JPH0640828B2 - Crude drug manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention utilizes tissue-cultured flaxseed plants to obtain callus obtained by culturing flaxseed plants in order to comprehensively utilize crude drug components of plants belonging to the genus Linum. It relates to a method of harvesting and using it as a crude drug.

(b) Conventional Technology Flax (Linum usitatissimum) and its seed flaxseed have been used as ancient herbal medicines. That is, the root has been used as a complement, an active blood drug, the stems and leaves as an analgesic and a hemostatic agent, and the flax seed has been used as an anti-itch for skin and a hair-growth agent, a gastrointestinal drug, and an antidote analgesic agent. 1983 edition).

In addition, flaxseed phospholipids have a strong effect of lowering serum cholesterol level and are also effective in the prevention of arteriosclerosis (Japanese Patent Laid-Open Publication No. Sho.
60-126224).

In addition, flaxseed oil obtained from flaxseed is used for industrial purposes such as paints in Japan, but in Europe and the United States, it is used for health foods containing linolenic acid as a main nutrient source.

In this way, flax can utilize all of seeds, stems, leaves, and roots as a health food or a herbal medicine, and can also utilize the whole plant comprehensively.

(c) Problems to be solved by the invention However, flax is rarely cultivated in Japan at present, and seeds are exclusively imported to collect linseed oil. However, crude drugs obtained from imported seed flaxseed can only be obtained through a limited process, and roots, raw stems and leaves are also difficult to obtain.

The present invention is intended to provide a method for obtaining a culture containing a necessary component by tissue culture on a scale of factory production,
According to this invention, any crude drug component of flax can be obtained at any time. In other words, the active ingredient of root, the active ingredient of stem and leaf, or the active ingredient of flaxseed can be obtained separately or collectively.

In addition, there are many inventions such as Psycho (Japanese Patent Publication No. 56-52557) as a method for producing a crude drug by culturing plant tissue, but a method for culturing flax callus and producing a crude drug from the harvest is still unknown. Has not been done. Further, the technology for producing a tocopherol from a tissue culture of an oil and fat producing plant is soybean (Japanese Patent Laid-Open No. 59-154998), safflower (Japanese Patent Laid-Open No. Sho 59-154998).
No. 60-149393), etc., but a method for producing a lipid component by tissue culture of a flax plant has not yet been known.

(d) Means for Solving the Problem That is, the present invention is a method for producing a herbal medicine, which comprises culturing a flax plant in a tissue culture in an artificial medium to obtain a culture. More specifically, the tissue of a flax plant is light- or dark-cultured in an artificial medium containing auxin and cytokinin to form callus cells, and the callus cells are then exposed in the artificial medium having a component concentration of 1/2. Cultivated to differentiate into adventitious buds, or callus cells with a concentration of 1/2 and auxin-free in the artificial medium to produce differentiation-induced embryoid bodies, from which either A method for producing a crude drug, which comprises extracting a crude drug component.

Linum usitatiss is a plant of the genus Flax used in the present invention.
Examples include imum and Linum angustifolium, but are not limited thereto.

Here, the flax usually used as a crude drug will be described in detail below as an example.

A generally known method can be applied to induce callus of the genus Flax. In other words, tissue slices of flax seedlings and plants with strong growth, such as leaf, stem, shoot apex, root, flower tissue slices and seed embryos may be cultivated. Workability is good if tissue sections are taken from leaves and stems.

The medium used for culturing callus is an artificial medium based on general inorganic salts or a modified medium thereof, such as Murashige-Skoog (MS) medium, White medium, Heller medium, and Rinsmeier-Skoog (LS) medium. Any of medium, niche medium, Hyponex medium, etc.,
Further, any known medium can be used without being limited to these media, and their basic composition may be appropriately modified. The medium contains vitamins such as thiamine hydrochloride, nicotinic acid, pyridoxine hydrochloride, inositol,
Glucose as sugar, such as pantothenate and biotin
Organic substances such as sucrose and sorbitol, such as glycine, asparagine, and glutamine, can be added as amino acids as appropriate. Further, auxins such as 2,4-D, indoleacetic acid, indolebutyric acid, and naphthaleneacetic acid as plant hormones, and cytokinins such as kinetin, benzyladenine, and zeatin are appropriately combined and used,
Natural extracts such as coconut milk, yeast extract, malt extract, peptone, casein degradation product, and banana juice may be added as appropriate. These media may be liquid media or solid media containing agar.

As the culture conditions in the present invention, the temperature is 15 to 30 ° C., p
H3.5-8.5, either static or shake culture in dark or light conditions can be adopted. The light condition may be natural light or artificial light. The subculture is performed every 1 to 6 weeks according to the growth of the culture, and divided appropriately.

The fully grown culture is transferred to a differentiation induction medium to promote differentiation. That is, the concentration of auxin is decreased to increase kinetin, or plant growth hormone is removed, or the salt concentration is decreased to cause rooting or germination or form embryoid bodies.

When a callus is undifferentiated and is in a rapid growth phase when a crude drug component is produced by tissue culture, the crude drug component contained in the callus at this time is diluted and has a lower concentration than the active ingredient contained in the original plant. However, when callus is induced to differentiate, the active ingredient increases, and when the culture method is devised, the concentration becomes higher than in the original plant. In the present invention, it is preferable to culture callus of a flax plant with a differentiation-inducing medium to enhance and collect active ingredients, and when it is desired to comprehensively obtain effective lipids and all other ingredients, embryoid bodies (adventitious embryos), leaves ， When you want to get a lot of stem components, adventitious buds, and when you want to get a lot of root components, grow adventitious roots.

(e) Example Example 1 a. Callus induction After rinsing linseed seeds with running water overnight, immerse it in 70% ethyl alcohol solution for a short period of time, sterilize it by immersing it in 5% sodium hypochlorite solution for 20 minutes, wash with sterilized water, and repeat this operation again. After that, thoroughly wash with sterilized water, place the sterilized seeds on a culture pin in which sterilized 1% agar solution is solidified, and
Germinate at 5 ° C for 4 days, and then give seedlings (2 to 3 cm) under conditions of daylight and night darkness for 5 days to obtain 0.5 to 1 cm tissue pieces aseptically, and darken at 25 ° C. MS medium (in addition to predetermined inorganic salts, inositol 100, thiamine hydrochloride 0.1, nicotinic acid 0.5, pyridoxine hydrochloride 0.
5, citric acid 150 mg / each, sucrose 30 g / added, pH was adjusted to 6 and agar 9 g / added, 2,4-D1
ppm, 0.2 ppm of benzyladenine was added). Callus formation was observed in 10 days, and after 1 month, it grew to a size of 10 to 15 mm. The callus was cut off and replanted every 3 weeks, and after 2 months, callus in a rapid growth stage with vigorous growth was obtained.

b. Induction of Differentiation This callus was divided into 5 to 10 mm, placed on MS medium whose concentration was reduced to 1/2, and subjected to light culture to obtain adventitious buds. On the other hand, the embryo medium was obtained by subculturing in a liquid medium obtained by removing auxin from a 1 / 2-concentration MS medium every 3 weeks and performing light culture. In addition, callus during the rapid growth phase
(Naphthalene acetic acid 0.5 ppm as auxin, benzyladenine 0.1 ppm as cytokinin) After dark culture for 3 weeks on agar medium, the result of dark culture by subculturing every 3 weeks on 1/2 concentration MS dehormonal liquid medium , Got rooted callus.

c. Crude drug effect Collect the adventitious bud, embryoid body and adventitious root callus obtained in b,
After washing away the medium components, it was squeezed to obtain juice, and the herbal drug effects were compared, and the results shown in Table 1 were obtained.

d. Table 2 shows the phospholipids of the embryoid body obtained in Extraction Component b.

It can be seen from Tables 1 and 2 that the components of the culture are almost the same as the crude drug components of flax plants.

(f) Effect of the Invention According to the present invention, it is possible to obtain a crude drug for flax and its components, which have been difficult to produce up to now, at any time on a scale of factory production, and artificially necessary parts of flax, especially leaves. ，
The crude drug components of stem and seed are optionally obtained.

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Claims (1)

[Claims] 1. A tissue of a plant belonging to the genus Linum is lightly or darkly cultured in an artificial medium containing auxin and cytokinin to form callus cells, and the callus cells are then artificially mixed at a component concentration of 1/2. To make an adventitious bud that is differentiation-induced by light culture in a medium, or to make an embryoid body that is differentiation-induced by light-culturing the callus cell in the artificial medium having a component concentration of 1/2 and no auxin, A method for producing a crude drug, which comprises extracting a crude drug component from any one of them.