use of food-derived quercetin to reduce triglycerides and hypertension. Japanese Patent Application Laid-Open No. 2002-171934 discloses a method for producing a dietary supplement using onion skins as a raw material, which is dried with hot air at 100 to 160°C to improve its poor taste, has a high quercetin content, and is highly advantageous for preventing high blood pressure, etc.

[0031] In addition, JP 2020-121952 A confirmed a significant reduction in triglycerides in mice using quercetin glycoside extracted from tea leaves. [Prior art documents] [Patent documents]

[0032] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-280734 [Patent Document 2] Japanese Patent Application Laid-Open No. 2002-171934 [Patent Document 3] Japanese Patent Publication No. 2020-121952 [Non-Patent Document 1] Food Science and Technology (54), 5-18, 2016-03: National Agriculture and Food Research Organization: Preventive function of quercetin on lifestyle-related diseases Summary of the Invention [Problem to be solved by the invention]

[0033] There is a need for methods to effectively utilize the active ingredients in Ryukyu pine, such as procyanidin, which has antioxidant properties and promotes lipid metabolism.

[0034] In JP 2010-280734 A, various biological materials were subjected to extraction treatment to search for materials that provide lipase inhibitory activity or antioxidant components, but the active ingredients that exert these effects have not yet been identified, so the effects were verified.

[0035] Japanese Patent Application Laid-Open No. 2002-171934 discloses an extract containing a high amount of quercetin, which is made from onion skins as a raw material. However, quercetin is also a component that is effective in small amounts, and there are concerns about the safety and stability of high concentrations.

[0036] JP 2020-121952 A describes an extraction method for isolating quercetin glycoside from dried tea leaves, but this also has safety and stability issues at high concentrations, and cannot be widely used as a food product.

[0037] The present invention aims to realize a neutral fat suppressing composition using Ryukyu leaves that is safe and stable and can be used in foods by finding multiple active ingredients contained in Ryukyu pine that effectively exert neutral fat suppressing effects, and making them exhibit a synergistic effect, rather than extracting only a portion of the active ingredients contained in Ryukyu pine at a high concentration, thereby enhancing the neutral fat suppressing effect as a whole, and by obtaining an extract composition from Ryukyu pine needles with a well-balanced composition composition in which no component is concentrated at a high level. [Means for solving the problem]

[0038] The present invention can solve the above-mentioned problems by the following means.

[0039] In claim 1 of the present invention, Ryukyu pine needles are roughly crushed to 0.5-30mm, then rapidly cooled from room temperature to -10℃ within 30 minutes, vacuum dried to sublimate water, crushed uniformly, and extracted with 70% or more ethanol.The method for producing a neutral fat-suppressing composition using Ryukyu pine needles is characterized in that the procyanidin content in the composition extracted by the method is 1.0 μg / mg to 2.0 μg / mg and the quercetin content is 0.1 μg / mg to 2.5 μg / mg.

[0040] The Ryukyu pine needles are made from the leaves of Ryukyu pine (Pinus luchuensis) grown in Okinawa Prefecture.

[0041] The coarse crushing treatment may be any treatment that can roughly crush the Ryukyu pine needles to about 0.5 mm to 30 mm, preferably about 0.5 to 10 mm.

[0042] The vacuum drying process may be a conventional freeze-drying process, which involves drying the frozen product under reduced pressure. Freeze-drying dries the product without heating, preventing the active ingredient from evaporating along with the water content, and preventing oxidation of the active ingredient, thereby preventing deterioration of the active ingredient. Preferably, quick freezing is preferred.

[0043] The crushing treatment may be any treatment that can crush the material quickly and uniformly without applying heat, and commercially available crushing treatment devices can be used.

[0044] The extraction with ethanol may be an organic solvent extraction treatment using ethanol as a solvent.

[0045] The neutral fat-lowering composition is a composition that can suppress an increase in the amount of neutral fat in the blood when a high-fat diet or the like is ingested.

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[0047] The rapid freezing process involves cooling the Ryukyu pine needles from room temperature to -10°C within 30 minutes.

[0048] The vacuum drying involves sublimating water from frozen Ryukyu pine needles in a vacuum.

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[0050] The crushing process can be carried out using any method that can crush freeze-dried Ryukyu pine needles. A bead crusher is preferred. This crusher crushes samples by violently colliding stainless steel beads with the sample, resulting in a crushing action. This crusher crushes samples uniformly in a short time without applying heat.

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[0054] The procyanidin content is preferably about 1.0 μg / mg to 2.0 μg / mg. A commercially available procyanidin product containing 1.46 μg / mg is available.

[0055] delete

[0056] The quercetin content is preferably about 0.1 μg / mg to 2.5 μg / mg, and a commercially available quercetin product containing 2.2 μg / mg is available.

[0057] delete

[0058] delete [Effects of the Invention]

[0059] The present invention has the following effects. (1) A composition using Ryukyu pine leaves that has a neutral fat suppressing effect can be realized. (2) The active ingredients can be extracted reliably and without deterioration during the extraction process of Ryukyu pine needles. . (3) A neutral fat-lowering composition can be realized that effectively utilizes the effects of procyanidin, an active ingredient in Ryukyu pine needles. (4) A neutral fat-lowering composition can be realized that effectively utilizes the effects of quercetin, an active ingredient in Ryukyu pine needles. (5) The active ingredients of Ryukyu pine needles, procyanidins and quercetin, each exert their respective mechanisms of action effectively, making it possible to realize a neutral fat-lowering composition containing them in a well-balanced manner. (6) The active ingredients of Ryukyu pine needles, procyanidins and quercetin, effectively exert their respective mechanisms of action, making it possible to create foods and beverages that contain a balanced amount of these ingredients. (7) A neutral fat-lowering composition can be realized in which the active ingredients of Ryukyu pine needles are not concentrated to a high level, and the effects of each active ingredient are fully exerted, thereby enhancing the synergistic effect. (8) It is possible to realize a food or drink containing a neutral fat-lowering composition that contains the active ingredients of Ryukyu pine needles in a balanced manner and is highly safe and stable. [Brief explanation of the drawings]

[0060] [Figure 1] FIG. 1 is a flow chart showing a method for producing a neutral fat-lowering composition according to the present invention. [Figure 2] FIG. 1 shows a graph of blood triglyceride levels in test mice according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0061] Next, one embodiment of the present invention will be specifically described with reference to the drawings.

[0062] FIG. 1 is a flow diagram showing a method for producing a neutral fat-lowering composition according to the present invention. S-1) Rough crushing process Ryukyu pine needles were collected from Okinawa Prefecture and roughly crushed to pieces of 2 cm or less.

[0063] S-2) Quick freezing process Coarsely crushed Ryukyu pine needles were frozen in a quick-freezing device (air blast freezer) so that the product temperature passed through the maximum ice crystal formation zone (-5 to -1°C) within 30 minutes.

[0064] S-3) Vacuum drying process The frozen Ryukyu pine needles were vacuum dried using a freeze-drying device.

[0065] S-4) Grinding process The vacuum-dried Ryukyu pine needles were crushed in a bead crusher.

[0066] S-5) Organic solvent extraction process Crushed Ryukyu pine needles were subjected to organic solvent extraction using 70% ethanol.

[0067] S-6) Filtration process The organic solvent extract was filtered through a 0.22 μm filter.

[0068] S-7) Neutral fat suppressing composition A neutral fat-lowering composition in the form of a yellow powder was obtained.

[0069] [Measurement of active ingredient content] The contents of procyanidin and quercetin, which are active ingredients expected to have a neutral fat suppressing effect in Ryukyu pine needles, were measured.

[0070] The content in 1 mg of powder was measured using ultra-high performance liquid chromatography (UPLC). ----------------------------------------------------------- Active ingredient content (μg / mg) ----------------------------------------------------------- Procyanidin 1.54 ----------------------------------------------------------- Quercetin 0.12 ----------------------------------------------------------- The ratio of procyanidins to quercetin in Ryukyu pine needles is approximately 13:1.

[0071] Regarding procyanidins, the content of commercially available functional foods (1.46 μg / mg). The lipolytic effect of quercetin has been confirmed even at 1 μM.

[0072] [Anti-obesity animal experiments] The above neutral fat-lowering composition was administered to mice in the following four groups, and whole blood samples were taken to measure neutral fat levels. Each group consisted of four mice. Group A: Normal diet + PBS administration group Group B: Normal diet + Ryukyu pine needle extract administration group Group C: High-fat diet + PBS administration group Group D: High-fat diet + Ryukyu pine needle extract administration group

[0073] The rats were allowed to freely consume the respective diets (normal diet or high-fat diet), and PBS or Ryukyu pine needle extract was orally administered to each group using an oral tube for two months.

[0074] Two months after the start of administration, blood samples were taken from all mice, and blood glucose levels, serum lipids, etc. were measured, and neutral fat levels were also measured. Figure 2 shows a graph showing the neutral fat levels in the blood of test mice according to the present invention. .

[0075] From the results of each measurement in this experiment, a significant difference was found in triglyceride levels between Group C (high-fat diet + PBS) and Group D (high-fat diet + Ryukyu pine needle ethanol extract).

[0076] These results suggest that the ethanol extract of Ryukyu pine needles is effective in suppressing the uptake of neutral fats. It was suggested that the ingredients were contained.

[0077] Furthermore, there was no significant difference between Group A (normal diet + PBS) and Group B (normal diet + Ryukyu pine needle ethanol extract), indicating that the inhibitory effect only occurs when a large amount of fat is ingested.

[0078] The neutral fat-suppressing composition of the present invention does not exhibit its neutral fat-suppressing effect with a normal diet, but exhibits its effect only when ingested with a high-fat diet. In other words, the neutral fat-suppressing effect of the Ryukyu pine needle extract of the present invention is not affected under normal conditions, but is exhibited only under abnormal conditions, thereby realizing a neutral fat-suppressing composition.

[0079] Furthermore, the effective freeze-drying process ensures that the active ingredients can be extracted without the need for evaporation or other processes, and since no heat is applied, there is no deterioration, and each active ingredient is extracted effectively and in a balanced manner. In other words, the Ryukyu pine needle extract of the present invention has a blending ratio of procyanidins and quercetin that exhibits the unique neutral fat suppressing effect described above, and each active ingredient acts effectively through a synergistic effect, preventing any one active ingredient from becoming highly concentrated, resulting in a well-balanced, safe, and reliable neutral fat suppressing composition. [Explanation of symbols]

[0080] S-1 Rough crushing process S-2 Quick freezing process S-3 Vacuum drying process S-4 Crushing process S-5 Organic solvent extraction process S-6 Filtration process S-7 Neutral fat suppression composition

Claims

[Claim 1] A method for producing a neutral fat suppressing composition using Ryukyu pine needles, characterized in that the Ryukyu pine needles are roughly crushed to 0.5 to 30 mm, the roughly crushed Ryukyu pine needles are rapidly cooled from room temperature to -10°C within 30 minutes, vacuum dried to sublimate water in a vacuum, uniformly ground, and then extracted with 70% or more ethanol, resulting in a composition having a procyanidin content of 1.0 μg / mg to 2.0 μg / mg and a quercetin content of 0.1 μg / mg to 2.5 μg / mg.

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