JP2703783B2 - Triterpenoid production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to the production of triterpenoids such as burionophosphoric acid contained in plants belonging to the Cucurbitaceae family such as luffa and watermelon, and the tissue of Cucurbitaceae plants using a specific medium. The present invention relates to a method for producing by tissue culture.

[Conventional technology]

Known compounds belonging to triterpenoids include plant-derived crude drug components such as saponin and glycyrrhizin, and are important compounds as pharmaceuticals or raw materials thereof. Recently, it has been revealed that plant-derived triterpenoids have various physiological activities. It is known that such plant-derived triterpenoids are contained in the leaves or roots of many plants, and among them, brionolic acid and its derivatives are contained in the roots of Cucurbitaceae plants. It is clear. This brionolic acid and its derivatives have a structure different from that of triterpenoids derived from other plants, and are expected to have a unique physiological activity, and are also attracting attention as raw materials for medicine.

However, when bryonolic acid and its derivatives are produced, when plants are used as raw materials, cultivation requires a large amount of cost and the productivity must be extremely low. Therefore, the present inventors have already carried out studies on the production of triterpenoids by loofah tissue culture and reported that loofah callus produces brionolic acid and its derivatives (Plant and Cell Physiology 25 , 1571- 1574 (1984);
Organic Magnetic Resonance 22 , 93-100 (1984)].
However, the amount was still not enough.

[Means for solving the problem]

Therefore, as a result of examining various culture conditions of calli of Cucurbitaceae plants such as luffa and watermelon, it was found that the productivity was greatly changed by hormones contained in the medium. That is, it was found that the productivity of triterpenoids was dramatically improved by reducing the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) used in the medium to 5 × 10 ⁻⁸ M or less. The invention has been completed.

That is, according to the present invention, the tissue of Cucurbitaceae plant is 2,
A method for producing triterpenoids is provided, wherein tissue cultivation is performed using a medium containing 4-dichlorophenoxyacetic acid at a concentration of 5 × 10 ⁻⁸ M or less to produce triterpenoids.

The tissue culture of the present invention is performed using a Cucurbitaceae plant, and specifically, as a corresponding plant, a loofah (Luffa cy
lindrica) and watermelon (Citrullus vulgaris).

The use ratio of 2,4-D contained in the medium used in the present invention must be 5 × 10 ⁻⁸ M or less. In the medium
When the content of 2,4-D is 5 × 10 ⁻⁸ M or more, the amount of triterpenoid produced is low even when tissue culture is performed using such a medium. Is 5 ×
Tissue culture is performed using a medium of 10 ⁻⁸ M or less. Among the above ranges, a range of 10 ⁻⁸ M or less is particularly preferable.

In the present invention, the use ratio of 2,4-D in the medium is 5%.
× 10 ⁻⁸ M or less, and by adding at least one of naphthalene acetic acid (NAA), indole acetic acid (IAA) and indole butyric acid (IBA) at a concentration of 10 ⁻⁸ M to 10 ⁻⁴ M, to obtain a triterpenoid. Can be further improved. The use ratio of hormones such as NAA, IAA and IBA is 10 ⁻⁸ M to 10 ⁻⁴ M, and within the range, 10 ⁻⁶ M to 10 ⁻⁴ M.
The range of ^-5 M is particularly preferred, and these hormones may be used alone or in combination.

As other components of the medium used in the present invention, an inorganic component and a carbon source are essential components, to which vitamins and amino acids are added as necessary.

As the inorganic components of the medium, nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, enso, iodine,
Inorganic salts containing elements such as cobalt can be given. Specifically, potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, chloride Magnesium, sodium sulfate,
Compounds such as ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride can be exemplified.

Examples of the carbon source of the medium include carbohydrates such as glucose and sucrose and derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

As the vitamins in the medium, biotin, thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide and riboflavin ( Vitamin B ₂ ) and the like.

The plant hormones in the medium include 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NA
A), indoleacetic acid (IAA), indolebutyric acid (IBA)
In addition, auxins such as derivatives thereof can be exemplified.

The medium of the present invention usually contains the inorganic component in an amount of about 0.1 μM.
To about 100 mM, about 1 g / l to about 60 g / l of the carbon source, about 0.01 μM to about 100 μM of the plant hormones, and about 0.1 mg / l to about 150 mg / l of the vitamins. It is desirable.

As the medium used for tissue culture of cucurbits of the present invention, specifically, a conventionally known medium used for tissue culture of plants, for example, Murashige Skoog ('62) [Murashige & Skoog], Linsmeier Skoog (RM-1965) [Linsmaier & Skoog], White ('63) [White], Gamborg [Gamborg] B-5, Mitsui M-9 A medium prepared by adding the plant hormone according to the present invention and the above-mentioned carbon source to the medium or the like of a niche niche (Nitsch Nitsch), and further adding the above-mentioned vitamins and the like as needed, can be exemplified. In the present invention, among these, a medium prepared using a Rinsmeier Skoog or Murashige Skoog medium is particularly preferred. The composition of the above-mentioned conventionally known medium is described in, for example, "New Plant Tissue Culture" by Takeuchi, Nakajima and Furuya, pp. 386-391, Asakura Shoten, 1979.

The medium that can be used in the present invention is a liquid medium or a solid medium containing usually 0.5 to 1% of agar, gelatin or the like, but in the present invention, it is preferable to use a liquid medium.

In the present invention, a tissue or cell of a plant belonging to Cucurbitaceae is tissue-cultured using the above-mentioned medium to obtain a cultured cell or a cultured tissue containing a triterpenoid.

As the tissue of the plant used in the tissue culture of the present invention, specifically, other than tissue pieces such as roots, leaves, stems, seeds, and flower buds of the plant, the tissue culture according to the present invention or other conventional tissue Examples include cultured cells or cultured tissues of the plant obtained by the culture method. In the present invention, among them, it is particularly preferable to use a callus or a suspension cell obtained by tissue-culturing a plant tissue in advance and culture it with a medium according to the present invention. The callus or suspension cells as a raw material are grown and cultured using the medium of the present invention to obtain callus or suspension cells containing a large amount of triterpenoids. In addition, tissues containing leaves and stems that have been differentiated from callus or suspension cells and have greenery, or hairy roots that are infected with an adventitious root or a hairy root disease fungus (eg, Agrobacterium rhizogenes) and emerged thereby can also be used.

In the present invention, as a method for separating triterpenoids from cultured cells containing triterpenoids, for example, a method described in literature [Plant and Cell Physiology 25 , 1571 to 1574 (198)
4)].

〔Example〕

Hereinafter, the method of the present invention will be described more specifically with reference to examples.

Example 1 Seeds of luffa (Luffa cylindrica) were aseptically treated, and cotyledon and stem sections were cut from aseptically germinated seedlings.
This was transplanted to Rinsmeyer Skoog's agar medium supplemented with 2,4-D to a ^{concentration} of 10 ^-6 M, and cultured in a dark place at 27 ° C. Loofah callus obtained after 5-7 weeks was 2,4-
D was added to a liquid medium of Rinsmeyer Skoog supplemented to 10 ^-7 M, and cultured with shaking at 27 ° C in a dark place, followed by subculture for 3 years or more.

100 mg (dry weight) of this callus was added to 2,4-D at 10 ^-8 M, NA
A was added to 10 ^-6 M and IAA to 10 ^-8 M, and transplanted to a 500 ml Sakaguchi flask containing 300 ml of Rinsmeier Skoog's liquid medium containing 3% glucose as a sugar source, and kept at 27 ° C. in a dark place. For 3 weeks with shaking. The obtained callus (4.2 g, dry weight) was dried, chloroform was added thereto, and the mixture was subjected to reflux extraction in a hot water bath. This was filtered and the filtrate was concentrated to give 110 mg of needle crystals of brionolic acid. The obtained crystals were confirmed to be brionolic acid by gas chromatography (Shimazu GC4C, column: 1% OV-17 1m, 27 ° C) using a sample.

Comparative Example 1 The procedure of Example 1 was repeated, except that a 500 ml Sakaguchi flask containing 300 ml of Rinsmeier Skoog's liquid medium to which 2,4-D was added to 10 ^-7 M was used. The results are shown in Table 1.

[Effects of the Invention] According to the present invention, triterpenoids,
Above all, particularly, a large amount of bryonolic acid can be efficiently produced.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication (C12P 5/02 C12R 1:91)

Claims (4)

(57) [Claims] 1. A medium containing 2,4-dichlorophenoxyacetic acid at a concentration of 5 × 10 ⁻⁸ M or less for producing triterpenoids by tissue culture of a plant belonging to Cucurbitaceae. To produce triterpenoids. 2. The culture medium further contains one or more of naphthalene acetic acid, indole acetic acid and indole butyric acid at 10 ^-8 M
2. The production method according to claim 1, wherein the addition is carried out at a concentration of ^10-4 M. 3. The plant belonging to Cucurbitaceae is Luffa cyli (Luffa cyli).
3. The production method according to claim 1, wherein the production method is ndrica). 4. The method of claim 3, wherein the triterpenoid is bryonolic acid and / or
4. The production method according to claim 1, wherein the production method is a derivative thereof.