JP2007254322A - Cryptomeria japonica leaf extract and food or drink containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a Cryptomeria japonica leaf extract which is obtained by selectively extracting polyphenol compounds such as rutin from the leaves of Cryptomeria japonica, is highly safe, can easily orally be taken, and has an excellent anti-allergic action, and to provide a food or drink which contains the Cryptomeria japonica leaf extract and is suitable for treating or preventing allergic symptoms such as asthma and pollinosis, especially inflammation or the like caused by allergic diseases. <P>SOLUTION: This Cryptomeria japonica leaf extract is characterized by extracting the water extract of Cryptomeria japonica leaves with an extracting solvent containing the first organic solvent to remove ingredients soluble in the first organic solvent, and then at least twice extracting the water extract of the Cryptomeria japonica leaves with an extracting solvent containing the second organic solvent to obtain the ingredients soluble in the second organic solvents. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cedar leaf extract obtained by selectively extracting polyphenols such as rutin from cedar leaves, and a food and drink containing the cedar leaf extract.

  Allergic diseases such as asthma, hay fever, atopic dermatitis, allergic rhinitis are diseases classified as type I allergy (anaphylaxis type). In allergic reactions up to the onset of the disease, IgE antibodies, mast cells, It is known that FcR-positive cells are involved, and the IgE antibody concentration in the serum or tissues of patients with allergic diseases is known to be higher than that in healthy individuals.

  In the onset of type I allergic diseases, the antigen (allergen) -specific invasion of antigen (allergen) starts production of IgE antibody specific to the antigen (allergen), and the antigen-antibody reaction is triggered by the invasion of the antigen (allergen) again. , Chemical mediators, and Th2-type cytokines such as IL-4, IL-5, IL-6, and IL-13 are released and induce eosinophil infiltration to cause inflammation in the target tissue. .

  Conventionally, as inhibitors of type I allergic symptoms, histamine antagonists, release inhibitors, synthesis inhibitors, autonomic nerve agents, steroids, and the like are known as chemical mediators. There is a problem that side effects such as action and sleepiness occur, and as a method of treating type I allergy, there is known a desensitization therapy or the like in which an injection containing allergen as a main ingredient is regularly administered. This treatment method requires a long-term hospital visit of 2 to 3 years and has a problem that the burden is large.

  As a more efficient method of suppressing allergic symptoms, a method of suppressing IgE antibody production that occurs in the first stage can be considered. Many IgE antibody production inhibitors have been proposed so far, and not only synthetic drugs but also IgE antibody production inhibitors composed of highly safe natural plant-derived extracts have been proposed (Patent Document 1). And 2).

On the other hand, cedar ( Cryptomeria japonica D. Don) leaf extract is known to be effective in preventing hay fever (see Patent Documents 3 and 4). Leaf tea etc. are used.

The cedar leaves contain various terpenoids, flavonoids, etc. Among them, sesquiterpene, which is one of the terpenoids, has been confirmed to have antiallergic action. .
However, the terpenoids such as sesquiterpenes have a problem that they are hardly soluble in water and difficult to process as food.

In addition, in the conventional method for producing a cedar leaf extract that extracts cedar leaves with hot water, in addition to the active ingredients contained in the cedar leaves, components with low pharmacological action were also extracted. When an extract is blended into a health food or the like without a purification step, there are problems that the product value is impaired and the physiological activity is low.
Therefore, in order to efficiently ingest the active ingredient in the cedar leaf extract, it has been desired to develop a cedar leaf extract containing a large amount of the active ingredient and having an excellent antiallergic action.

JP 2003-28111 A Japanese Patent Laid-Open No. 2004-43359 JP-A-9-20671 Japanese Patent Laid-Open No. 2001-141

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention is a cedar leaf extract obtained by selectively extracting polyphenols such as rutin from cedar leaves, having high safety, easy oral intake, and excellent antiallergic action, and An object of the present invention is to provide a food or drink suitable for treatment or prevention of allergic symptoms such as asthma and hay fever, particularly inflammation due to allergic diseases, including leaf extract.

In order to solve the above-mentioned problems, the present inventors have conducted intensive studies. As a result, among the water extracts of cedar leaves, a fraction that does not substantially contain sesquiterpene and has an excellent IgE antibody production inhibitory effect is obtained. I found it.
Among the components of the cedar leaf, it has been completely known that components other than the sesquiterpene, which has conventionally been regarded as the active body of anti-allergic action of the cedar leaf, specifically inhibit IgE antibody production. This is a new finding of the present inventors.

This invention is based on the said knowledge by this inventor, and as a means for solving the said subject, it is as follows. That is,
<1> After extracting and removing components soluble in the first organic solvent using an extractant containing the first organic solvent from the water extract of cedar ( Cryptomeria japonica D. Don) leaves, A cedar leaf obtained by extracting at least twice a component soluble in the second organic solvent using an extractant containing the second organic solvent with respect to the water extract of cedar leaf It is an extract.
<2> The cedar leaf extract according to <1>, wherein the second and subsequent extractions performed using the extractant containing the second organic solvent are fractionation treatment by column chromatography.
<3> The second organic solvent according to any one of <1> to <2>, wherein the second organic solvent is at least one of ethyl acetate, a mixture of chloroform and methanol (molar ratio 2: 1), and methanol. Cedar leaf extract.
<4> Any one of <1> to <3>, wherein the content of the second organic solvent in the extractant containing the second organic solvent used for the second and subsequent extractions is 60% by mass or more. Is a cedar leaf extract.
<5> The cedar leaf extract according to any one of <1> to <4>, wherein the sesquiterpene content is 5% by mass or less.
<6> The cedar leaf extract according to <5>, wherein the first organic solvent is a solvent capable of dissolving sesquiterpene.
<7> The cedar leaf extract according to <6>, wherein the first organic solvent is at least one of n-hexane, n-pentane, petroleum ether, and ether.
<8> The cedar leaf extract according to any one of <1> to <7>, containing at least one of rutin, (−)-epigallocatechin gallate, and (−)-epigallocatechin gallate. .
<9> A food or drink comprising the cedar leaf extract according to any one of <1> to <8>.
<10> An anti-inflammatory agent comprising the cedar leaf extract according to any one of <1> to <8>.
<11> An antiallergic agent comprising the cedar leaf extract according to any one of <1> to <8>.
<12> The antiallergic agent according to <11>, which is used for any of treatment and prevention of asthma.
<13> The antiallergic agent according to <11>, which is used for either treatment or prevention of hay fever.

  According to the present invention, a cedar leaf extract, which is selectively extracted from a cedar leaf, such as rutin, is safe, easy to ingest, and has an excellent antiallergic effect, and the cedar leaf A food or drink suitable for treatment or prevention of allergic symptoms such as asthma and hay fever, especially inflammation due to allergic diseases, etc. can be provided.

(Cedar leaf extract)
The cedar leaf extract of the present invention is obtained by extracting and removing components soluble in the first organic solvent using an extractant containing the first organic solvent with respect to the cedar leaf water extract. A component soluble in the second organic solvent can be extracted at least twice by using an extractant containing the second organic solvent with respect to the water extract of cedar leaves.
The cedar leaf extract includes any one of an extract, a dried product obtained by drying a diluted solution of the extract, or a roughly purified product or a purified product thereof.

  The cedar leaf used as the raw material for the cedar leaf extract is not particularly limited as long as it is a leaf portion cut from cedar, and can be appropriately selected according to the purpose. Preferable examples are leaves cut from cedar during the month.

Here, examples of the cedar include cedar ( Cryptomeria japonica (L.fil) D. Don), Ayasugi ( Cryptomeria japonica D. Don f. Lycopodioides Hort.), And Cryptomeria japonica D. Don. var. araucarioides Henk. Et Hochst) , Oogonsugi (Cryptomeria japonica D. Don var. aurea Hort.) Okinasugi, Jindaisugi (Cryptomeria japonica D. Don var. Zindai Hort.), Taisho Tama cedar, Mankichisugi (Cryptomeria japonica D. Don f . Mankitiana Hort.), Midorisugi (Cryptomeria japonica D. Don f. viridis Hort.), Mejirosugi (Cryptomeria japonica D. Don f. albo -spicata Hort.), and Yoresugi (Cryptomeria japonica D. Don f. torta Makino) , etc. Is mentioned.

The cedar leaf that is the raw material for extraction can be any of cut, pulverized, dried, and dried pulverized cedar leaves, but is preferably dried and pulverized immediately after collection. .
Examples of the method for drying the cedar leaves include a method of drying by sun-drying after cutting, a method of heat drying using a dryer, and the like. These may be used alone or in combination of two or more. be able to. Among these, after drying in the sun, a method of drying with a dryer is preferable. For example, after drying in the sun for 48 to 72 hours, using a dryer under a temperature condition of 100 to 120 ° C. A method of drying for 30 minutes is preferred.

The water extract of cedar leaves for obtaining the cedar leaf extract is an extract obtained by extracting the cedar leaves with water, preferably a hot water extract. .
Examples of the water include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, and those subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like. Is preferred.

As extraction temperature for obtaining the water extract of the said cedar leaf, 10-110 degreeC is preferable and 100-110 degreeC is more preferable. Moreover, it is preferable to perform extraction while heating.
Moreover, as extraction time of the said cedar leaf extract, 10 minutes or more are preferable and 40 to 60 minutes are more preferable.

  The first organic solvent used for obtaining the cedar leaf extract is not particularly limited as long as it is hydrophobic and can dissolve sesquiterpene, and can be appropriately selected according to the purpose. However, for example, n-hexane, n-pentane, petroleum ether, ether and the like are preferable, and n-hexane is more preferable. These may be used individually by 1 type and may use 2 or more types together.

As the amount of the extractant containing the first organic solvent, the mixture of the extractant containing the first organic solvent and the cedar leaf water extract contains the cedar leaf water extract. The amount is preferably adjusted to be 30 to 90% by mass, and more preferably adjusted to be 40 to 60% by mass.
The mixture of the extractant containing the first organic solvent and the water extract of cedar leaves is, for example, allowed to stand for a certain period of time after mixing, so that the components soluble in the first organic solvent can be added to the first solvent. It can extract to the extractant containing the one organic solvent.
As extraction temperature for extracting the component which can be melt | dissolved in said 1st organic solvent, 15-30 degreeC is preferable and 20-25 degreeC is more preferable.
In addition, the extraction time of the component that can be dissolved in the first organic solvent is preferably 10 minutes or more, and more preferably 10 to 20 minutes.
In addition, in order to shorten the extraction time, at least one of stirring and heating of the mixture may be performed as necessary.
The extraction agent containing the first organic solvent after the extraction can be separated from the aqueous layer by a known method.

  The second organic solvent used to obtain the cedar leaf extract is not particularly limited as long as it is hydrophobic and can be appropriately selected according to the purpose. For example, ethyl acetate, chloroform and methanol (Molar ratio 2: 1) and methanol are preferred, and ethyl acetate is more preferred. These may be used individually by 1 type and may use 2 or more types together.

As the amount of the extractant containing the second organic solvent, the mixture of the extractant containing the second organic solvent and the water extract of cedar leaves contains the water extract of cedar leaves. It is preferable to adjust so that it may become 30-90 mass%, and it is preferable to adjust so that it may become 40-60 mass%.
The mixture of the extractant containing the second organic solvent and the water extract of cedar leaves is, for example, allowed to stand for a certain period of time after mixing, so that the components soluble in the first organic solvent can be added to the first organic solvent. It can extract to the extractant containing the one organic solvent.
As extraction temperature for extracting the component which can be melt | dissolved in this 2nd organic solvent using the extractant containing said 2nd organic solvent, 15-30 degreeC is preferable and 20-25 degreeC is more preferable.
Further, the extraction time of the component that can be dissolved in the second organic solvent is preferably 10 minutes or more, and more preferably 10 to 20 minutes.
In addition, in order to shorten the extraction time, at least one of stirring and heating of the mixture may be performed as necessary.
The extraction agent containing the second organic solvent after the extraction can be separated from the aqueous layer by a known method.

  The extraction performed using the extractant containing the second organic solvent is preferably performed at least twice, and for the second and subsequent times, for example, fractionation treatment by column chromatography is preferable. Specifically, the water extract of cedar leaves is adsorbed on an adsorbent such as silica gel, and unnecessary components such as sesquiterpene are removed using a solution containing the first organic solvent or a high content of the first organic solvent. Then, elution is performed using a second organic solvent or a solution containing a high content of the second organic solvent to obtain a purified product. The solvent for eluting the purified product is preferably a solution containing 60% by mass or more of the second organic solvent.

  The cedar leaf extract (cedar leaf extract) obtained as described above is subjected to treatments such as dilution, concentration, drying and purification according to a conventional method, and the diluted or concentrated solution of the extract, the extract It is good also as a dry product of these, these rough refined products, and purified products. In obtaining a dried product of the extract, a pharmaceutically acceptable carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity. In addition, the cedar leaf extract may be mixed with the carrier or any other auxiliary agent and formulated into an arbitrary dosage form such as powder, granule, tablet, etc. for easy storage and handling. it can.

The cedar leaf extract of the present invention has, as active ingredients, rutin represented by the following structural formula (1), (−)-epigallocatechin represented by the following structural formula (2), and the following structural formula (3 )-(-)-Epigallocatechin gallate represented by) and substantially free of sesquiterpene. As content of this sesquiterpene, it is preferable that it is 5 mass% or less with respect to the total mass of the cedar leaf extract of this invention.

  The cedar leaf extract of the present invention obtained as described above has at least one of an IgE antibody inhibitory action and an action of inhibiting eosinophil infiltration at an inflammatory site, and utilizes these actions. Thus, it can be used as an active ingredient of an anti-inflammatory agent or an antiallergic agent.

There is no restriction | limiting in particular as a usage method of the said cedar leaf extract, Although it can select suitably according to the objective, For example, it can use as a health food, a pharmaceutical, a quasi-drug, etc.
Moreover, it can use suitably as a component of the food / beverage mentioned later by making it contain in a general food and a general drink.

(Food)
The food and drink of the present invention is a food and drink containing the cedar leaf extract of the present invention.
Here, the food and drink are those which are less likely to harm human health and are taken by oral or gastrointestinal administration in normal social life. It is not limited to the category of goods and the like, and means, for example, a wide range of foods that are taken orally, including general foods, health foods, health functional foods, quasi drugs, pharmaceuticals, and the like.

  The food and drink of the present invention may be a blend of the cedar leaf extract of the present invention in any food and drink that does not impede its activity, or nutrition based on the cedar leaf extract. It may be a supplement.

  In producing the food and drink of the present invention, for example, sugars such as dextrin and starch; proteins such as gelatin, soybean protein and corn protein; amino acids such as alanine, glutamine and isoleucine; Arbitrary adjuvants, such as saccharides; soybean oil, fats and oils, such as a medium chain fatty acid triglyceride, can be added and it can formulate in arbitrary dosage forms.

  The food and drink that may contain the cedar leaf extract is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include soft drinks (effervescent drinks, fruit drinks, flavored drinks, flavored drinks). Syrups, lactic acid beverages, coffee beverages, tea-based beverages, soy milks, etc.), nutritional beverages, alcoholic beverages, etc. Confectionery such as ice cream, ice sherbet and shaved ice; seasonings such as sauce, sauce and spices; dairy products such as processed milk and fermented milk; salad oil, tempura oil, margarine, mayonnaise, shortening, whipping Fats and oils and processed foods such as creams and dressings; buckwheat noodles, udon, harsame, gyoza skin, cucumber skin, Chinese noodles, instant noodles Noodles; fish and livestock processed foods such as kamaboko, ham, sausage; curry, stew, oyakodon, rice bowl, miscellaneous rice, Chinese rice bowl, bonito, tempura, eel rice, hayashi rice, oden, mabodorf, beef bowl, meat sauce, egg Examples include retort pouch foods such as soup, omelet rice, dumplings, shumai, hamburger, meatballs; various forms of beverages and foods, and health and nutritional supplements.

  The addition amount of the cedar leaf extract of the present invention in the food and drink varies depending on the type of food and drink to be targeted, and cannot be unconditionally defined, but is added within a range that does not impair the original taste of the food or drink. That's fine.

  The food and drink of the present invention can be taken orally on a daily basis, and due to the action of the added cedar leaf extract, it causes allergic symptoms such as asthma and hay fever, especially allergic diseases. Inflammation and the like can be easily improved.

  The cedar leaf extract and food and drink of the present invention are preferably applied to humans, but may be applied to animals other than humans as long as the respective effects are exhibited. it can.

  Hereinafter, although the Example of this invention is described, this invention is not limited to this Example at all.

(Production Example 1)
-Manufacture of cedar leaf tea extract-
600 g of cedar leaf tea (manufactured by Kanekoen Co., Ltd.) was extracted for 40 minutes while heated in hot water to obtain 2 L of hot water extract. As shown in FIG. 1, 2 L of n-hexane was added to the obtained hot water extract and mixed, and two-layer distribution was performed to obtain an aqueous layer and an n-hexane layer. n-hexane extraction was performed three times in total. To the obtained aqueous layer, 2 mL of ethyl acetate was added, and extraction was performed three times in the same manner. Ethyl acetate was distilled off from the obtained ethyl acetate layer under reduced pressure to obtain 10.3 g of an ethyl acetate extract of cedar leaves. Got.
Next, the obtained ethyl acetate extract was dissolved in a small amount of ethyl acetate, and silica gel chromatography was performed to obtain a 10% ethyl acetate / n-hexane fraction (Fr.1), a 20% ethyl acetate / n-hexane fraction. Fraction (Fr.2), 50% ethyl acetate / n-hexane fraction (Fr.3), 70% ethyl acetate / n-hexane fraction (Fr.4), and 100% ethyl acetate fraction (Fr.3). .5) was fractionated into 5 fractions.
As a result of comparison by thin layer chromatography, as shown in FIG. 1-Fr. Among the five fractions of No. 5, in particular, 12% by mass of Fr. 4 and 39% by mass were obtained. 5 was found to be substantially free of terpenoids.

-Identification of compounds-
The obtained Fr. 4 and Fr. About 5, the HPLC analysis was performed on the following measurement conditions, and the component contained in each fraction was identified. As a result, the Fr. It was confirmed that 4 contained (-)-epigallocatechin gallate 385.9 mg and rutin 11.8 mg. The Fr. 5 confirmed that 128.9 mg of (−)-epigallocatechin gallate and 21.3 mg of (−)-epigallocatechin gallate were contained.
"Measurement condition"
HPLC column: PEGASIL-B ODS (SEN-SHU Pak) (4.6 mm × 250 mm)
Flow rate: 1.0 mL / min Column temperature: 40 ° C
Mobile phase solvent: ethyl acetate / acetonitrile / 0.06% sulfuric acid = 2/12/86 (v / v / v)

(Example 1, Comparative Example 1)
-Preparation and administration of asthma model mice-
Six 7-week-old female BALB / c mice were administered subcutaneously with a mixture of ovalbumin (OVA) 0.1 mg and aluminum hydroxide 1.6 mg on the 0th and 7th days of the study, Asthma model mice were prepared by nasally administering 0.01 mg of the above ovalbumin on the 14th, 15th and 16th days.
The asthma model mice were bred by daily oral administration of 0.5 mL of the test sample from day 0 of the test.
In addition, as a test sample, as Example 1, Fr. 4 was dissolved in an equal volume mixture of water and methylcellulose at a concentration of 3.92 g / mL. As Comparative Example 1, an equal volume mixture of water and methylcellulose was used.

-Evaluation-
The asthma model mouse was dissected on the 18th day, serum and lung lavage fluid were collected, IgE antibody titer in serum, total leukocyte count in alveolar lavage fluid, and eosinophil infiltration into alveolar cavity were measured. It measured by the following method. The results are shown in FIGS.

<IgE antibody titer in serum>
IgE antibody titer was detected by adding serially diluted serum to a 96-well plate coated with rat anti-mouse IgE heavy chain (Nihon Becton Dickinson Co., Ltd.), using biotin-labeled OVA and streptavidin-labeled HPRT. The color was developed with TMB Microwell Peroxidase (manufactured by Funakoshi Co., Ltd.), the reaction was stopped with 2N sulfuric acid, the absorbance at 450/540 nm was measured, and the obtained OD value was defined as the IgE antibody titer. The IgE antibody titer is an OD value obtained by 10-fold dilution measurement.

<Total leukocyte count in alveolar lavage fluid and eosinophil infiltration into alveolar space>
The alveolar lavage fluid was prepared by washing the lungs with 1 mL of physiological saline and collecting them. The total white blood cell count and eosinophil count in the alveolar lavage fluid were measured. The total white blood cell count is stained with Turku's solution (manufactured by Wako Pure Chemical Industries) and measured using a hemocytometer. Eosinophils are stained with Giemsa's solution (manufactured by Merck) and observed under an optical microscope. The number was measured.

  From the results of Table 1, in the asthma model mouse of Example 1 to which the cedar leaf extract of the present invention was orally administered, IgE antibody production was suppressed and infiltration of eosinophils in the alveolar lavage fluid was suppressed. I found out.

  The cedar leaf extract of the present invention is highly safe, easy to ingest, suppresses IgE antibody production by ingestion, is suitable for treatment or prevention of inflammation and allergic diseases, and is taken orally It can be widely applied to general foods, health foods, functional health foods, quasi drugs and pharmaceuticals.

1 is a process diagram for obtaining a cedar leaf extract of Production Example 1. FIG. FIG. 2 is a diagram showing the results of thin layer chromatography of the cedar leaf extract obtained from Production Example 1. FIG. 3 is a graph showing IgE antibody titers in sera of asthma model mice of Example 1 and Comparative Example 1. FIG. 4 is a graph showing the total white blood cell count in the alveolar lavage fluid of the asthma model mice of Example 1 and Comparative Example 1. FIG. 5 is a graph showing the amount of eosinophil infiltration into the alveolar space of the asthma model mice of Example 1 and Comparative Example 1.

Claims (9)

After extracting and removing components soluble in the first organic solvent using an extractant containing the first organic solvent from the water extract of the cedar ( Cryptomeria japonica D. Don) leaves, the cedar leaves A cedar leaf extract obtained by extracting at least twice a component soluble in the second organic solvent using an extractant containing the second organic solvent with respect to the water extract.   The cedar leaf extract according to claim 1, wherein the second and subsequent extractions performed using the extractant containing the second organic solvent is a fractionation treatment by column chromatography.   The cedar leaf extract according to any one of claims 1 to 2, wherein the second organic solvent is at least one of ethyl acetate, a mixture of chloroform and methanol (molar ratio 2: 1), and methanol.   The cedar leaf according to any one of claims 1 to 3, wherein the content of the second organic solvent in the extractant containing the second organic solvent used for the second and subsequent extractions is 60% by mass or more. Extract.   The cedar leaf extract according to any one of claims 1 to 4, wherein the content of the sesquiterpene is 5% by mass or less.   The cedar leaf extract according to claim 5, wherein the first organic solvent is a solvent capable of dissolving sesquiterpene.   The cedar leaf extract according to claim 6, wherein the first organic solvent is at least one of n-hexane, n-pentane, petroleum ether, and ether.   The cedar leaf extract according to any one of claims 1 to 7, comprising at least one of rutin, (-)-epigallocatechin, and (-)-epigallocatechin gallate. A food or drink comprising the cedar leaf extract according to any one of claims 1 to 8.

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