JPH0759196B2 - Production method of lichen components by lichen plant tissue culture - Google Patents

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a method for producing a lichen component by lichen plant tissue culture, and more particularly to a method for culturing an undifferentiated symbiont derived from a lichen plant tissue to produce a lichen component.

[Prior Art] Lichen plants are a group of plants which are symbiotic organisms composed of certain fungi and algae and which occupy a unique botanical position. Moreover, when the lichen plant is observed under a microscope, the internal structure of the lichen plant is the skin layer (the outermost tissue of the lichen body that protects the lichen body, hyphae are gathered and fused with each other), and the algal layer ( Hyphae surrounding and holding the algae that make up the lichen), medulla (the mycelium is loosely intricate and is the basis of the lichen), false roots (protruding from the lower cortex of the lichen, lichen) It has a very large structural characteristic. (However, false roots may not have arisen from the lower cortex.)

The so-called lichen component, which is a metabolite of lichen plants, is completely different from the components of many higher and lower plants, and is a chemically special limited department. According to Asahina's classification, the following lichen components are listed (Asahina and Shibata, "Chemistry of Lichen Components," Kawade Shobo (194
8 years))).

A. Aliphatic Lichen Components Group 1: Acids a. Monobasic lactone acid, b. Dibasic acids, c. Tribasic acids Group 2: Neutral substances exhibiting Liebermann reaction (Zeoline compounds) Group 3: Polyhydric alcohols B. Aromatic lichen components Group 1: Pulvinic acid derivatives Group 2: Depsides a. An orthine-based compound, b. Ortin / Betalutin mixed compound, c. Betaertine compounds Group 3: Depsidones a. An orthine-based compound, b. Betaertine compounds Group 4: Quinones a. An oxyanthraquinone derivative, b. Phenanthrenequinone derivatives Group 5: Xanthone derivatives Group 6: Diphenylene oxide derivatives Group 7: Nitrogen-containing compounds (diketopiperatin derivatives) C.I. Carbohydrate Group 1: Polysaccharides

More specifically, the following compounds are typical examples of the lichen component. I. Depsidones Valiolaric acid, norstictic acid, pannarin, fusodaric acid, fisodoic acid, psoromoic acid, fumarprotocetralic acid, loparic acid, α-colatolic acid, serratic acid, nutictic acid, alectronic acid. Procisetralic acid etc. II. Depsides Hypotharnolic acid, Tamunolic acid, Ponic acid, Homolithic acid, Palpatic acid, Palpatolic acid, Microfilinic acid, Spherophorin, Petramic acid, Difructaic acid, Atranoline, Lecanoic acid, Antiacetic acid, Eperic acid, Optusate acid, Oripetolic acid, Peomyces acid, perlatriphosphoric acid, tenioline, unpyricaric acid, etc. III. Diphenylene oxides Strepticillin, didymic acid, usnic acid, calicin, pinastrinic acid, lysocarpic acid, purpinic acid (methyl purpinate), etc. V. Anthraquinones Rhodocladonic acid, endocrotin, parietin, etc. VI. Phenoanthrenequinones, such as telephoric acid. VII. Fatty acids Rangiformic acid, caperate acid, etc. VIII. Triterpene Zeolin, ursolic acid, etc. IX. Tetronic acids, such as protricesteric acid. X. Xanthones such as thiophanic acid.

Regarding the physiological significance of the lichen component, since the growth of the lichen plant is slow, it is a defense against the attack of microorganisms and the feeding of small animals, and unlike other fungi, it grows in the sun. Therefore, it is considered to be protection against ultraviolet rays. Therefore, since ancient times, lichen components have been used for applications resulting from these functions. For example, dyes, antibiotics, fragrances and the like.

However, in addition to the slow growth of lichen plants, their growth is likely to be restricted by the natural environment such as season, climate, temperature and latitude, and the artificial environment such as sulfur dioxide concentration and soot and smoke concentration. Natural cultivation is very difficult and unsuccessful. Moreover, since many lichen plants have very similar outer shapes and completely different components, it requires skill in selecting materials, and therefore it is difficult to collect them from nature. On the other hand, there is an attempt to separate fungi and algae constituting the lichen body and to produce lichen components from the separated fungi (Yoshimura, "Primary Color Japanese Lichen Botanical Encyclopedia", page 319, nursery company). However, in this method, the effect produced by the symbiosis of algae and fungi is ignored, so the desired lichen component may not be produced.

Recently, studies on plant tissue culture have been conducted as a method for producing plant components. Plant tissue culture
Compared to natural plants that grow yearly or monthly, it grows at a much faster rate, so it is possible to produce the target ingredients in a short time, and unlike natural cultivation, the effects of weather etc. It has the advantage that it does not receive a lot of labor and does not require much labor for collection, and that it allows planned production on an industrial scale.However, although research on artificial culture of lichen plants is ongoing, There are no reports of success.

[Problems to be Solved by the Invention] A technical problem to be solved by the present invention is to artificially and efficiently produce lichen components produced by natural lichen plants.

[Means for Solving the Problems] The inventors of the present invention have conducted various studies to solve the above technical problems, and as a result, have identified at least one fungal cell from the same individual lichen plant that has been differentiated. An undifferentiated symbiont induced by culturing a tissue part containing at least one algal cell and culturing the same retains almost the same lichen component-producing ability as a natural lichen plant, and It was found that by culturing the differentiation symbiont in a medium suitable for its growth, it can be artificially and efficiently proliferated while retaining the ability to produce lichen components, and the present invention has been completed based on these findings.

As described above, the usual culture method or plant culture method has already been applied for the purpose of artificially growing lichen plants. However, in those conventional methods, the bacterial cells are emphasized exclusively, and the growth of the bacterial cells themselves has been focused. Unlike these conventional methods, the present inventors attach importance to the relationship between fungi and algae, create a state in which both exist as an undifferentiated symbiont, and culture the tissue to obtain the desired lichen component. We have established an industrial method that produces stable, rapid, and high-yield products. That is, it is said that there is novelty in the constitution of the present invention in culturing an undifferentiated symbiont in a tissue, and because of such novel constitution, stable and rapid industrial production of a desired lichen component is possible. A huge effect has been achieved. To the best of the knowledge of the inventors, it is considered that the undifferentiated symbiont itself is a novel entity, and no attempt has been made to tissue-culture such an undifferentiated symbiont. Prior to the present invention, it was also unclear whether the lichen component could be produced appropriately.

[0012] The gist of the present invention is to culture a differentiated tissue containing at least one fungal cell and at least one algal cell collected from a lichen plant of the same individual in a medium suitable for growing the tissue. A lichen component is produced by culturing the induced undifferentiated symbiont of a lichen plant having the same ability to produce a lichen component as that of a natural lichen plant in a medium suitable for the undifferentiated symbiont to produce the lichen component. And a method for producing a lichen component, characterized in that the lichen component produced from the obtained culture is obtained.

The method of the present invention is a universal method that can be applied to various lichen plants, as is clear from the examples described later. That is, the method of the present invention, for each family of lichen plants exemplified below, is generally applicable: Telostistaceae, centipedeaceae, mossaceae, sarcogaceae, anti-mossaceae, plumaceae, candleae, Bombycidae, Amanitacephalus, Honenokeceae, Pleurotus, Bombycidae, Singingaceae, Rhizomaaceae, Helitrigolidaceae, Salamidaceae, Asterociliaceae, Syringa moss, Pleurotus genus, Pleurotus mossaceae, Pleurotus spp. , Lichinaceae, Sarcophagidae, Rhizobiaceae, Ranunculaceae, Anophylliaceae, Ranunculaceae, Acer moss, Coralliaceae, Pingoraceae, Leopardaceae, Ranunculaceae, Ranunculaceae, Rhizomiaceae, Rhizomiaceae, Pokeweed family , Matte department etc.

The term "uncultivated symbiotic organism" as used herein means a system which does not have a characteristic differentiation structure of lichen plants, but exhibits a symbiotic effect between lichen algae and lichen fungi, and at least one alga cell. A system consisting of at least one bacterial cell. The term "symbiotic effect" refers to a synergistic effect that acts between lichens and lichens and promotes the growth of both and the production of metabolites. Including move.
It is considered to be the movement of a trace amount of physiologically active substance.

[Action] The undifferentiated symbiont of the lichen plant used in the present invention is obtained by using the lichen plant as a raw material and inducing it. An example of a specific operation procedure in the case of inducing the undifferentiated symbiont from Acasarogase, which belongs to the family Lecanoridae, is as follows.

First, a lichen body of the family Acacellus gallaceae is thoroughly washed with deionized sterile water, and then cut into a suitable size into small pieces. At this time, it is necessary that the small piece contains both the alga part and the fungal part. The small pieces are placed on an appropriate medium, for example, a solid medium such as Mulanges-Coog's medium, and cultured under constant temperature conditions of 0 to 40 ° C., usually in the light.

By such culture, an undifferentiated symbiont is formed from the surface of the lichen body around the 3rd week. Therefore, this is aseptically transplanted onto a new medium having an appropriate composition, and the temperature is 0 to 40 ° C., preferably 20. Culturing is usually continued in the light at a constant temperature of ~ 35 ° C. It is preferable to suspend the undifferentiated symbiont thus obtained in a liquid medium and to carry out so-called liquid culture such as shaking culture or aeration-agitation culture in the liquid medium. This is because liquid culture is suitable for culturing on an industrial scale, and in liquid medium, the transfer of substances between lichen algae and lichen fungi in the symbiont is carried out rapidly, so that the symbiotic effect of solid culture Remarkably expressed than when used,
This is because the growth becomes active.

As the medium used for culturing the undifferentiated symbiont, a natural or synthetic, organic or inorganic medium is used.
For example, various well-known inorganic synthetic media may be used as a base, and organic substances such as nutrients, carbon sources, various natural extract substances, etc. may be added thereto within a range that does not impair the symbiotic effect. Typical examples of the above-mentioned inorganic synthetic medium include white medium, Hildebrand medium, Rinsmeier-Skoog medium, Murashige-Skoog medium and the like. In addition, those with improved composition of these media can also be used.

As the above nutrients, thiamine hydrochloride, pyridoxine hydrochloride, vitamins such as nicotinic acid, amino acids such as glycine and asparagine, and hexavalent alcohols such as inosit and sorbit may be used. As the carbon source, carbohydrates (sucrose, glucose, maltose, etc.),
Organic acids (acetic acid, etc.) and alcohols (methanol, glycerin, etc.) can be used. Examples of the various natural extract substances include casein hydrolyzate, coconut milk, yeast extract, malt extract, and the like, which may be used alone or in an appropriate combination. The photopic culture method employed in the present invention is a culture method performed by setting a culture vessel at 10,000 lux or less, and examples of the light source include sunlight, fluorescent lamps, incandescent lamps, mercury lamps and the like.

To separate and collect lichen components from the undifferentiated lichen symbiont that has been cultured and propagated as described above, a known method may be used. For example, when the lichen component is separated and collected by the solvent extraction method, an example of the operation procedure will be described below by taking the above-mentioned Acacia ogasse as an example. First, the culture solution is filtered to collect undifferentiated symbionts, and the mixture is incubated at 60 ° C for 24 hours or
Dry at 0 ° C. for 3 hours to remove water. Then
Acetone extraction is carried out by the Soxhlet extraction method, hot digestion method or cold immersion method. In this case, polar solvents other than acetone (for example, methanol, ethanol, etc.) can also be used. The resulting acetone extract is concentrated and the acetone is distilled off. Partition the concentrate between water and ethyl acetate. In this case, organic solvents other than ethyl acetate (e.g. chloroform, methylene dichloride, n-
Hexane, ethyl ether, benzene, methyl acetate, n
-Pentane, cyclohexane, petroleum ether etc.) can also be used. Then, an ethyl acetate layer and an aqueous layer are separated, and ethyl acetate is distilled off from the obtained ethyl acetate layer to obtain an ethyl acetate extract. From this ethyl acetate extract, the target lichen component usnic acid can be obtained by column chromatography or thin layer chromatography.

The usnic acid thus obtained is 20
It has a melting point of around 3 ° C., then various solvent systems, such as hexane / ether / formic acid = 130/80/20 (volume ratio, the same below), hexane / ethyl acetate / formic acid = 50/40 /
7, silica gel G thin layer chromatography with benzene / dioxane / formic acid = 90/45/4 etc.
It matches perfectly with the spot of the commercial preparation usnic acid. Further, the infrared absorption spectrum and the nuclear magnetic resonance spectrum also agree with the spectrum of the standard product. As a result, it can be identified as usnic acid.

[0022]

EXAMPLES Next, the present invention will be specifically described with reference to examples.

Example 1 Acasarogasses belonging to the family Lecanoraceae, Salogasseceae, collected in Kyoto City was cut into small pieces of about 1 cm in length, washed thoroughly with water, and then immersed in sterile distilled water several times in a sterile box. And wash. The lichen pieces of Acacia ogasse thus obtained are aseptically placed on a synthetic agar medium having the following composition. As a medium, thiamine hydrochloride 0.1 ppm, pyridoxine hydrochloride 0.5 ppm, nicotinic acid 0.5 ppm, glycine 2 ppm, and isositol 100 ppm were added to Murashige-Skoog's inorganic salt medium to adjust to pH 6.0, and agar 1.
Use a medium that has been sterilized as usual by adding 0% W / V. A small piece of Acacia ogasse placed on such a medium is cultivated at a culturing temperature of 25 ° C. under light irradiation of 2000 lux. A green undifferentiated symbiont develops around the third week. Approximately 1 g (fresh weight) of the obtained undifferentiated symbiont was transplanted into 100 ml of a liquid medium obtained by removing agar from the above medium, and cultured on a shaker at a shaking speed of 80 rpm at a culture temperature of 25 ° C. under light irradiation of 2000 lux. 1
Continue the culture for a month. After 1 month, the culture solution is filtered and about 5g
An undifferentiated symbiont (raw weight) is obtained. After grinding this with a milk needle,
A Soxhlet extractor for 3 hours of extraction with acetone for 8 hours
Repeat times. The resulting acetone extract is concentrated to about 50 ml, transferred to a separating funnel, water of the same volume and 100 ml of ethyl acetate are added, and after shaking, the ethyl acetate layer is separated. The extraction operation was repeated several times, and the collected ethyl acetate extract was concentrated to distill off ethyl acetate to obtain an ethyl acetate extract. The result of thin-layer chromatography of this ethyl acetate extract was
Shown in the figure. Solvent: hexane / ether / formic acid = 1
Color development: 30/80/20 (volume ratio): 10% by weight sulfuric acid was sprayed and then heated at 110 ° C. for 5 seconds. The presence of usnic acid, salicic acid, protocetralic acid, and norstictic acid was confirmed by comparison with a standard. In addition, in FIG. 1, the preparations a) saratinic acid, b) stictic acid, c) protocetralic acid, d) fumar protocetralic acid, e) scumarate acid, f) norstictic acid, g) thiophanic acid, h ) Usnic acid and i) calicin.

Example 2 A lichen that was collected in Hirakata City and was cut in the same manner as in Example 1 except that the Rhododendronaceae, which belongs to the family Liliaceae, was cut into small pieces of about 0.5 cm ² in width and was used. The components were obtained, and the analysis results are shown in FIG. The presence of usnic acid, protocetolaric acid and fumar protocetolaric acid was confirmed by comparison with a standard.

Example 3 The same procedure as in Example 1 was carried out except that Moechiidae moss belonging to the family Lecanophoridae was collected in Hirakata City and was cut into small pieces of about 0.5 cm ² in width, which were used. A lichen component was obtained by the analysis, and the analysis results are shown in FIG. The presence of thiophanic acid and stictic acid were confirmed by comparison with the standard.

Example 4 A lichen component was obtained in the same manner as in Example 1 except that the peduncle of Coeda red moss belonging to the family Lecanophoraceae, which was collected in Kyoto City, was used, and the analysis results are shown in FIG. .
The presence of usnic acid and scamate acid was confirmed by comparison with the preparation.

Example 5 Lichens of the order Lecanora, belonging to the family Lepidoptera, collected in Kyoto, were cut into minute pieces of about 0.5 cm ² in width, and the same procedure as in Example 1 was carried out except that this was used. The components were obtained, and the analysis results are shown in FIG. The presence of calicin, stictic acid, and norstictic acid was confirmed by comparison with a standard sample.

Examples 6 to 29 In the same manner as in Example 1, microparticles obtained from the following lichen plants were tissue-cultured, and the obtained undifferentiated symbiont was cultured in a liquid medium. The presence of the lichen component described in the known literature was confirmed in the culture. Example species name generic name gathering place 6 Matsugegoke Parmeliaceae Kyoto 7 Karakusagoke Parmeliaceae 〃 8 key Parmeliaceae Parmeliaceae 〃 9 Senshigoke Senshigoke 〃 10 Parmeliaceae Parmeliaceae 〃 11 Quick shift shy Parmeliaceae Parmeliaceae 〃 12 mud side beard lichen Usnea 〃 13 tsuzuic Rekara Tachigoke beetle Karatachigoke 〃 14 Iva Kara Tachigoke Karatachigoke 〃 15 Akahigegoke Usnea 〃 16 Tanah Merah helmet moss Kabutogoke Toyama Prefecture 17 Koganekinori Honekinori Nagano Prefecture 18 thorns beard lichen Usnea Kyoto 19 Chidji Les Mallotus Gejigeji moss Gejigejigoke 〃 20 shy black Bosi moss Kuroboshigoke Hirakata 21 Ebiragoke Kabutogoke Shiga Prefecture 22 Togehakutengoke Parmeliaceae Kyoto City 23 〃 24 Inutsumegoke Tsumegoke 〃 25 Yamahikonori Yamahikonori Furano 26 Yokowa beard lichen Usnea 〃 27 Haimatsugoke Eirantai 〃 28 Tsunomatagoke Yamahikonori 〃 29 Naga beard lichen Usnea 〃

[Effects of the Invention] As is clear from the above, the present invention makes it possible for the first time to efficiently and artificially produce lichen components produced by natural lichen plants.

[Brief description of drawings]

FIG. 1 shows a thin layer chromatogram of lichen components obtained in Examples of the present invention and that of a standard sample.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12P 17/06 7432-4B 17/18 D 7432-4B // (C12N 5/00 C12R 1:91 ) (C12P 1/00 C12R 1:89) (C12P 1/00 C12R 1:91) (C12P 1/00 C12R 1: 645) C12R 1:91)

Claims (1)

[Claims] 1. Production of a lichen component similar to that of a natural lichen plant, which is induced by collecting a small piece of a natural lichen plant containing the same individual fungal cells and an algal cell and culturing the same in a medium suitable for the growth of these cells. The undifferentiated symbiont of the lichen plant having the ability is cultured in a medium suitable for the undifferentiated symbiont to produce the lichen component to produce the lichen component, and the lichen component produced from the obtained culture is used. A method for producing a lichen component characterized by obtaining.