JP2016073224A - Cultured ginseng callus and ginsenoside highly containing cultured ginseng extract, and application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide cultured ginseng callus that content of ginsenoside, which is a kind of saponin, is high, ginsenoside highly containing cultured ginseng extract extracted from the callus, and foods, medical supplies, and cosmetics.SOLUTION: Cultured ginseng callus is a cell aggregation cultured with a plant tissue culture method, and the hardness measured by a texture analyzer is 9-20 N. Use of Panax ginseng causes the cell aggregation to highly contain ginsenoside. Ginsenoside highly containing cultured ginseng extract extracted from the cultured ginseng callus also contains the ginsenoside as an active substance.SELECTED DRAWING: Figure 2

Description

The present invention relates to a cultured ginseng callus having a high content of ginsenoside, a kind of saponin, a high ginsenoside-containing cultured ginseng extract extracted from the callus, and uses thereof.

Ginseng, for example Panax ginseng CAMeyer, Panax japonicus CAMeyer, American ginseng (Panax quinquefolium L.), Panax notoginseng [Burk] FHChen, Siberian ginseng (Eleutherococcus senticosus ) Is a valuable Chinese herbal medicine and widely used. In addition, since these contain active ingredients such as saponin, panaxan, and sapogenin as secondary metabolites, the pharmacological effects of these ingredients have long since been nourished tonicity, longevity, blood glucose lowering, sedation Exciting action, diuretic action, etc. have been revealed. In addition, as active ingredients of these ginseng, panaxan, saponin, sapogenin, vitamin B group, β-sitosterol-D-o-glucoside, etc. have been reported. Particular attention has been paid.

  About the panaxan which has pharmacological effects, such as a hypoglycemic effect, it extracts from the ginseng efficiently, and the report about the ginseng extract containing many panaxanes is made. (See Patent Document 1)

  On the other hand, as an active ingredient of the above ginseng, saponin, which is a kind of glycoside, contains ginsenoside as its main component, fatigue recovery effect, mental stability effect, blood circulation promotion effect, anti-inflammatory effect, cancer cell proliferation As a functional substance with various bioregulatory actions such as inhibitory action, antithrombotic action, antidiabetic action, memory learning function improving action, etc., it is highly used such as additives added to health drinks etc. or itself as pharmaceuticals It is attracting attention as a valuable component.

  In addition, ginsenoside as a carrot saponin is classified into various types depending on the type of aglycone contained, and it has been found that the composition and content of ginsenoside vary greatly depending on the classification. Therefore, in order to provide ginsenoside stably and in large quantities, it is preferable to use cultured ginseng rather than extracting from cultivated ginseng.

  Therefore, by cultivating ginseng by a plant tissue culture method, an undifferentiated cell mass (callus) is produced, and it is proliferated to obtain cultured ginseng callus, from which ginsenoside is extracted. ing. However, although an effective amount of ginsenoside can be stably extracted, the form of ginseng callus containing a large amount of ginsenoside has not yet been clarified.

JP-A-8-245411

An object of the present invention is to provide a callus containing a large amount of ginsenoside as an active ingredient in cultured ginseng callus in view of the above-mentioned problems when ginseng callus is cultured to obtain ginseng callus. is there.

Thus, as a result of repeated studies on cultured ginseng callus containing a large amount of ginsenoside, the present inventors have found that there is a correlation between the hardness of callus and the ginsenoside content, and the present invention has been completed.
That is, the present invention provides a cultured ginseng callus characterized in that it is a cell mass cultured by a plant tissue culture method and has a hardness of 9 to 20 N as measured by a texture analyzer. It is preferable to use ginseng.

  Furthermore, this invention provides the high ginsenoside containing cultured ginseng extract extracted from the said cultured ginseng callus.

  Furthermore, the present invention provides foods, pharmaceuticals and cosmetics containing the above-described cultured ginseng callus or high ginsenoside-containing cultured ginseng extract.

According to the present invention, it is possible to obtain a cultured ginseng callus having a high content of ginsenoside, which is a kind of ginseng saponin, which is an active ingredient in medicinal ginseng, so that it can be used as a selection standard for cultured ginseng callus. Therefore, the cultured ginseng extract obtained by extracting from such cultured ginseng callus also has the effect of being able to be efficiently blended into foods such as health drinks, pharmaceuticals, and cosmetics.

It is a photograph which shows the cultured carrot callus obtained in the Example of this invention. It is a graph which shows the relationship between the hardness (N) and ginsenoside content (mg / g) of the cultured ginseng callus obtained from the various ginseng strains shown in Table 1.

Hereinafter, the present invention will be described in detail.
The cultured ginseng callus used in the present invention is obtained from a ginseng tissue such as medicinal ginseng by a normal tissue culture method.

As an example of the tissue culture method in the present invention, for example, the following method can be employed.
First, the plant tissue used as the tissue culture material is sufficiently washed. Next, the plant cells washed with water are immersed in 70% by weight ethanol for about 10 to 60 seconds to sterilize the material with alcohol, and further, 1 to 5% by weight sodium hypochlorite aqueous solution, calcium hypochlorite aqueous solution, etc. Disinfect with a disinfectant. The treatment time with the disinfectant varies depending on the size and type of the material, but it is desirable that the treatment time be in the range of 5 to 60 minutes in view of adverse effects on the cells.

The plant tissue after sterilization is sufficiently washed with sterilized water in a clean bench, then cut into sections of an appropriate size (about 10 to 20 mm) using a scalpel, and allowed to stand on a callus induction agar medium. Agar medium used for callus induction is generally a basic medium composed of major inorganic components, trace inorganic components, and sugar, with growth regulators, vitamins, amino acids, and natural extracts added as needed. Things can be used.

As the medium, a solid medium or a liquid medium can be used. Examples of the gelling agent for preparing the solid medium include agar and gellan gum.

Callus induction is generally performed under conditions of 20 to 30 ° C. There is no problem with the light irradiation under dark, weak light, or strong light, but dark or weak light is desirable from the viewpoint of growth speed. Callus is observed around 10 to 20 days after the start of culture, and callus of a size that can be subcultured can be obtained in 30 to 40 days.

  The cultured ginseng callus used in the present invention is a callus obtained from ginseng tissue or a callus obtained by growing this callus by liquid culture. In addition, the ginseng used in the present invention is not limited to the type or strain thereof as long as it belongs to the family Urugiaceae, and is not limited to Panax notoginseng, Panax ginseng, Chiksetsu. Examples of carrots known per se include ginseng (Panax japonicus CAMeyer), American ginseng (Panax quinquefolium), and Ezokogi (Acanthopanax senticosus).

  Moreover, when the cultured carrot callus obtained by the normal plant tissue culture method is used for liquid culture and proliferated, the callus can be proliferated indefinitely by liquid culture. Liquid culture conditions need not be exceptional. Examples of the medium include Murashige-Skoog medium, White medium, and Au. El. O. L. Gamborg B5 medium, Nitsch medium, Heller medium, Morel medium, and the like can be used. If necessary, additives such as casein degrading enzyme, soybean powder, corn steep liquor, and vitamins can be appropriately added to these media.

  The callus (cell group or cell mass) obtained by the liquid culture is collected by a known means such as filtration. These cells exist from a single cell as the smallest unit to a cell mass as the largest unit. The obtained cell mass does not become extremely large by shaking or stirring in liquid culture, and the maximum diameter is about 10 to 20 mm.

  The cultured carrot callus of the present invention is characterized by the hardness in the state obtained as described above. That is, the hardness of the hydrous callus (raw callus) obtained by culturing and the amount of ginsenoside contained as an active ingredient have a correlation.

  The hardness of cultured ginseng callus can be measured using a texture analyzer, and the hardness of cultured ginseng callus containing a large amount of ginsenoside is in the range of 9-20N, preferably in the range of 9-12N. It is. When the cultured carrot callus has a hardness of less than 9N, the water content in the cell tends to increase, and it is estimated that the accumulated amount of ginsenoside, which is hardly soluble, decreases. In addition, when the hardness exceeds 20 N, the water content in the cell tends to decrease, and therefore it is presumed that the activity of various synthases in the cell decreases and the amount of ginsenoside itself synthesized decreases. Absent. In addition, the hardness measurement in this invention is performed using the texture analyzer (product name: TA.XT plus) by Eihiro Seiki Co., Ltd.

The cultured ginseng extract obtained by extracting the cultured ginseng callus of the present invention contains a high content of ginsenoside, and as the ginsenoside, Rb ₁ , Rb ₂ , Rb ₃ , Rc, Rd, Re, Rf, Rf ₁ , Rg ₁ , Rg ₂ , Rg ₃ , Rg ₅ , Rk ₁ , Rk ₃ , Rh ₁ , Rh ₂ , Rh ₄ , Rg ₂ , Ro and the like are included as active ingredients. The cultured ginseng callus in the present invention contains ginsenoside as an active ingredient in an amount of 5 to 15 mg, preferably 10 to 15 mg, per 1 g of cultured ginseng callus.

  When extracting the extract from the cultured carrot callus of the present invention, the carrot callus itself after culture may be used, but the callus is sun-dried or heat-dried, preferably 30 to 80 ° C, more preferably 50 to 70 ° C. It can also be used after being gradually dried under warm air. When drying is performed at a high temperature exceeding 80 ° C., the ginseng-specific scent may be lost, or the contained active ingredients may be decomposed. When drying is performed at a low temperature of less than 30 ° C., it takes a long time to complete the drying, so there is a possibility that it may be spoiled during the drying process, but it is not necessarily limited to a drying temperature of 30 to 80 ° C. It is not a thing.

  When the cultured carrot callus is dried, the water content of the dried callus (including carrot itself) is preferably adjusted to 10% by weight or less, particularly preferably 5% by weight or less. The reason is that when the moisture content exceeds 10% by weight, the storage stability tends to decrease. However, when the storage stability is not required, the moisture content is not necessarily 10% by weight or less. Needless to say.

The cultured carrot callus prepared as described above can be directly or pulverized using a pulverizer, a stone mortar, etc., and then fractionated into a powder particle size by a sieve or the like to obtain a callus powder having a desired particle size. It can also be granulated by pulverization after freeze-drying. Thus, by making callus into powder or granules, it becomes easier to extract the active ingredient and the extraction time can be shortened, which is preferable in the manufacturing process.

  The cultured ginseng extract of the present invention is obtained by extracting from the cultured ginseng callus obtained as described above, and contains a large amount of ginsenoside as an active ingredient. Usually, when producing an extract, the cultured ginseng callus is dried as described above, and powdered or granulated as necessary, and then an extraction solvent is added to extract the ginseng extract. Examples of the extraction solvent include water and / or alcohols. The alcohols preferably include lower alcohols having 1 to 4 carbon atoms, and specifically include methanol, ethanol, propanol, isopropanol, butanol and the like. Alcohol may be used as one kind or a mixture of two or more kinds, and may be used by mixing with water at an appropriate ratio, for example, ethanol / water (50/50 weight ratio).

The amount of the extraction solvent is not particularly limited, but is usually 3 to 200 parts by weight, preferably 5 to 100 parts by weight, more preferably 10 parts per 1 part by weight of powdered or granular callus (in terms of dry matter). ~ 50 parts by weight. Below this range, extraction of the active ingredient amount as an extract becomes insufficient.On the other hand, when this range is exceeded, an increase in the extraction amount of ginsenoside cannot be expected, and the time required for filtration and concentration treatment described later becomes long. It is not efficient.

As temperature at the time of extracting an extract, it is 5-80 degreeC normally, Preferably it is 15-75 degreeC, More preferably, it is 50-70 degreeC. Extraction is possible even at a low temperature of less than 5 ° C., although the extraction time becomes longer. On the other hand, if the extraction temperature exceeds 80 ° C., the ginseng-specific fragrance may be lost or the ginsenoside contained may be decomposed. Furthermore, in order to extract, the method of performing by stirring or shaking an extraction solvent as needed other than the method of performing on stationary conditions can also be employ | adopted. The extraction time is not particularly limited and is usually 0.5 to 2 hours although it varies depending on the extraction conditions (extraction temperature and the like).

The cultured ginseng extract thus obtained contains ginsenoside at a high content, and the amount of ginsenoside extracted per 1 g of the extract solid content is usually preferably 20 mg or more.

The cultured ginseng extract of the present invention is converted into a high ginsenoside-containing cultured ginseng extract as it is or, if necessary, by further performing optional treatment such as filtration, centrifugation, and concentration. Examples of the filtration treatment include ultrafiltration using a membrane filter or filtration purification using diatomaceous earth filtration.

The cultured ginseng callus and cultured ginseng extract obtained by the present invention are prepared as they are or by carrying out formulation treatment or processing treatment with any carrier or additive to prepare pharmaceuticals, cosmetics, foods (beverages), etc. be able to. For example, carriers and additives include pharmaceutically acceptable disintegrants, excipients, storage amounts, buffering agents, swelling agents, etc., food hygiene acceptable, cosmetic ingredients, etc. It can be used, and it can be prepared in any form such as liquid, emulsion, suspension, cream, solid, powder or granule.

Hereinafter, the present invention will be specifically described with reference to examples and comparative examples. However, the present invention is not limited to these examples, and various applications can be made without departing from the technical scope. is there.

<Examples and Comparative Examples>
The cultured carrot callus of the present invention was obtained as follows. The roots of various cultivated ginseng strains were thoroughly washed with tap water, immersed in 70% by weight ethanol for 1 minute, and then immersed in a 1% by weight sodium hypochlorite aqueous solution for 30 minutes for sterilization.

Next, the sterilized cultivated ginseng root was thoroughly washed with sterilized water in a clean bench, and the vicinity of the root formation layer was collected using a cork borer. The collected sample was cut into sections having a length of about 10 mm and left on an MS agar induction medium.

In the MS agar induction medium, 2 ppm of 2,4-D as auxin, 0.1 ppm of kinetin as cytokinin and 3% by weight of sucrose are added to basic MS medium components, and the pH is 5.8 with sodium hydroxide solution. Adjusted. Thereafter, 0.9% by weight of agar was added and dissolved by heating, and 50 ml was dispensed into a 100 ml Erlenmeyer flask, and steam-sterilized at 121 ° C. for 20 minutes in an autoclave (TOMY, LBS325) was used. The culture was performed at 25 ° C. under dark conditions for about 40 days to obtain solid culture callus.

The obtained solid culture callus was selected by visual observation, and was subcultured for about 1 year in a cycle of about 28 days under dark conditions at 25 ° C. In the subculture, the culture was performed using a medium in which 2,4-D in the MS agar induction medium was replaced with indolebutyric acid. The concentration was the same 2 ppm.

Callus selected by subculture for one year as described above were transferred to a liquid medium in order to increase the amount of callus. The liquid medium was prepared by adding 2 ppm of indolebutyric acid, 0.1 ppm of kinetin and 3% by weight of sucrose to the MS medium, and adjusting the pH to 5.8 with a sodium hydroxide solution. Then, 300 ml was dispensed into a 1 liter flask and used after being autoclaved at 121 ° C. for 20 minutes in an autoclave (manufactured by TOMY, LBS325).

The culture was performed at a rate of 80 rpm on a shaker having a callus of 23 g, a culture temperature of 25 ° C., a dark place, and a shaking condition of 7 cm in amplitude. Subculture was carried out for about 1 year at a cycle of about 28 days, and cultured ginseng callus in the present invention was obtained.

As auxins used in the present specification, indoleacetic acid (IAA), indolebutyric acid (IBA), naphthaleneacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and 2, 4,5-trichlorophenoxyacetic acid (2,4,5-T) and the like. Examples of cytokinins used in the present specification include kinetin, zeatin, benzyladenine and thidiazuron.

  The cultured ginseng callus obtained as described above was measured for its hardness and ginsenoside content by the following method. The results are shown in Table 1. The relationship between the hardness and ginsenoside content in Table 1 is shown in FIG.

<Callus hardness measurement>
After the cultured ginseng callus obtained from each ginseng strain was washed with water, callus having a similar callus shape and a diameter of about 10 mm was visually selected. The callus hardness of the selected callus was measured using a texture analyzer (product name: TA.XT plus, manufactured by Eiko Seiki Co., Ltd.). Note that a 20 mmφ cylindrical plunger was used for the measurement, and the maximum load value when pushing into 50% of the callus thickness at a speed of 10 mm / sec in an environment of room temperature (about 25 ° C.) was defined as callus hardness. The number of measurement samples was n = 3.

<Measurement of ginsenoside content>
The cultured ginseng callus obtained from each ginseng strain was dried at 70 ° C. for 8 hours and finely pulverized with a coffee mill to obtain powdered callus (particle size: about 0.1 to 1 mm). After weighing 10 mg of the obtained powdered callus and adding 1 ml of 80 wt% methanol, ultrasonic extraction of ginsenoside as an active ingredient was performed using an ultrasonic cleaner (product name: T780, manufactured by Elma). did. The extraction conditions were room temperature (25 ° C.).
After completion of the extraction treatment, the mixture was filtered through a membrane filter (manufactured by Advantech Toyo Co., Ltd., average membrane pore size 0.45 μm), and the filtrate (cultured carrot extract) was LS / MS / MS (manufactured by Waters, trade name: ACQUITY UPLC H- The ginsenoside content was measured using Class).

As is clear from the results in Table 1 and FIG. 2, the NK strain, NKW strain, and NHL2 strain with low callus hardness have very low ginsenoside content, and other ginseng strains with callus hardness of 9N or more have a large amount of ginsenoside. It is clear that it contains.
From the above results, it was found that the softer the ginseng callus, the lower the content of ginsenoside as an active ingredient, and the higher the callus hardness of 9N or higher, the higher the ginsenoside content.

1 cultured carrot callus

Claims (4)

  A cultured carrot callus, which is a cell mass cultured by a plant tissue culture method and has a hardness of 9 to 20 N by a texture analyzer.   The cultured ginseng callus according to claim 1, wherein the cell mass is ginseng.   A high ginsenoside-containing cultured ginseng extract extracted from the cultured ginseng callus according to claim 1.   A food or medicine and a cosmetic comprising the cultured ginseng callus or the high ginsenoside-containing cultured ginseng extract according to claim 1 or 3.

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