JPS62210991A - Production of crude drug - Google Patents

Abstract

PURPOSE:To obtain a crude drug from an optional part in an optional period on a factory production scale, by tissual cultivation of a plant of the genus Linum in an aritificial culture medium and collecting the crude drug of Linum usitatissimum from the resultant culture. CONSTITUTION:A tissual piece, preferably budding leaf, etc., of a plant of the genus Linum, e.g. Linum usitatissimum, etc., is cultivated in an artificial culture medium, e.g. at 15-30 deg.C and 3.5-8.5pH under dark or light condition. The resultant calli are then cultivated in a differentiation inducing culture medium to grow cupules, adventitious buds. etc. Components are then extracted therefrom to afford the aimed crude drug.

Description

[Detailed description of the invention] +8) Industrial application field The present invention is directed to tissue culturing plants of the genus Linum and harvesting the resulting callus in order to comprehensively utilize the medicinal components contained in plants of the genus Linum. and how to use it as a herbal medicine.

(b) Prior Art Linum (Linum usitatissimum) and its seeds, flaxseed, have been used as herbal medicines since ancient times. In other words, the roots are used as a supplement and blood stimulant, and the stems and leaves are used as analgesics and hemostasis.
Flaxseed is also an anti-itching agent for the skin, a hair tonic, and a gastrointestinal medicine.

It has been used as a detoxifying analgesic (Yakusha's "Illustrated Encyclopedia of Japanese Medicinal Plants" 1981 edition).

Furthermore, the phospholipids in flaxseed have a strong effect of lowering serum cholesterol levels and are also effective in preventing arteriosclerosis (Japanese Patent Application Laid-open No. 126224/1983).

Furthermore, linseed oil obtained from flaxseed is used in industrial applications such as paints in Japan, while in Europe and the United States it is used in health foods whose main nutritional source is lilunic acid.

In this way, the seeds, stems, leaves, and roots of flax can all be used as health foods or herbal medicines, and it is also possible to use the entire plant body comprehensively.

(C) Problems to be Solved by the Invention However, at present, flax is hardly cultivated in Japan, and the seeds are exclusively imported to extract flaxseed oil. However, the herbal medicine obtained from imported flaxseed seeds can only be obtained through a limited number of processes, and the roots and raw stem leaves are also difficult to obtain.

The present invention aims to provide a method for obtaining a culture containing necessary components on a factory production scale by Mi weave culture.According to this invention, any crude medicinal component of flax can be obtained at any time. can. That is, it becomes possible to obtain the active ingredients of the root, the active ingredients of the stems and leaves, or the active ingredients of the flaxseed separately or collectively.

In addition, there are many inventions such as ginseng (Japanese Patent Publication No. 31917/1983) and Saiko (Japanese Patent Publication No. 52557/1983) as methods for producing crude drugs by culturing plant tissue. There is still no known method for producing crude drugs from harvested products.

In addition, a technology for producing tocopherols from tissue culture of oil-producing plants was developed by soybean (Japanese Patent Application Laid-Open No. 59-154).
998), Safflower (Japanese Unexamined Patent Publication No. 149393/1983)
However, there is still no known method for producing lipid components by tissue culture of plants of the genus Linum.

(Means for solving the problem d1, that is, the present invention is a method for producing crude drugs, which is characterized by tissue-cultivating plants of the genus Linum in an artificial medium to obtain a cultured product.

The plants of the genus Linum used in the present invention include Linumusi
tatissimum, Linum angusti
Examples include, but are not limited to, folium.

Here, flax, which is usually used as a herbal medicine, will be explained in detail below as an example.

General known methods can be applied to induce callus of the genus Linus. That is, tissue sections of budding or actively growing flax plants, such as leaves, stems, and shoot tips.

Tissue sections of roots, flowers, or seed embryos may be cultured, but it is easier to collect tissue sections from leaves and stems of sprouts, which are easiest to culture.

The medium used for culturing callus is a general artificial medium based on inorganic salts or a modified medium thereof, such as Murashige-Skoog (MS) medium, White's medium, Heller's medium, Linsmeyer-Skoog (LS). ), Etchi's medium, Hyponesox medium, etc., and any known medium can be used without being limited to these media, and the basic composition thereof may be modified as appropriate. The medium contains vitamins such as thiamine hydrochloride, nicotinic acid, pyridoxine hydrochloride, inositol,
Pantothenate, biotin, etc., glucose as a sugar,
Amino acids such as sucrose and sorbitol, as well as glycine, asparagine, and glutamine.

Organic substances such as the following may be added as appropriate. In addition, as plant hormones, auxins such as 2.4-D, indoleacetic acid, indolebutyric acid, naphthaleneacetic acid, etc., and cytokinins such as kinetin, benzyladenine, zeatin, etc. are used in appropriate combinations, and coconut milk, yeast extract, malt extract, peptone , casein decomposition products, banana juice, and other natural extracts may be added as appropriate. These media may be liquid media or solid media with agar added.

The culture conditions in the present invention include temperatures of 15 to 30°C, p.
H3,5-8.5. Either static or shaking culture in dark or light conditions may be employed. The lighting conditions may be natural light or artificial light. Transplanting is performed every 1 to 6 weeks according to the growth of the culture, and the culture is divided as appropriate.

Sufficiently grown cultures are transferred to differentiation-inducing medium to promote differentiation. That is, the concentration of auxin is decreased and kinetin is increased, or the plant growth hormone is removed, or the salt concentration is decreased to cause rooting or germination or to form embryonic bodies.

MLv! When producing crude drug ingredients by A culture, if the callus is undifferentiated and in a rapid growth phase, the crude drug ingredients contained in the callus at this stage are dilute and have a lower concentration than the active ingredients contained in the original material. However, when the callus is induced to differentiate, the active ingredient increases, and if the culture method is devised, the concentration becomes higher than that in the original material. In the present invention, it is preferable to culture the callus of plants of the genus Linum in a differentiation-inducing medium and harvest the active ingredients while increasing the amount of active ingredients. If you want to get a lot of stem ingredients, you can produce adventitious buds, and if you want to get a lot of root ingredients, you can produce adventitious roots.

(81 Examples Example 1 a. Callus-inducing flaxseed seeds were washed with running water overnight, immersed for a short time in 70% ethyl alcohol solution, then sterilized by immersion in 5% sodium hypochlorite solution for 20 minutes, and then soaked in sterile water. After repeating this operation once more, wash thoroughly with sterilized water, place the sterilized seeds on a culture bottle filled with sterilized 1% agar solution, and
Germination was carried out for 4 days at 5°C, and germination was continued for 5 days under conditions of daylight and nighttime darkness to obtain sprouts (2-3 cm), which were aseptically cut into tissue pieces of 0°5-1cffI and incubated in the dark at 25°C. By the way, MS medium (in addition to specified inorganic salts, inositol 100, thiamine hydrochloride 0.1, nicotinic acid 0.5, pyridoxine hydrochloride 0.5, citric acid 150 mg/j! each, 1!
After adding 30g/l, adjust the pH to 6, add 9g/l of agar, 2.4-Dlppm, 0°2ppm of benzyladenine.
(added with m). Callus formation was observed in 10 days, and the size grew to 10 to 15 mm after 1 month. The callus was separated and transplanted every 3 weeks, and after 2 months, a callus in the rapid growth period with vigorous growth was obtained.

Induction of b1 differentiation This callus was divided into 5-1Q mm pieces, plated on MS medium with a concentration reduced to 1/2, and cultured in the light to obtain adventitious buds. On the other hand, embryoids were obtained by subculturing every 3 weeks in a liquid medium in which auxin was removed from a 1/2 concentration MS medium and culturing in the light. Furthermore, the callus in the rapid growth stage was treated with 1/2 concentration of MS (
Naphthalene acetic acid o, sppm as auxin, benzyladenine as cytokinin 0.1 ppm) After 3 weeks of dark culture on agar medium, subcultured in MS dehormone liquid medium at 1/298 degrees every 3 weeks and dark culture. Rooted callus was obtained.

C0 herbal medicine effect Harvest each callus of adventitious buds, embryonic bodies, and adventitious roots,
After thoroughly washing away the medium components, the effects of the herbal medicines were compared, and the results shown in Table 1 were obtained.

*Duke method, the average value of each 5 times, the value is the time until bleeding stops. Filter the juice, loosely soak the filtrate in gauze, apply it lightly to the puncture site for 30 seconds after puncturing, remove it and measure. Distilled water is also used. Same.

d. Extracted components Table 2 shows the phospholipids of the embryos obtained above.

Table 2: Solvent extraction of phospholipids from embryoid bodies* Hexane extraction, acetone insoluble portion PC: Phosphatidylcholine PE: Phosphatidylethanolamine PI: Phosphatidylinositol Components of culture from Tables 1 and 2 It can be seen that it is almost equivalent to the medicinal components of flax plants.

(f) Effects of the invention According to the present invention, it is possible to obtain flax herbal medicines and their components, which have been difficult to produce up to now, at any time on a factory production scale, and also to artificially produce the herbal medicines in the necessary parts of flax. Ingredients are optionally available.

Claims (3)

[Claims] (1) A method for producing crude drugs, which comprises tissue culturing a plant of the genus Linum in an artificial medium to obtain a culture. (2) Claim No. 3 (2) Using a culture that has been induced to differentiate
The manufacturing method described in section 1). (3) The manufacturing method according to claim (1), which involves extracting and concentrating crude drug components from a culture.