WO2006112283A1

WO2006112283A1 - Anti-fatigue agent

Info

Publication number: WO2006112283A1
Application number: PCT/JP2006/307529
Authority: WO
Inventors: Takashi Kahara; Naofumi Umigai; Masahiro Takahashi; Katsura Funayama
Original assignee: Riken Vitamin Co., Ltd.
Priority date: 2005-04-13
Filing date: 2006-04-10
Publication date: 2006-10-26
Also published as: JPWO2006112283A1

Abstract

An anti-fatigue agent or beverage comprising crocetin represented by the formula below or a pharmacologically acceptable salt thereof as the active ingredient. The anti-fatigue agent and beverage are useful for the prevention of or recovery from fatigue.

Description

Specification

Anti-fatigue

Technical field

[0001] The present invention relates to an anti-fatigue agent and a food or drink containing crocetin or a pharmacologically acceptable salt thereof as an active ingredient.

Background art

[0002] Fatigue is defined as a temporary qualitative or quantitative decline in physical and mental work ability that occurs when physical or mental stress is applied continuously. ing. And when it doesn't recover spontaneously and you can't get out of the state of being tired all the time, you get close to a condition called chronic fatigue or actually get sick.

[0003] In order to prevent or recover from fatigue, it is generally said that it is effective to sufficiently replenish energy sources and take a rest or sleep. Energy sources are the three major nutrients: carbohydrates, lipids, and proteins from the diet. Vitamin B, vitamin B, vitamin B, vitamin B

These nutrients are converted into energy by the action of vitamin B groups such as 1 2 6 1. this

2

In addition, vitamin C, vitamin E, etc. also have the function of preventing cell oxidization and improving blood flow. Therefore, it has been said that it is important to eat foods rich in these vitamins.

[0004] Further, as a preparation or food containing a compound effective for fatigue recovery, for example, a preparation containing L-calcintin L-tartrate (see Patent Document 1), piotin, carcin and pantothenic acid as active ingredients Containing fatigue improving agent (see Patent Document 2), anti-fatigue agent containing idebenone or its hydroquinone as an active ingredient (see Patent Document 3), organic acid having two or more carboxyl groups (taenoic acid) , Malic acid, tartaric acid, fumaric acid, adipic acid and succinic acid) or their salts as active ingredients (see Patent Document 4), L-carcin and histidine-related dipeptides (carnosine) contained in livestock meat , Anserine, valenine) and taurine have been proposed for physical strength enhancement materials for fatigue recovery and foods using the same (see Patent Document 5). In addition, ascorbic acid, acetylyl L-carcin, tetrahydrobiopterin (BH4), dehvdroepiandrost erone sulfate (DHE A-S) (see Non-Patent Document 1) is also considered effective for fatigue recovery. However, research on fatigue has not yet been fully studied and elucidated in terms of theory, and it has been confirmed that it has a remarkable effect on prevention or recovery of fatigue, and there are few compounds that have been put to practical use. This is the actual situation.

[0005] On the other hand, with recent advances in research on the functionality of carotenoid pigments, singlet oxygen scavengers (see Patent Document 6), collagen production promoters (see Patent Document 7), skin immune activation, In addition to its action on the skin as an agent (see Patent Document 8), physiological activities such as cholesterol-lowering action, tumor formation-inhibiting effect, and liver toxicity-inhibiting effect have been reported (see Non-Patent Document 2). ). However, the anti-fatigue effect of crocetin was not clear!

Patent Document 1: Japanese Patent Laid-Open No. 4-128223

Patent Document 2: Japanese Patent Laid-Open No. 7-233070

Patent Document 3: Japanese Patent Laid-Open No. 10-53520

Patent Document 4: JP-A-10-175856

Patent Document 5: Japanese Patent Laid-Open No. 2001-46021, Claim 2

Patent Document 6: Japanese Patent Laid-Open No. 5-320036

Patent Document 7: Japanese Patent Laid-Open No. 7-285846

Patent Document 8: Japanese Patent Laid-Open No. 11-246396

Non-Patent Document 1: Masaaki Tanaka, “Death and Fatigue”, History of Medicine, 2003, No. 204, No. 5, p. 362-364

Non-Patent Document 2: Masayo Chika, “About the Carotenoid Pigment without Bitter”, Bulletin of Fukuoka Women's Junior College, 1 999, No. 57, p. 31—34

Disclosure of the invention

Problems to be solved by the invention

[0006] An object of the present invention is to provide an anti-fatigue agent containing a compound having an anti-fatigue effect as an active ingredient and a food or drink used for the prevention or recovery of fatigue. Means for solving the problem

[0007] As a result of intensive research to solve the above problems, the present inventors have found that the carotenoid color It was found that crocetin, a kind of element, has an anti-fatigue effect. Based on these findings, the present inventors have further researched and completed the present invention.

That is, the present invention

(1 set

[Chemical 1]

An anti-fatigue agent, characterized by containing crocetin represented by the formula or a pharmacologically acceptable salt thereof as an active ingredient,

(2) Formula

[Chemical 2]

A food or drink characterized by containing as an active ingredient crocetin represented by or a pharmacologically acceptable salt thereof,

(3) The food or drink according to (2) above, which is labeled for use in preventing or recovering from fatigue, and

(4) Formulas for manufacturing food and drink for the purpose of preventing or recovering from fatigue

[Chemical 3]

Of crocetin represented by the formula or a pharmacologically acceptable salt thereof

About.

The invention's effect [0008] According to the present invention, it is possible to provide an anti-fatigue agent and food and drink that are effective in preventing or recovering from fatigue.

Crocetin used in the present invention has an excellent anti-fatigue effect as compared with citrate, which has been reported to be effective for recovery from fatigue in the conventional furrows.

Brief Description of Drawings

[0009] [Fig. 1] Fig. 1 is a graph showing changes in body weight during the breeding period of rats of a control group and each sample administration group.

FIG. 2 is a graph showing the swimming time of rats in the control group and each sample administration group.

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] Crocetin used as a raw material in the present invention has the formula

[Chemical 4]

It is a compound represented by these. This crocetin is usually obtained by hydrolyzing crocin (a digentiobiose ester of crocetin), which is a carotenoid yellow pigment. Crocin is the power of gardenia augusta MERRIL var. It is used well.

[0011] In the present invention, there is no limitation on the method for extracting crocin as well as the above-mentioned plant basic power

The dried fruit strength of crushed gardenia can also be extracted using water, alcohol (eg, methanol, ethanol, etc.), or a mixture thereof. For example, when water / alcohol mixture is used, the extraction condition is preferably about 1 to 18 hours at room temperature (about 0 to 30 ° C) to 50 ° C, and about 2 to 4 hours at about 30 to 40 ° C. Is more preferable. The extraction operation is usually repeated several times.

[0012] The hydrolysis of crocin may be carried out in accordance with a conventional method, and is usually acid, alkali or suitable. It is performed by the action of a proper hydrolase. Examples of the acid include hydrochloric acid, sulfuric acid, and phosphoric acid, and examples of the alkali include sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate. Examples of hydrolases include β-darcosidase.

[0013] Industrially, crocin is preferably hydrolyzed with alkali.

The hydrolysis may be performed with stirring and stirring or heating. Preferably, the mixture is heated to about 20 to 70 ° C., preferably about 40 to 60 ° C. with stirring for about 1 to 24 hours, preferably about 3 to 5 hours. In this way, the hydrolysis can be accelerated by hydrolysis.

[0014] When the hydrolysis of crocin is an alkali hydrolysis, an appropriate amount of an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid such as cuenic acid is usually added to the reaction solution after completion of the hydrolysis. The liquid is adjusted to a pH of about 4.0 or less, preferably about pH 1.0 to 3.0, or the reaction solution is made of an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, or quenate, for example. In addition to an aqueous solution of an organic acid such as the above, crocetin is precipitated by adjusting the liquidity to a pH of about 4.0 or less, preferably about pH 1.0 to 3.0. Thereafter, the crocetin can be recovered as a paste-like solid by centrifuging the mixed liquid in which crocetin is precipitated or filtering through a filter paper or a filter cloth.

[0015] When the hydrolysis of crocin is hydrolysis with an acid, crocetin usually precipitates simultaneously with the hydrolysis, so that the reaction solution is obtained as a suspension. After completion of the reaction, the resulting suspension is centrifuged or filtered through a filter paper or a filter cloth to recover crocetin as a pasty solid.

[0016] The crocetin (pasty solid) obtained as described above usually has acid, neutralized salt and impurities derived from the raw material attached to the surface of the solid, so that the impurities are removed. A cleaning process is performed for the purpose. The treatment may be performed using a known method, for example, washing the pasty solid with a sufficient amount of water. Next, using, for example, a shelf-type ventilation dryer or a vacuum dryer, the solid material, for example, washed with water, is preferably dried at a temperature not exceeding about 50 ° C in an atmosphere of nitrogen gas, and remains in the solid material. Remove water. [0017] However, even the crocetin thus obtained is produced by hydrolysis and other substances such as lipids and their degradation products, polyphenols such as chlorogenic acid, and the like. It is preferable to further purify the crocetin since it may contain saccharides such as glucose and gentiobiose that are not completely removed. Here, the method for purifying the crocetin is not limited, and known methods such as column chromatography and recrystallization are used.

[0018] The crocetin used in the present invention is preferably crocetin having a purity of about 70% by mass or more, and preferably crocetin having a purity of about 90% by mass or more, more preferably about 95% by mass or more. Most preferably. Here, the purity of crocetin is calculated based on the color value of pure crocetin. The color value is a value measured by [Color value measurement method] described in Examples below.

In the present invention, pharmacologically acceptable salts of crocetin include, for example, alkali metal salts such as sodium and potassium, alkaline earth metal salts such as magnesium and calcium, pyridine, dimethylamine, jetylamine, ethanol And pharmaceutically acceptable salts of organic amino compounds such as min.

[0020] Since crocetin, which is effective in the present invention, has an anti-fatigue effect, it is used as an anti-fatigue agent containing the crocetin or a pharmacologically acceptable salt thereof as an active ingredient, or for the purpose of preventing or recovering from fatigue. It is useful as a product.

[0021] The anti-fatigue effect of crocetin can be evaluated by the method described in Non-Patent Document 1, for example. In other words, when rats are kept under water immersion restraint, it is impossible to take sufficient sleep and resting postures for rats that avoid water, and they cannot always rest mentally and physically. It becomes. The body weight of rats under such conditions is measured daily, and the presence or absence of an anti-fatigue effect is evaluated by comparing the fluctuations in body weight between the rats in the control group and the samples.

[0022] Fatigue level can be evaluated by measuring swimming time in a state where a weight is loaded on a rat. As a result of evaluating the degree of fatigue by this method, crocetin showed an excellent anti-fatigue effect as shown in Examples described later.

[0023] The anti-fatigue agent and anti-fatigue food and drink of the present invention are the crocetin or a pharmacologically acceptable salt thereof as it is or a pharmaceutically acceptable additive, food material, food. The raw material of the product and, if necessary, food additives and the like are mixed as appropriate, and are prepared as preparations such as liquids, powders, granules, tablets, microcapsules, soft capsules or hard capsules and foods and drinks according to conventional methods. In addition, a crocetin-containing composition such as an oil or fat composition mainly composed of crocetin or a pharmacologically acceptable salt thereof, an oZw type emulsion, a wZo type emulsion or a solubilized solution is produced according to a conventional method. The crocetin-containing composition may be added to a food or drink to produce the food or drink of the present invention.

[0024] The food and drink may take any food form such as a solid food, a cream-like or jam-like semi-solid food, a gel food, and a beverage. Examples of food and drink include soft drinks, drops, candy, chewing gum, chocolate, gummi, yogurt, ice cream, pudding, jelly confectionery, cookies, margarine, shortening, mayonnaise and dressing. The above food and drink are intended for the prevention or recovery of specified health foods with a label indicating that they are used for the prevention or recovery of fatigue, or the accumulation of fatigue and various illnesses that accompany it. Useful as a health food.

[0025] Examples of additives, food materials, food raw materials and food additives used in the preparation of the above-mentioned preparations and foods and beverages include excipients (for example, lactose, dextrin, corn starch, crystalline cellulose, etc.), lubricants (Eg, magnesium stearate, sucrose fatty acid ester, glycerin fatty acid ester, etc.), disintegrant (eg, carboxymethylcellulose calcium, anhydrous calcium hydrogen phosphate, calcium carbonate, etc.), binder (eg, starch paste, hydroxypropyl) Cellulose fluid, gum arabic fluid, etc.), solubilizers (eg, gum arabic, polysorbate 80, etc.), sweeteners (eg, sugar, fructose, glucose liquid sugar, honey, aspartame, etc.), colorants (eg, j8—force Mouth lotions, food tar pigments, riboflavin, etc.), preservatives ( For example, sorbic acid, methyl oxybenzoate, sodium sulfite, etc.), thickeners (for example, sodium alginate, sodium carboxymethylcellulose, sodium polyacrylate, etc.), acid-detergents (for example, BHT (dibutylhydroxytoluene)) , BHA (dibutylhydroxyl-sol), ascorbic acid, tocopherol, etc.), fragrance (for example, heart force, strawberry fragrance, etc.), acidulant (for example, citrate, lactose, DL-malic acid, L-apple) Acid), seasonings (eg DL-alanine, L-alanine, 5'-sodium inosinate, sodium L-glutamate), emulsifiers (eg Lysine fatty acid esters, sucrose fatty acid esters, etc.), pH adjusters (eg, citrate, trisodium citrate, etc.), vitamins, minerals, amino acids and the like.

[0026] In the case of the above-mentioned preparation, the content of crocetin or a pharmacologically acceptable salt thereof varies depending on its purpose and use, and is not uniform, but the purity is 100% by weight with respect to the total weight of the preparation. in terms of, usually about 0.0001 to 50 weight ^_0/0, preferably from about 0.001 to 20 wt%, more preferably about 0.01 to 10 wt%.

[0027] In the case of the above food and drink, the content of crocetin or a pharmacologically acceptable salt thereof is usually about 0 in terms of crocetin having a purity of 100% by mass with respect to the total mass of the food or drink. . 00003-10 weight ^_0/0, preferably <is about 0.01 to 5 weight ^_0/0.

[0028] When the above-mentioned preparations and foods and drinks are taken orally, the daily dose of crocetin or a pharmacologically acceptable salt thereof is about 0.1 l in terms of crocetin with a purity of 100% by mass. It is in the range of ˜500 mg, preferably about 1 to 200 mg, more preferably about 2 to 50 mg. This dose may be taken in one or several divided doses. However, the actual dose should be determined in consideration of the purpose and the situation of the intaker (gender, age, health status, etc.).

[0029] Further, in the case of parenteral preparations such as injections and infusions, about 0 mass% of crocetin or a pharmacologically acceptable salt thereof is converted into crocetin with a purity of 100% by mass per day for an adult. Olg ~: LOOmg, preferably about 0.1 to 50 mg, more preferably about 0.1 to 15 mg.

[0030] Further, the anti-fatigue agent of the present invention can be used in combination with other drugs and supplements intended to recover from fatigue. Examples of such drugs and supplements include vitamins (eg, vitamin B, vitamin B, vitamin B, vitamin B, vitamin E, etc.), amino acids (eg,

1 2 6 12

Taurine, funoles noretiamine, norin, leucine, isoleucine, glutamine, anoleginine, L—cystine, etc.), glucuronolatathone, ursodeoxycholic acid, glucuronic acid, gamma oryzanol or garlic extract components (eg And oxoamidin powder).

[0031] Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited thereto. Example

[0032] [Preparation of crocetin]

To 300 g of the dried fruits of ground gardenia, 600 mL of a methanol / water mixed solution [(1: 1) (v / v)] was added and stirred at room temperature for 3 hours, followed by suction filtration. Add 600 mL of methanol / water mixture ((1: 1) (vZv)) to the extraction residue, stir at room temperature for 30 minutes, and then perform suction filtration twice to obtain a total of about 1800 mL of extract as filtrate. . This extract was concentrated at ₆₀ ° C. under reduced pressure using a rotary evaporator to obtain about lOOg of a concentrate containing crocin (color value = 573).

The obtained concentrate was mixed with 34 g of a 40 mass% sodium hydroxide aqueous solution and subjected to a hydrolysis reaction at 50 ° C. for 3.5 hours with stirring. After the completion of the reaction, the reaction solution was acidified by adding it to 40 mL of a 4% by mass phosphoric acid aqueous solution, and then allowed to stand at room temperature for about 3 hours.

The precipitated precipitate is recovered by centrifugation (10,000 X g, 10 minutes), further washed with 200 mL of water and centrifuged twice, and the resulting pasty solid is obtained at 50 ° C for 8 hours. Vacuum dried for hours.

The above method was repeated 5 times, and the resulting crystals were collected into one to obtain about 12 g of crude crocetin. The color value of this was about 12,500.

[0033] [Preparation of purified crocetin]

To about 10 g of the crude crocetin obtained above, 180 mL of dimethylformamide was added and dissolved at 80 ° C. Insoluble matter was filtered through quantitative filter paper (No. 5C, Advantech Toyo Co., Ltd.), and the filtrate was allowed to stand at 10 ° C for 3 days. Next, the mother liquor containing the produced crocetin crystals is filtered through a glass filter No. 3, washed with 200 mL of methanol, and the crystals are vacuum-dried at 50 ° C. to obtain a purified crocetin product (hereinafter referred to as purified crocetin). 1. 6g was obtained.

The above method was repeated 20 times, and the obtained crystals were collected into one to obtain about 32 g of purified crocetin. The color value of this product was 34,200.

[0034] [Purity of purified crocetin]

The purified crocetin was further recrystallized from dimethylformamide, and the resulting crocetin crystals (color value 35, 700) were designated as “pure crocetin”, and the purity of the purified crocetin was expressed by the following formula. Calculated. [Equation 1] Purity of purified crocetin = (color value of purified crocetin / color value of pure crocetin) X 1 00 The calculated purity of purified crocetin was 95.8%.

The color value was measured by the following method with reference to “Voluntary Standards for Food Additives Other than Chemical Synthetic Products (Second Edition)”, edited by Japan Food Additives Association, “Kuchinashi Ajiri”.

[0035] [Color value measurement method]

Weigh accurately the sample so that the absorbance to be measured is in the range of 0.3 to 0.7, and dissolve in Kolthoff's buffer (50 mM Na CO—50 mM Na B O, pHlO. 0).

2 3 2 4 7

Set to OmL. If it is difficult to dissolve, dissolve by sonication. Weigh exactly 10 mL and add Kolthoff buffer solution (50 mM Na CO—50 mM Na B O, pHlO. 0).

2 3 2 4 7

Make 50 mL to make the test solution. Mr. Kolthoff buffer (50 mM Na CO—50 mM

twenty three

Na B O, pHlO. 0) as a control, with a liquid layer length of lcm and a maximum absorption area around 420 nm.

2 4 7

Measure the absorbance A and determine the color value using the following formula.

[0036] [Numeric 2] ι ο «(A X 2 5 0)

Color value (E,) II

Sample collected (g)

[0037] [Test example]

The submerged rats were allowed to swim with a weight loaded, and the effect of sample administration on the swimming time was examined. At the same time, a group that did not receive the sample as a control was also tested.

(a) Experimental materials

(1) Sample

Crocetin administration group sample: Purified crocetin (color value: 34, 200) Weigh accurately 20.00 g, pulverize in an agate mortar, add 0.5 mass% sodium carboxymethylcellulose aqueous solution little by little, and mix accurately. The test solution was 10 mL.

Quenic acid administration group sample: Quenic acid (special grade reagent; Wako Pure Chemical Industries, Ltd.) 20.00 g was accurately weighed and dissolved by adding physiological saline to make exactly lOOmL as a test solution. Here, the crocetin administration group sample was used in the examples of the present invention, and the citrate administration group sample was used in the comparative example.

[0038] (2) Experimental animals

5-week-old male SD rats (SPF) were preliminarily raised for 7 days and then subjected to the experiment. Rats are kept in the SPF barrier breeding room (lighting time from 8:00 to 18:00, ventilation rate: 18 times Z) at room temperature 24 ± 3 ° C and relative humidity 55 ± 15% throughout the pre-breeding period and experimental period. Each of the solid feed (MF) and sterilized distilled water was ingested freely.

[0039] (b) Experimental method

(1) Normal breeding period

Based on the body weight data at the start of the study, rats were divided into groups of 5 animals by random sampling by rank. The day of grouping was defined as dayO, and the sample solution was administered by oral gavage once a day from dayO to day6 at a rate of lmL per lOOg of rat body weight. During this time, the body weight, food intake and water intake of the rats were measured daily.

[0040] (2) Water immersion period

From day 7 to day 12, the cage was filled with water (water temperature 23 ± 3 ° C) to a depth of 1.5 cm, and the rats were raised so that they could not rest. (The sample solution was administered by oral gavage once a day from 1 & 7 to (1 & 12 at a rate of lmL per lOOg of body weight of the rat. The last day of administration was 30 minutes before the swimming test. Food intake and water intake were measured daily.

[0041] (3) Weight load swimming test

A weight swimming test was performed on dayl2. Put water (water temperature 23 ± 1 ° C) to a height of 40cm in a tank, tie a weight of 8% by weight of the weight to the base of the tail with a string, and forcibly swim the rat. Time was measured.

[0042] (c) Results

The measurement results of body weight are shown in FIG.

During the normal breeding period, the rats in the crocetin-administered group (Example) showed a steady increase in body weight, and the rate of increase was greater than that in the control group. The rats in the citrate group also showed a steady increase in body weight, and the rate of increase was similar to that in the control group.

During the water immersion period, the rats in the crocetin-administered group (Example) lost a small amount of body weight. Although a small trend was observed, it did not significantly decrease during the period. On the other hand, weight loss was observed in rats in the control group and the citrate-treated group. As a result of testing the difference in the mean value between the control group and the crocetin-treated group by Dunnet method, the difference was statistically significant on both days (risk rate 5). %)Met. This result shows that the anti-fatigue agent of the present invention is useful for fatigue during insomnia.

[0043] Based on the measurement results of the swimming time, the average difference between the control group and each administration group was tested by the Dunnet method, and the results are shown in FIG.

The rats in the crocetin-administered group (Example) recorded a swimming time of 144.8 ± 37.2 seconds, compared to the rats in the control group (swimming time; 65.6 ± 12.4 seconds). It was statistically significant (risk rate 5%). On the other hand, the rats in the kenic acid group recorded a swimming time of 84.4 ± 14.4 seconds, and the swimming time was prolonged compared with the rats in the control group, but the difference was not statistically significant. I helped.

This result shows that the anti-fatigue agent of the present invention is useful for fatigue operation. Industrial applicability

[0044] According to the present invention, it is possible to provide an anti-fatigue agent and food and drink that are effective in preventing or recovering from fatigue. Crocetin or a pharmacologically acceptable salt thereof used in the present invention has an excellent anti-fatigue effect as compared with citrate, which has been reported to be effective for recovery from fatigue in the conventional furrow.

Claims

The scope of the claims

Expression

An anti-fatigue agent comprising crocetin represented by the formula or a pharmacologically acceptable salt thereof as an active ingredient.

Expression

[Chemical 2]

Or a pharmacologically acceptable salt thereof as an active ingredient.

[3] The food or drink according to claim 2, which is labeled as being used for prevention or recovery of fatigue.

[4] To produce foods and drinks aimed at preventing or recovering from fatigue,

Expression

[Chemical 3]

Or a pharmacologically acceptable salt thereof.