JPS6034B2 - Tissue culture method for plants of the family Murasakiceae - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing the tissue of a plant of the family Murasaceae containing a naphthoquinone pigment such as shikonin.

More specifically, the present invention relates to a method for efficiently producing a large amount of naphthoquinone compounds and other useful ingredients by tissue culturing plants of the family Prunusaceae using a liquid column having a specific composition. The roots of the purple plant, a plant belonging to the purple family, include
It contains naphthoquinone-based compounds such as shikonin (R=-OH) represented by the following formula (R=-OH, -OCOCH3, etc.), and has traditionally been called "purple root" and used in Chinese medicine.

In other words, the ointment obtained by extracting shikonin and other substances from Shikonin using oils such as sesame oil is called Shiunsuga.
It is used for various skin diseases, cuts, burns, hemorrhoids, etc.
It is known to have effects such as increased vascular permeability and granulation formation. However, the medicinal ingredients such as shikonin that can be extracted from the purple root are only available in small quantities, and there are other problems such as the cultivation of purple violet takes time and depends on the natural environment and the fertility of the plant, and its stable supply is at risk. There is.

On the other hand, Mamoru Tabata, Hajime Mizukami, and others proposed the use of ``phytochemistry'' to propagate the cells and tissues of plants in the Physaceae family using tissue culture methods.
try) Volume 13, page 927, "Pharmaceutical Journal" No. 95
No. 1376 page, “Phytochemistry” (Ph
It is reported in Volume 16, page 1183 of YBchemist, Volume 17, page 95 of the same volume.

According to this method, regardless of the season or weather,
It is very advantageous because it can propagate plants of the family Murasakiceae. However, the methods disclosed in these publications all use agar as a solid medium, and are unsuitable for mass production. Therefore, the present inventors
We investigated a method of growing callus in a similar manner using a liquid tower suitable for mass production, and first, we developed a method of growing callus in the form of a liquid medium without adding agar to the medium used by Tabata et al. (Linsmeyer-Skoog medium). It was used for tissue culture of Japanese purple grass, but although the callus proliferated to some extent, the amount of pigments such as shikonin produced was small, and the amount produced also varied widely, making it impossible to secure a stable yield.

The present inventors have conducted further studies on liquid substrates that are passed through tissue culture of plants belonging to the family Murasaceae and produce large amounts of naphthoquinone compounds such as shikonin. ``We discovered that the growth is rapid, a large amount of naphthoquinone compounds are produced, there is little variation in the amount produced, and stable production can be ensured, leading to the completion of this invention.''

That is, the present invention relates to a method for culturing tissues of plants belonging to the family Prunusaceae, which is characterized by using a liquid medium containing cellulose fibers. The liquid medium used in this invention may be any of the various liquid media conventionally used for plant tissue culture, as long as it contains cellulose fibers.

That is, a normal culture medium contains a carbon source or energy source, inorganic salts, a nitrogen source, growth factors such as vitamins, and the like. Examples of carbon sources or energy sources include carbohydrates such as sucrose and their derivatives, organic acids such as fatty acids, primary alcohols such as ethanol, and amino acids such as aspartic acid. Examples of inorganic salts include calcium chloride and magnesium sulfate. , iron sulfate, calcium dihydrogen phosphate, and the like. Examples of the nitrogen source include nitrogen-containing compounds such as ammonium ions, nitrate ions, amino acids, and decomposition products of complex proteins such as beptone. Specifically, the liquid bases used in this invention include "Linsmeyer-Skoog medium, Pleides camellia, Gamborg medium, Nitsuchi Nitsuchi medium and their modified bases, and these include cellulose-based Used in combination with fiber.

Examples of cellulose-based fibers include bleached yarn, absorbent cotton, gauze, furnace paper, etc., and any material can be used as long as the material is cellulose-based, and its form is not particularly limited. Examples include short fibers, long fibers, woven fabrics, and nonwoven fabrics. The ratio of cellulose fibers in the liquid medium is about 1 to 25, especially about 10 to 25, per 1 of the liquid medium.
If it is set on the 15th evening, particularly excellent effects can be obtained. In addition, auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid,
It is preferable that a compound such as indole acetic acid is contained at a concentration of 10-7 to 10-4M, preferably 0.5 x 10-6 to 10-5M. In this case, cytokinins such as kinetin and zeatin are contained at a concentration of 10- It is suitable for the growth of callus and the production of naphthoquinone compounds to coexist in the liquid column at a concentration of 7 to 10-4M, especially 10-6 to 1.5×18-5M.

Further, nutrients such as yeast extract, malt extract, tomato juice, casamino acid, coconut milk, and vitamin mixture may be added to the liquid medium as necessary. Another preferred embodiment of the present invention includes a method of using a liquid medium in which liquid paraffin and/or fats and oils are further added to the liquid medium, thereby further improving the yield of shikonin and the like.

As the liquid paraffin, liquid paraffin specified in the 9th revised Japanese Pharmacopoeia is suitable, and as the oil or fat, both natural oil and synthetic oil can be used.

The proportion of liquid paraffin and/or oil/fat is about 10 to 1,000, particularly about 300 to 500, per night of liquid.
' is preferred.

When such liquid paraffin and/or oil is added, most of the produced shikonin pigment is transferred from the callus surface to the organic layer consisting of liquid paraffin and/or oil. Preferred examples of the tissue culture method of the present invention include the following methods.

That is, after sterilizing tissue pieces collected from the plant body of a plant belonging to the family Murasakiceae, such as seeds, growing points, leaves, stems, seeds, etc., the tissue pieces are solidified with agar or placed on a Ssmeyer-Skoog solid column. After about 7 to 30 days at 10 to 35o0,
A part of the tissue piece is turned into a callus. When the callus thus obtained is subcultured, the growth rate gradually increases and stable callus is obtained. This callus is added to the above-mentioned liquid column and shaken for tissue culture. In the present invention, light is not necessarily necessary, and culture in a feeding area is preferable for the growth of callus.

The culture temperature is preferably 10 to 35 degrees, particularly 23 to 2,800 degrees. If it is less than 1000 or more than 3500, the growth rate of callus is low.

The plants to which this invention is applied are not particularly limited to plants of the family Purple, but among them, purple
Penn mountain h ehi enemy rorhizon Sieb. e
tZucc. ) is desirable, and as mentioned above, callus containing naphthoquinone compounds such as shikonin and other medicinal ingredients can be obtained.

To extract active ingredients such as shikonin from callus, it is possible to adopt the method conventionally used for extracting purple roots.According to this invention, since a liquid medium is used, mass culture and tank culture are possible. There are two methods for separating callus from the culture medium: decantation,
It is industrially advantageous because simple operations such as furnace filtration can be employed.

Cellulose fibers can be reused after being separated from callus by an appropriate method. In addition, callus multiplies quickly, and pigments such as shikonin are produced in large amounts, with little variation in the amount produced, and a large amount of pigments can be produced stably and reliably.

Hereinafter, particularly preferred examples of the present invention will be explained in detail with reference to Examples.

However, the present invention is not limited to these Examples in any way. Examples 1 to 7 and Comparative Example 3 Into a 300ml Erlenmeyer flask, Linsmayer-Skoog medium (30% sucrose, 10-6M indoleacetic acid,
80' (including 10-5M kinetin) and the additives shown in Table 1 were added and sterilized at 120°C for 10 minutes.

After cooling, moist purple callus (obtained in advance by static culture method or liquid culture method) was placed for 1.5 days and cultured with rotation on a rotary shaker at 2500℃ for 14 days (amplitude 2).
5 side, 10 pm). After culturing, the purple callus was collected by furnace filtration, cellulose fibers were removed, and the callus was heated at 35oC.
After drying at 0 for 24 hours, the weight was measured. In addition, the content of naphthoquinic pigments such as shikonin in the dried callus or in the organic layer was measured. The measurement method is based on ``Phytochemist'' by Hajime Mizukami et al.
ry) The method described in Volume 16, pages 1185-1186 was followed.

The results are shown in Table 1 (however, each value is for liquid medium 1
It is shown as the yield per evening. )Table 1 Rice 1g each/flask added rice Rice 30 each Z-flask added rice Rice 3x3 skin woven fabric

Claims (1)

[Scope of Claims] 1. A method for cultivating tissue of plants belonging to the family Murasaceae, which comprises using a liquid medium containing cellulose fibers. 2. The tissue culture method according to claim 1, wherein the liquid medium is a liquid medium to which liquid paraffin and/or oil is added together with cellulose fibers. 3. The tissue culture method according to claim 1 or 2, wherein the plant of the family Murasakiceae is Murasaki.