JP7763451B2 - Method for producing neutral fat suppressing composition using Ryukyu pine needles - Google Patents

Description

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.

The Ryukyu pine (Pinus luchuensis) is a member of the pine family and is endemic to the Ryukyu Islands, found in abundance throughout Okinawa Prefecture. Ryukyu pine is used for pulp and woodworking within Okinawa Prefecture, but the needles that fall on roads and the ground are unused, so new ways of using Ryukyu pine needles are being sought.

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

Obesity is a condition in which more fat than normal accumulates in adipose tissue, and is a risk factor for diseases such as diabetes, high blood pressure, and hyperlipidemia.

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.

Lipids exist in the body as substances such as neutral fats (triglycerides, TG) and cholesterol. Furthermore, when neutral fats and cholesterol combine and bind to proteins to make them more water-soluble, they form substances called LDL and HDL.

Fat in food is absorbed by the intestines and initially becomes a substance rich in triglycerides, which then enters the bloodstream. Once it circulates through the bloodstream and reaches fat cells, the triglycerides are stored in the fat cells.

Adipose tissue, as subcutaneous fat, plays a role in maintaining body temperature and protecting 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.

While triglycerides and cholesterol are essential for the body, if there is too much of them, the walls of blood vessels become narrow and fragile, which can cause various problems throughout the body. It is also said that if triglycerides are too high, they can cause damage to the pancreas.

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

JP 2010-280734 A discloses a lipase inhibitor containing an extract of 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 umbellata, Senna serrata, Butterfly leopard, and Ryukyu pine, which has lipase inhibitory activity and is obtained by adding diatomaceous earth and extracting with 50% ethanol at an extraction temperature of 70-90°C.

By using extracts from biological materials, lipase inhibitory activity or antioxidant components are provided. 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 and freckles, as well as the risk of developing malignant neoplasms (cancer), and other diseases. The following test results have been disclosed.

After cutting each plant to an appropriate size, it was either hot air dried at 65°C for 12 hours or freeze-dried, then 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, and 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 times. The extract was then filtered through a 0.45μm membrane filter.

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

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

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.

The main physiological functions of procyanidins are as follows:

1) Antioxidant effect 2) Drug metabolism promoting effect It has been reported that it suppresses cell damage and inflammation through its drug metabolism promoting effect.
3) Inhibition of liver damage 4) Promotion of glucose metabolism Procyanidins and foods containing large amounts of them have been reported to promote glucose metabolism and have the effect of maintaining and improving health, including preventing and improving hyperglycemia.
5) Lipid metabolism promoting effect Although various reports have been made on the lipid metabolism promoting effect and obesity/fat accumulation suppression effect of procyanidins, all of these experiments were 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) Effects on Digestive Enzymes: Proanthocyanidins have been reported to inhibit lipase activity, which is involved in lipid metabolism. For example, procyanidins have been shown to exhibit lipase inhibitory activity in both in vitro and in vivo studies, and the inhibitory activity increases depending on the degree of polymerization from dimers to pentamers. Furthermore, in animal experiments, lipid loading tests have shown that triglyceride absorption is suppressed by one hour of pretreatment with procyanidins.

Procyanidin is known to be 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 hoped that it can be put to practical use in foods with functional claims, which could also contribute to society.

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

Quercetin is a flavonoid found widely in vegetables, fruits, tea, etc. It is known to have a wide range of physiological functions, including potent antioxidant activity, inhibiting platelet aggregation and adhesion, dilating blood vessels, and anti-cancer effects.

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

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

In terms of foods, asparagus, which contains a lot of rutin (quercetin-3-rutinoside), as well as red lettuce, onions, bell peppers, and romaine lettuce, have been reported to have high quercetin contents. Onions are known to have a particularly high quercetin content.

In Japan, the main source of quercetin is green tea, with significant amounts also being consumed from onions, asparagus, tomatoes, etc. The quercetin content of Japanese onions is said to be approximately 10-50 mg/100 mg.

In mouse studies,
Effect of quercetin on diabetes improvement: By feeding 0.5% quercetin-containing feed 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).

Quercetin's effect on improving obesity and metabolic syndrome When quercetin was added to a Western-style diet at a ratio 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 improved (Non-Patent Document 1).

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 30 or more) hypertensive patients (Non-Patent Document 1).

As such, there is hope for elucidating the effectiveness of quercetin ingested from food, as well as effective intake methods and mechanisms of action.

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.

Furthermore, in JP 2020-121952, a mouse test using quercetin glycoside extracted from tea leaves confirmed a significant reduction in neutral fat levels.

JP 2010-280734 A Japanese Patent Application Laid-Open No. 2002-171934 Japanese Patent Application Laid-Open No. 2020-121952 Food Science and Technology (54), 5-18, 2016-03: National Agriculture and Food Research Organization: Preventive function of quercetin on lifestyle-related diseases

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

JP 2010-280734 A is currently in the process of researching materials that provide lipase inhibitory activity or antioxidant components through extraction processing of various biological materials. However, the active ingredients that exert these effects have not yet been identified, and the effects of these activities have been verified.

JP 2002-171934 A discloses an extract containing a high amount of quercetin, made from onion skins. However, quercetin is also an ingredient that is effective in small amounts, and there are concerns about the safety and stability of high concentrations.

JP 2020-121952 A describes an extraction method for isolating quercetin glycosides from dried tea leaves, but this also has safety and stability issues at high concentrations, preventing it from being widely used as a food product.

Rather than extracting only a few of the active ingredients contained in Ryukyu pine at high concentrations, we have identified multiple active ingredients contained in Ryukyu pine that effectively exert their triglyceride-lowering effects, and are able to exert a synergistic effect between them, thereby enhancing the overall triglyceride-lowering effect.By creating an extract composition from Ryukyu pine needles with a balanced composition of ingredients so that no single ingredient is concentrated at a high level, we aim to create a triglyceride-lowering composition using Ryukyu leaves that is safe and stable and can also be used in food.

The present invention can solve the above problems by the following means.

In claim 1 of the present invention, there is provided a method for producing a neutral fat-lowering composition using Ryukyu pine needles, which comprises roughly crushing Ryukyu pine needles to 0.5 to 30 mm, rapidly cooling the roughly crushed Ryukyu pine needles from room temperature to -10°C within 30 minutes, vacuum drying the needles to sublimate water in a vacuum, uniformly pulverizing the needles, and then extracting the resulting composition with 70% or more ethanol , wherein the resulting composition has 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.

These Ryukyu pine needles are made from the leaves of the Ryukyu pine (Pinus luchuensis) grown in Okinawa Prefecture.

The coarse crushing process can be any process that can roughly crush the Ryukyu pine needles to approximately 0.5 mm to 30 mm. Preferably, it is approximately 0.5 to 10 mm.

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.

The grinding process can be any process that can grind the material quickly and uniformly without applying heat. Commercially available grinding equipment can be used.

The extraction with ethanol may refer to an organic solvent extraction process using ethanol as the solvent.

This neutral fat-suppressing composition is a composition that can suppress an increase in blood neutral fat levels when a high-fat diet is ingested.

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

This vacuum drying process involves sublimating moisture from frozen Ryukyu pine needles in a vacuum.

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Any method can be used for the crushing process as long as it can crush freeze-dried Ryukyu pine needles. A bead crusher is preferred. This bead crusher crushes the sample by violently colliding stainless steel beads with the sample, resulting in impact and abrasive action, allowing for uniform crushing in a short time without applying heat.

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The procyanidin content is preferably approximately 1.0 μg/mg to 2.0 μg/mg. Commercially available procyanidin products contain 1.46 μg/mg.

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The quercetin content is preferably approximately 0.1 μg/mg to 2.5 μg/mg. Quercetin products available commercially contain 2.2 μg/mg.

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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) In the extraction process of the components of Ryukyu pine needles, the active ingredients can be extracted reliably and without deterioration.
(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) It is possible to realize a neutral fat-lowering composition in which the active ingredients of Ryukyu pine needles, procyanidins and quercetin, are contained in a well-balanced manner, with their respective action mechanisms being effectively exerted.
(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 synergistic effects.
(8) It is possible to realize a food or drink containing a neutral fat-lowering composition that contains a balanced amount of active ingredients from Ryukyu pine needles and is highly safe and stable.

FIG. 1 is a flow chart showing a method for producing a neutral fat-lowering composition according to the present invention. FIG. 1 shows a graph of blood triglyceride levels in test mice according to the present invention.

Next, one embodiment of the present invention will be described in detail using drawings.

FIG. 1 is a flow diagram showing a method for producing a neutral fat-lowering composition according to the present invention.
S-1) Coarse Crushing Step Ryukyu pine needles from Okinawa Prefecture were collected and roughly crushed to 2 cm or less.

S-2) Rapid freezing process The roughly crushed Ryukyu pine needles were frozen in a rapid 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.

S-3) Vacuum drying process The frozen Ryukyu pine needles were subjected to a vacuum drying process using a freeze-drying device.

S-4) Crushing step The vacuum dried Ryukyu pine needles were crushed using a bead crusher.

S-5) Organic Solvent Extraction Step The crushed Ryukyu pine needles were subjected to organic solvent extraction using 70% ethanol.

S-6) Filtration Step The organic solvent extract was filtered through a 0.22 μm filter.

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

[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.

The contents in 1 mg of powder were measured using ultra-high performance liquid chromatography (UPLC).

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Active ingredient content (μg/mg)
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Procyanidin 1.54
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Quercetin 0.12
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The ratio of procyanidin to quercetin in Ryukyu pine needles is approximately 13:1.

Regarding procyanidins, the content of commercially available functional foods (1.46 μg/
mg).
The lipolytic activity of quercetin has been confirmed to be effective even at a concentration of 1 μM.

[Anti-obesity animal experiments]
The above neutral fat-lowering composition was administered to four groups of mice, and whole blood was collected 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

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

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

The results of each measurement in this experiment showed a significant difference in triglyceride levels between Group C: high-fat diet + PBS and Group D: high-fat diet + Ryukyu pine needle ethanol extract.

These results suggest that the ethanol extract of Ryukyu pine needles contains active ingredients that inhibit the uptake of neutral fats.

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.

The triglyceride-suppressing composition of the present invention does not exhibit its suppressing effect when ingested with a normal diet, but only when ingested with a high-fat diet. In other words, the triglyceride-suppressing effect of the Ryukyu pine needle extract of the present invention is not affected under normal conditions, and the triglyceride-suppressing effect is only exerted under conditions that deviate from the normal state, resulting in a triglyceride-suppressing composition.

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.

S-1 Rough crushing process S-2 Rapid freezing process S-3 Vacuum drying process S-4 Pulverization process S-5 Organic solvent extraction process S-6 Filtration process S-7 Neutral fat suppressing composition

Claims (1)

A method for producing a neutral fat-suppressing composition using Ryukyu pine needles, comprising roughly crushing Ryukyu pine needles to 0.5 to 30 mm, rapidly cooling the roughly crushed Ryukyu pine needles from room temperature to -10°C within 30 minutes, vacuum drying the needles to sublimate water in a vacuum, uniformly pulverizing the needles, and then extracting the resulting composition with 70% or more ethanol, wherein the resulting composition has 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.