JPH07108217B2 - Tochu's plant tissue culture callus - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel plant tissue culture callus derived from a plant tissue piece of Eucommia ulmoides.

[Prior Art and its Problems] Most of the Du Chinese medicines currently used as crude drugs are naturally grown or cultivated in the mainland of China, etc.
Adult trees about 20 years old are felled, the bark is peeled off, and the resulting skin is used as a medicinal raw material.

However, it is hard to find a natural Tochu, and the tree age is 20.
Growing an old mature tree requires a vast area and a long time, and in addition, a great deal of labor is required for cutting trees and removing bark.

The plant tissue culture callus derived from the plant tissue piece of Tochu, if it itself contains a medicinal active ingredient present in the bark of Tochu, grows this derived callus,
By separating and collecting the medicinal active ingredient of Tochu from the obtained callus, it is possible to obtain the active ingredient of Tochu in-vitor without spending the above-mentioned enlarged area, great labor and long time. .

The present invention has been made from the above viewpoints, and an object thereof is to provide a plant tissue culture callus containing the medicinal active ingredient of Tochu.

[Means for Solving Problems] To achieve the above object, the plant tissue culture callus of Tochu according to the present invention is characterized in that it is formed by culturing a plant tissue piece of Tochu and contains a medicinal active ingredient of Tochu. To do.

Here, as the part of the plant tissue piece of Tochu to be cultured,
Since it is necessary to use an organ that can easily secure a large amount of material (explant), the current year branch (long branch), especially its stem node and epicotyl, are often used. However,
The site to be cultured is not limited to these.

The culture of the plant tissue piece of Kakanaka consists of so-called induction culture in which callus is induced from the plant tissue piece and so-called proliferation culture in which the induced callus is propagated. Induction culture of callus is preferably carried out in a solid medium such as agar, and growth culture of callus is preferably carried out by repeated subculture in a liquid medium.

As a solid medium, an ordinary medium for callus induction culture can be used. For example, a composition based on B-5 (Gamborg) 1966 shown in Table 1 or WPM (Woody plant medium) 1982 shown in Table 2 can be used. As the (growth regulating substance), a substance to which auxin and cytokinin are added is often used. As auxins, NAA (naphthalene acetic acid), IAA (indole acetic acid), 2,4-D (2,4-dichlorophenoxyacetic acid), CPA (4-chlorophenoxyacetic acid), IBA (indole butyric acid), etc. are often used. .
Further, as cytokinins, 6-BA (N ⁶ -benzyladenine), kinetin (N ⁶ -furfurylaminopurine), 2ip (N ⁶ -r, r-dimethylallylaminopurine),
Zeatin and the like are often used. As the liquid medium, an ordinary medium for callus growth culture can be used, and, for example, a composition obtained by omitting agar from the composition of the above solid medium is often used.

In the callus induction culture and the callus clonal growth culture, the culture temperature is usually preferably 25 ° C. ± 2 ° C., and in the growth culture, a medium in which plant hormones are added to the base composition of the liquid medium at a plurality of concentration stages. Is used to carry out subculture several times to several tens of times in units of about 1 month.

Of the callus induction culture and the callus clonal expansion culture, at least the latter culture is preferably performed in the light. This photopic culture can be performed for 10 to 24 hours, 10
It is a culture method by placing the culture under light irradiation of 0 to 20000 lux, preferably 1000 to 10000 lux.

However, neither the culture conditions nor the composition of the medium are limited.

Thus, the plant tissue culture callus derived / proliferated from the plant tissue pieces of Tochu is a medicinal active ingredient present in the bark of Tochu, especially pinoresinol di-O-β-D-glucoside (Pinoresinol) having a remarkable antihypertensive effect. −di−
O-β-D-glucoside). This ingredient, for example, was found in Tochu plant tissue culture callus (150.6 g) at about 0.
Contains 45 mg (It is known that this component contains about 0.04% in the bark of Tochu.) In addition, Aucubin, Syringin, Liriodendrin are also present in this callus.
Etc. are included. Isolation and immobilization of the above-mentioned components of callus can be performed by solvent extraction, thin layer chromatography (TL
C), nuclear magnetic resonance spectrum (NMR), ultraviolet absorption spectrum (UV), infrared absorption spectrum (IR), mass spectrum (MS) and the like.

EFFECTS OF THE INVENTION According to the present invention, it was found that the plant tissue culture callus derived from the plant tissue pieces of Tochu contains the medicinal active ingredient present in the bark of Tochu. . Therefore, by proliferating this induced callus, and separating and collecting the medicinal active ingredient of Tochu from the obtained callus, a vast area, a great amount of labor and labor as in the case of collecting the medicinal active ingredient from the mature tree of Tochu. The active ingredient of Tochu can be obtained in-vitro without spending a long time. Therefore, the callus according to the present invention is an extremely useful substance as a material for crude drugs.

Further, in this invention, the plant tissue culture callus derived from the plant tissue piece of Tochu, since it is obtained by using the current-year branch instead of the adult tree of Tochu, as a raw material, the mature tree about 20 years old is felled, The conventional troublesome work of peeling off the bark and using the obtained skin portion as a raw material is not necessary.

Moreover, it is difficult to find a natural Tochu tree, and the tree age is 20.
A large area and a long time are required to grow an old mature tree, but the present invention can solve such a problem.

[Embodiment] Next, the present invention will be specifically described with reference to its embodiment.

a) Formation of callus Carrying seedlings of 3 years old into a greenhouse, cutting the growing current year's branches to a length of about 30 cm, and cutting this cut piece with 2% antiformin (sodium hypochlorite solution) With
Sterilization was performed by rotating for 20 minutes. Cut this sterilized product into a number of small pieces with a length of about 3 cm using pruning scissors, and place these pieces on a solid medium in a test tube (pear tree)
did. This solid medium is B-5 (Gamborg) 1 shown in Table 1.
On 966 agar medium, 0.5 mg / NAA (naphthalene acetic acid)
It was prepared by adding 0.6 mg / 6-BA (N ⁶ -benzyladenine), respectively. The culture temperature was maintained at 25 ° C ± 2 ° C. In this way, Toshinaka's callus was induced in the test tube.

Next, this induced callus was clonally propagated by liquid culture. That is, using a 300 ml conical flask, as a liquid medium, the plant hormone was added at a concentration of 15 steps to the composition obtained by omitting agar from the composition of the solid medium.
Using the medium of the 15th section, continuous subculture was performed 5 times in units of about 1 month. This culture was performed at a temperature of 25 ° C ± 2 ° C for 16 hours,
It was carried out by a light culture in which the culture was placed under light irradiation of 3000 lux. Thus, the callus of Tochu was clonally propagated by liquid culture.

b) Analysis of callus-containing components Tochu plant tissue culture callus thus obtained (150.6 g)
According to the flow sheet shown in FIG. 1, methanol extraction, suspension of the extract in water, polystyrene resin MCI gel column chromatography of the water-soluble product, and the silica of fractions Fr.2 and Fr.3 were analyzed. Sequentially applied to gel column chromatography, fraction F
r.3-2c (4.5 mg) was obtained. Also, CHCl ₃ : MeOH: H ₂ O = 7: 2.
The fraction Fr.3 was subjected to silica gel thin layer chromatography using a developing solvent of 5: 0.2. The developed state is shown in FIG.

In the developed state of FIG. 2, with respect to fractions Fr.1-1 and Fr.2-1, their Rf values by silk gel thin-layer chromatography and the color reaction by heating in a 20% sulfuric acid aqueous solution were tested for By comparison with that, fraction Fr.1-1 was identified as aucubin,
Fraction Fr.2-1 was identified as Syringin.

Regarding the fraction Fr.3-2c, its Rf value by silica gel thin layer chromatography, the ¹ H-NMR spectrum shown in FIG. 3, and the ¹³ C-NMR spectrum shown in FIG. 4 should be compared with those of the authentic sample. By this fraction Fr.3
-2c was identified as Liriodendrin.

Fraction Fr.3-2c contained 100.5,111.0,115.8,1118.3,135.0,14 as shown in the ¹³ C-NMR spectrum in FIG.
Substances showing weak signals are included in 6.0, 149.1, etc. By comparing the positions of these signals, that is, the chemical shift values, with those of the standard product, the above-mentioned contained substance was identified as pinoresinol di-O-β-D-glucoside. Liriodendrin and pinoresinol di-O-β-D- in the crystals of fraction Fr.3-2c (4.5 mg)
Since the content ratio with glucoside is about 10: 1, Tonochu's medicinal active ingredient, pinoresinol di-O-β-D-glucoside, is contained in Tochu's plant tissue culture callus (150.6 mg) at about 0.45 mg. It is supposed to be.

[Brief description of drawings]

The drawings show an embodiment of the present invention. Fig. 1 is a flow sheet showing a method for analyzing callus, Fig. 2 is a developed state of a fraction Fr.3 by silica gel thin layer chromatography, and Fig. 3 is a flow chart. Shows a ¹ H-NMR spectrum, and FIG. 4 shows a ¹³ C-NMR spectrum.

Front Page Continuation (72) Inventor Yasumi Kamino 1-6-14 Edobori, Nishi-ku, Osaka City, Osaka Prefecture Hitachi Shipbuilding Co., Ltd. (56) References Biological Abstracts Vol. 74 No. 13122

Claims (1)

[Claims] 1. A plant tissue culture callus of Tochu, which is formed by culturing a plant tissue piece of the current branch of Tochu and contains a medicinal active ingredient of Tochu.