WO2007004480A1

WO2007004480A1 - Method for increasing the production of secondary metabolite in plant

Info

Publication number: WO2007004480A1
Application number: PCT/JP2006/312859
Authority: WO
Inventors: Fawzia Afreen; Sayed Md. Akhter Zobayed; Toyoki Kozai
Original assignee: National University Corporation Chiba University
Priority date: 2005-06-30
Filing date: 2006-06-28
Publication date: 2007-01-11
Also published as: JP4686716B2; JP2007006778A

Abstract

Disclosed is a method for producing a large quantity of a plant-derived secondary metabolite rapidly. A method for increasing the production of a secondary metabolite in a plant comprising controlling at least one of the factors having influences on the growth of the plant after the seeding stage.

Description

Specification

Methods for increasing the production of secondary metabolites in plants

Technical field

[0001] The present invention relates to a method for increasing production of secondary metabolites in plants.

Background art

[0002] Secondary metabolism is a biochemical reaction common to many organisms, such as energy metabolism, amino acid 'primary metabolism such as protein / nucleic acid biosynthesis. ) In contrast to a limited range of organisms (Non-patent Document 1). There are few secondary metabolite that play an important role in sustaining life. On the other hand, alkaloids, terpenoids, phenols, antibiotics and pigments in various animals and plants and microorganisms. The physiological significance is not always clear! There is a case where a large amount of things are accumulated.

[0003] Various useful substances such as alkaloids, terpenoids, and flavonoids are known as secondary metabolites of plants. These substances are widely used in fields such as medicine, agriculture, and food. Plants themselves and their extracts have been developed and used as useful substances (Non-patent Document 2). However, the content in the plant body is very low, and the growth of the plant often takes several years from the late germination to the collection of secondary metabolites. In addition, the collection of plants can lead to environmental destruction, and the quality of the collected secondary metabolites is not necessarily uniform, and contamination with soil-derived harmful substances such as nickel and cadmium can occur. is there. Therefore, obtaining a sufficient amount of homogeneous and pure secondary metabolites quickly is an important issue in their use.

[0004] With the aim of resolving challenging issues, plant cell culture has been conducted since the establishment of the subculture method of plant cell culture by RJ Gotre and PR White (USA) in the late 1930s. Attempts have been made to produce the secondary metabolites of (Non-patent Document 2). A typical example of this effort is to optimize the culture conditions, which has been shown to improve productivity. Furthermore, when various chemical substances are added to the medium or stress is applied during cultivation, production efficiency is improved by creating an environment close to natural conditions. Has also been made. It has also been clarified that the production of secondary metabolites is induced by stimulation with an elicitor (Non-patent Document 2).

[0005] Methods for producing secondary metabolites by plant tissue culture can be divided into three types: cell suspension culture, organ culture, and substance conversion by fixed cells.

The suspension culture method for cells is an industrially effective method because it can be easily scaled up to a large culture tank. However, because plants produce and accumulate secondary metabolites in specific tissues, they are often not produced by culture methods using unbroken cells.

On the other hand, since organ culture uses sorted cells, it can be cultured while maintaining the ability to produce secondary metabolites. Crown gall cells generated by infection with agrobacterium (Agrobacterium tumefaciens) are a kind of tumor cells, and unlike normal callus, they can continue to grow without the addition of plant hormones. This property is effective for cell culture. Since 1985, it became clear that hairy roots induced by infection with Agrobacterium rhizogenes grew significantly faster without the addition of plant hormones and could be used for production of plant secondary metabolites. Research in the field has made significant progress.

[0006] In addition, by using an enzyme possessed by plant cells as a catalyst, substance exchange is performed to produce useful substances. At the same time as cultivating plant cells, it is possible to exchange precursors and perform substance exchange, or to continuously exchange substances by fixing cells.

[0007] However, none of the above methods sufficiently satisfy the requirements for the quantity, quality, purity and production rate of secondary metabolites. In addition, when an advanced system is used for in vitro culture using an incubator or a bioreactor, the cost becomes extremely high, and the system can be a promising method economically (Patent Document 1).

[0008] Some of the frequently used secondary metabolites of plants have pharmaceutical activity, but their production is still insufficient. For example, in Hypericum perforatum, the production of hypericin and the like is increased by adjusting the amount of CO and the amount of photons.

2

Have been reported (Non-Patent Documents 3 to 4). However, since these reports are only those using cultured tissues of Hypericum perforatum, the amount of secondary metabolites produced is extremely small and only small. [0009] In recent years, focusing on the fact that plant genetic traits are expressed by stress, secondary metabolites are induced according to the expression level, and cell functions are regulated. Some studies have attempted to solve the above problems (Non-patent Document 5). For example, Non-Patent Document 5 reveals that the production of hypericin and pseudohypericin of Hypericum perforatum increases by introducing an air flow into the in vitro system. However, since small non-tissue organisms such as callus cells were used in the methods in these studies, the amount of secondary metabolites obtained in a certain time is insufficient.

[0010] As described above, a factor that hinders mass production of plant secondary metabolites is the complexity of plant reactions to the external environment. For example, even when looking at the relationship with the amount of photosynthesis, there is a report that the production of secondary metabolites increases with an increase in the amount of photosynthesis when the light irradiation intensity is increased (Non-patent Document 4). Even in the case of cells that cannot be performed, the light irradiation intensity may increase with an increase (Non-patent Document 6).

In addition, as described above, secondary metabolites increase production in a poor environment where stress is applied to the plant, while growth of the plant body is impaired in a poor environment. The total production of secondary metabolites cannot be increased.

[0011] Patent Document 1: Japanese Patent Publication No. 2004-500053

Non-Patent Document 1: “Iwanami Biology Dictionary”, 3rd edition, Iwanami Shoten, Mar. 10, 1983, p. 957 Non-patent document 2: “Ito technique for IJ fe” ”http: //www.jpo .go.jp / shiryou / s— sonota / map / kagaku 17/2 / 2-6-1.htm

Non-patent literature 3: Zobayed et al, In Vitro Dev. Biol.- Plant. 40 (2004) 108-114 Non-patent literature 4: Briskin et al., Plant Physiol. Biochem. 39 (2001) 1075-1081 Non-patent literature 5: Zobayed et al., Plant Science 166 (2004) 333-340

Non-Patent Document 6: Zhong et al., Biotechnol. Bioeng. 38 (1991) 653-658

Disclosure of the invention

Problems to be solved by the invention

[0012] An object of the present invention is to provide a method for rapidly producing a large amount of plant-derived secondary metabolites. Means for solving the problem

[0013] As a result of intensive studies in view of the above problems, the present inventors have thought that it is desirable to apply stress at the tissue culture level and increase the production of secondary metabolites in plants. Surprisingly, we obtained the knowledge that secondary plants can increase production without giving stress to plants that have passed through the seedling stage, and completed the present invention as a result of further research. It reached to.

That is, the present invention relates to at least the following:

(1) A method for increasing the production of secondary metabolites in a plant, wherein at least one of the elements affecting the growth of the plant is controlled on the plant body that has passed through the seedling stage. Said method.

(2) The method as described above, wherein the plant is cultivated indoors.

(3) Factors affecting plant growth are photosynthetic photon flux density (PPF), carbon dioxide concentration, light wavelength, light period (including day length; the same applies to “light period”), temperature, humidity And one or more nutritional nutrients.

(4) The method described above, wherein the factors affecting plant growth are photosynthetic photon flux density and carbon dioxide concentration.

(5) The method as described above, wherein the photosynthetic photon flux density is 50 to: LOOO / z mol m ^_2 s ^_1 and the concentration of carbon dioxide is 300 to 2000 μmol mol ^_1 .

(6) The method further comprising applying stress to the plant.

(7) The method as described above, wherein the stress is UV-B irradiation.

(8) The said method whose plant is a Hypericaceae plant.

(9) The above-mentioned method, wherein the Hypericaceae plant is Hypericum perforatum.

(10) The method as described above, wherein the secondary metabolite is hypericin or a derivative thereof.

(11) The method as described above, wherein the plant is a plant belonging to the genus Glycyrrhiza.

(12) Physical strength Glycyrrhiza uralensis, 刖 § 方 fe.

(13) The method as described above, wherein the secondary metabolite is glycyrrhizic acid or a derivative thereof.

[0014] As described above, the present invention dramatically increases the production of secondary metabolites even under conditions suitable for plant growth in plants that have passed through the seedling stage, without applying stress. This is based on the knowledge that goes against the conventional common sense. The increase in production in this case also means an increase in the content of secondary metabolites in the plant rather than a quantitative increase simply due to the use of a larger plant. In addition, the growth rate of the plant in the present invention not only exceeds the growth rate in tissue culture, but also exceeds the growth rate when cultivated in the field or in a greenhouse. Therefore, the present invention does not use conventional plant tissue culture techniques, and rapidly produces high-concentration secondary metabolites in plants that have passed through the seedling stage in which the growth stage has advanced. This is possible and contributes to the low cost of producing secondary metabolites.

The invention's effect

More specifically, the present invention has the following excellent effects.

(1) The method of the present invention produces secondary metabolites in a plant by controlling at least one of the factors affecting the growth of the plant on the plant that has passed the seedling stage. The effect of increasing the production rate rapidly and reducing the production cost is brought about.

(2) Among the methods of the present invention, when the plant is cultivated indoors, it is possible to produce a secondary metabolite in the plant in a homogeneous and high-purity manner, and at the same time contribute to maintaining the environment. Also play.

(3) Among the methods of the present invention, factors affecting plant growth are one or two from photosynthesis photon flux density, carbon dioxide concentration, light wavelength, light period, temperature, humidity, and nutrient content. For those that are more than species, the production of secondary metabolites in the plant body is significantly increased.

(4) Among the methods of the present invention, if the factors affecting plant growth are the photosynthetic photon flux density and the concentration of diacid carbon, the production of secondary metabolites in the plant is synergistic. The effect which increases it automatically is produced.

(5) Among the methods of the present invention, those having a photosynthetic photon flux density of 50 to: LOOO / z molm ^ s- ¹ and a carbon dioxide concentration of 300 to 2000 mol mol ^_1 are contained in the plant body. There is an effect of synergistically increasing the production of secondary metabolites.

(6) Among the methods of the present invention, those further comprising applying stress to the plant, This has the effect of more significantly increasing the production of secondary metabolites in the plant body.

(7) Among the methods of the present invention, those in which the stress is UV-B irradiation exhibit an effect of further significantly increasing the production of secondary metabolites in the plant body.

(8) Among the methods of the present invention, when the plant is a Hypericaceae plant, the effect of remarkably increasing the production of secondary metabolites in the plant body of the Hypericaceae plant is achieved.

(9) Among the methods of the present invention, those that are Hypericaceae plant power are effective in significantly increasing the production of secondary metabolites in the plant body of Hypericum perforatum. .

(10) Among the methods of the present invention, when the secondary metabolite is hypericin or a derivative thereof, the hypericin or the derivative thereof is a hypericin or a derivative thereof having antidepressant activity or the like. There is an effect of remarkably increasing the production.

(11) Among the methods of the present invention, when the plant is a plant belonging to the genus Glycyrrhiza, the effect of remarkably increasing the production of secondary metabolites in the plant body of the genus Glycyrrhiza is achieved.

(12) Among the methods of the present invention, when the plant is Glycyrrhiza uralensis, the effect of remarkably increasing the production of secondary metabolites in the plant body of Gly cyrrhiza uralensis is exhibited.

(13) Among the methods of the present invention, when the secondary metabolite is glycyrrhizic acid or a derivative thereof, glycyrrhizic acid having antiviral activity or the like in a plant of the genus Glycyrrhiza, particularly Glycyrrhiza uralensis Or, it has the effect of significantly increasing the production of its derivatives.

Brief Description of Drawings

FIG. 1 is a diagram showing changes in weather conditions during the period when the test described in Example 1 was conducted.

FIG. 2 is a photographic diagram of Hypericum perforatum recovered in each test section in the test described in Example 1.

[FIG. 3] Leaves, stems, roots and plants of Hypericum perforatum in the test described in Example 1 It is a figure which shows the whole raw weight.

FIG. 4 is a graph showing the dry weight of leaves, stems, roots and the whole plant of Hypericum perforatum in the test described in Example 1.

FIG. 5 is a diagram showing the number of stem nodes in Hypericum perforatum in the test described in Example 1.

FIG. 6 is a graph showing the net photosynthetic rate (Pn) of Hypericum perforatum in the test described in Example 1. (A) shows a plant grown with controlled PPF and Z or CO concentrations.

2

(B) is for plants grown in a field.

FIG. 7 is a graph showing the concentrations of chlorophyll a (A) and b (B) and the chlorophyll aZb ratio (C) in the test described in Example 1.

FIG. 8 is a graph showing the concentrations of hypericin (A), pseudohypericin (B) and hyperforin (C) in leaf tissue in the test described in Example 1.

FIG. 9 is a diagram showing the correlation between the pure photosynthetic rate (Pn) and the hypericin (A) and pseudohypericin (B) concentrations in the test described in Example 1.

[Fig. 10] PPF (A) and CO concentrations (B) and total hypericity in the test described in Example 1.

2

FIG.

FIG. 11A is a photographic diagram showing an overall view after recovery of Glycyrrhiza ur alensis recovered in each test section in the test described in Example 2.

FIG. 11B is a photographic view showing the above-ground part of Glycyrrhiza ur alensis recovered in each test section in the test described in Example 2.

FIG. 12 is a photograph of the roots of Glycyrrhiza ural ensis recovered in each test plot in the test described in Example 2.

FIG. 13 shows raw and dry weights of leaves (a and b), stems (c and d), and roots (e and f) of Glycyrrhiza uralensis transplanted to a hydroponic system in the test described in Example 2. FIG.

FIG. 14 shows the raw weight and dry weight of leaves (a and b) and stems (c and d) of Glycyrrhiza uralensis transplanted to a plastic pot in the test described in Example 2. FIG. 15 is a graph showing changes in fresh root weight (a) and dry weight (b) of Glycyrrhiza uralensis transplanted to a plastic pot in the test described in Example 2.

FIG. 16 is a graph showing changes in the concentration of glycyrrhizic acid in Glycyrrhiza uralensis transplanted to a hydroponic system in the test described in Example 2 (a) or transplanted to a plastic pot (b).

FIG. 17 is a graph showing the concentration of glycyrrhizic acid of Glycyrrhiza uralensis irradiated with UV-B in the test described in Example 2.

FIG. 18 is a diagram showing the net photosynthetic rate in the test described in Example 2 when a high dose of UV-B is irradiated for 3 days.

FIG. 19 is a graph showing the net photosynthetic rate in the test described in Example 2 when irradiated with a low dose of UV-B for 15 days.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, “increasing production” means increasing production per unit time / unit area as compared with production under normal conditions in the outdoors or indoors (including indoors). To do.

“Factors affecting plant growth” refers to photosynthesis photon flux density, carbon dioxide concentration, light wavelength, light period, and other indicators that directly affect plant photosynthesis, as well as temperature and humidity. It also means other environmental indicators.

In the present invention, “adult seedling” means the final stage of the seedling stage. That is, it generally means a seedling in a stage immediately before transplantation to a stage suitable for transplantation. Therefore, “plants that have passed through the seedling stage” are plants that have passed through a stage suitable for transplantation, and are typically transplanted from a nursery bed or seedling pot to a main field or a larger pot. It means a plant body in the stage corresponding to the stage from immediately after to maturity. In addition, “plants that have passed the seedling stage” do not include cultured cells, cultured tissues, and calli. In the present invention, “stress” is desired not to occur in a normal environment. Elements that adversely affect plant growth, such as ultraviolet irradiation, insufficient soil moisture, excessive salinity, It means low air humidity, pests, dense planting, etc.

The method of the present invention provides at least one element that affects plant growth in plant cultivation. There is no particular limitation as long as the control of the seed element is performed on a plant that has passed through the seedling growing season.

Among the methods of the present invention, when the plant is cultivated indoors, the homogenous and high-purity production of the secondary metabolite in the plant body does not affect the environment, the environmental conditions, and the external origin If it is possible without being affected by factors and organisms, it is preferable because it has an effect.

Advantages when the method of the present invention is performed indoors are, for example, as follows.

1) Optimization of the environment for increased production and rapid production of plant secondary metabolites.

2) Removal or reduction of environmental, seasonal, geographic and political restrictions.

3) Realization of control of air temperature, humidity, carbon dioxide concentration and air velocity without being affected by the external environment.

4) Reduce the use of land and agricultural materials.

5) Increase in plant growth and secondary metabolite concentration.

6) Uniform quality of secondary metabolites and international standardization of product quality standards using secondary metabolites.

7) Inhibiting or promoting flowering when necessary.

8) Speed up production.

9) Universal characterization of the biochemical properties of secondary metabolites in specific plants.

10) Selection of excellent clones by genetic engineering and improvement of genetic traits.

11) Long-term preservation of excellent germ cells and realization of selected excellent germ cells by short time and low labor.

12) Avoid the use of pesticides such as insecticides, fungicides and herbicides, and avoid environmental pollution by reusing fertilizers.

13) Cultivation of genetically modified plants that are restricted to cultivation outdoors or in greenhouses.

The method of the present invention is preferably performed in a room, particularly in a building that is not easily affected by the external environment, photosynthetic photon flux density, carbon dioxide concentration, light wavelength, light period, temperature, humidity. It is more preferable that the treatment is performed in a room where one or more kinds of nutrients can be controlled. Most preferably, photosynthetic photon flux density, carbon dioxide concentration, It is performed in a room with facilities that can automatically control the wavelength, light period, temperature, and humidity of light.

[0020] Among the methods of the present invention, factors affecting plant growth include photosynthetic photon flux density, carbon dioxide concentration, light wavelength, light period, temperature, humidity, and nutrient content. If one or more of these factors affect plant growth, it is preferable because production of secondary metabolites in the plant can be significantly increased. The preferred amounts and extents of these elements are generally the preferred amounts and extents for plant growth and Z or photosynthesis.

[0021] In the present invention, preferably the photosynthetic photon flux density and concentration of carbon dioxide, their respective 50: a LOOO μ molm ^_2 s ^{_ 1} and 300~2000 μ mol mol ^{_ 1.} Further, as the light synthesis photon flux density, and more preferably ^{^{100~800 / ζ πιο1π 2 5 _ 1}} , 300~600 / ζ πιο1 m _2 s _ 1 it is even more preferred. The concentration of carbon dioxide, more preferably ^{500~1800 mol mol _ 1, 1000~1500 /} ζ πιο1 mol _ 1 is even more preferred. When the photosynthetic photon flux density and carbon dioxide concentration are within the above ranges, the production of secondary metabolites increases synergistically.

The preferred wavelength of light is 600ηπ! ˜700 nm, and the color is red. White light is next preferred. Note that the light having these colors includes not only single-color light but also multi-color light having light of these colors as a main component, that is, light having the largest proportion of these colors.

[0022] Among the methods of the present invention, the method further including applying stress to the plant is preferable because it has an effect of significantly increasing the production of secondary metabolites in the plant. Examples of stresses include, for example, ultraviolet radiation, excess or deficiency of soil moisture, excess salinity, low air humidity, natural sources such as pests, and artificial things such as dense planting.

[0023] Among these stresses, irradiation with ultraviolet rays, particularly UV-B, is more preferable because it has the effect of significantly increasing the production of secondary metabolites in the plant body. A suitable irradiation amount of UV-B is ^2.5 to 7.5 W m — ² as a total irradiation amount. As the total dose, more preferably 3. 0 to 7. OW m_ ^2, and most preferably 3. a 3~6. 5W m_ ^2. purple The number of days for irradiating the outside line may be changed within a range of 2 to 20 days. The irradiation intensity can be changed according to the change in the number of days.

[0024] Among the methods of the present invention, those in which the plant is a Hypericaceae plant are preferable because the production of secondary metabolites in the plant body of these plants is remarkably increased.

The leaves, stems, and flowers of these plants produce hypericin or its derivatives that are known to have antidepressant activity, antibacterial activity, antidiuretic activity, etc., and have recently also been known to have antitumor activity. Therefore, it has been used as a medical plant for a long time. Therefore, among the methods of the present invention, those that significantly increase the production of hypericin or a derivative thereof (such as pseudohypericin pseudonoinofolin: hyperforin etc.) in the body of a hypericaceae plant are To preferred. Hypericum perforatum (scientific name: Hypericum perforatum, English name: St Joh n's Wort) and hypericum erectum (scientific name: Hypericum erectum) are particularly preferably used.

[0025] In addition, among the methods of the present invention, when the plant is a plant belonging to the genus Glycyrrhiza, the production of secondary metabolites in the plant body of the genus Glycyrrhiza has the effect of significantly increasing. preferable. Glycyrrhiza genus plants that are Glycyrrhiza uralensis are preferred because they have the effect of significantly increasing the production of secondary metabolites in the plant body of Glycyrrhiza uralensis. Glycyrrhiza globra is also preferably used in the present invention.

The roots or rhizomes of these plants are used as a source of licorice containing glycyrrhizin having antiviral activity or the like, or as a food additive because of its sweetness. Are used respectively. Particularly in recent years, anti-HIV activity and anti-SARS activity have been found, and glycyrrhizic acid has attracted more attention. Therefore, among the methods of the present invention, those that significantly increase the production of glycyrrhizic acid or a derivative thereof in the plant body of the genus Glycyrrhiza are also preferable from the viewpoint of providing a medicine.

[Example]

[0026] In the following, the ability to explain the present invention in more detail by way of examples in any sense The present invention is not limited to these examples.

Example 1

Increased production of secondary f products in St John's Wort 1. Materials and Methods

1. 1 Plant, treatment and growth conditions

• Plant before transplanting ··· 30-day-old Hypericum perforatum seedlings (with 8 unfolded leaves) Seedling growth conditions: Closed system with artificial lighting (100 molm ^_2 _S ^_1 PPF)

Day length: 16L8D (16 hours light period 8 hours dark period)

Temperature: 27 ° C and 25 ° C in light and dark periods

[0027] · Transplantation ···· Transplanted in a plastic pot (diameter 5cm, height 7.5cm), and then left in a room with environmental condition control equipment.

Soil: Wet mixed soil (Yanmar Agricultural Machinery Co., Ltd.) 150g

Luminous intensity (PPF): 100, 300, 600 μmolm " ² s ^_1

CO concentration: 500, 1000, 1500 / z mol mol " ¹

2

Growth conditions: 7 days after 100 molm ^_2 s ^_1 PPF, 14 days after transplanting 300 μmolm " ² s ^_1

PPF, 600 molm ^_2 s ^_1 PPF 21 days after transplantation.

Day length: 16L8D (16 hours light period 8 hours dark period)

Temperature: 27 ° C and 25 ° C in light and dark periods

Relative humidity: 70 ± 5%

ΡΗ of nutrient solution 5.5

Test period ··· 45 days

Planting density · '· 178 shares m— ²

Fertilization: Hyponex (registered trademark, N: P: K = 6: 10: 5, Noyponex, Inc.) was carried out every 3 days with half the normal price.

Control: Plants cultivated in the field of Chiba University Horticultural Department on July 14 – August 28, 2004. Hyponex® was given daily as half the normal titer. Planting density is 12 ^m_2 . Changes in weather conditions are shown in Figure 1.

[0028] · Survey (1) Raw weight and dry weight were measured for stems, leaves and roots 45 days after transplantation. The number of stem nodes was also counted.

(2) Measurement of chlorophyll concentration

Porra et al. (1989), Biophysica Acta 975, 384, which was collected 45 days after transplanting the fifth leaf (0.020 ± 0.005 g) counted from the plant tip (shoot). -Based on 394.

(3) Net photosynthetic rate (Pn) measurement

The top force was the fifth, fully expanded leaf! On the 43rd day after transplantation, a portable photosynthesis system (LI-COR-6400 registered trademark, LI- COR Inc., USA) was used. .

Plants grown in a controlled environment were measured under each treatment condition (Table 1). For plants grown outdoors, the carbon dioxide concentration is 400 μmol.

At a temperature of 34 ° C (around noon on August 26, 2004) [Koo!

[0029] [Table 1] Treatment conditions for growing Hypericum perforatum in controlled environments (LL-HH) and outdoors (FC)

PPF ¹ co ₂ concentration treatment code (μιηοΐ s " ¹ ) (μιηοΐ mol" ¹ )

LL 100 500

LM 100 1000

LH 100 1500

ML 300 500

匪 300 1000

MH 300 1500

HL 600 500

H 600 1000

FC (control) 1770 ^Y 380 ^Y

zPhotosynthesis photon flux density (PPF) in an empty shelf

Y measured in the field (July 14, 2004-August 28, 2004) FPF and CC> 2 concentration

[0030] 1.2 Extraction and determination of hypericin, pseudohypericin and hyperforin Extraction and chemical analysis of hypericin, pseudohypericin and hyperforin

The method described in Zobayed et al. (2004), Plant Sci. 166, 333-340 was performed by a modified method. The outline is as follows.

The sixth and seventh fully developed leaves (fresh weight 0.25 g) were placed in yellow Eppendorf tubes (1.5 mL), immediately frozen in liquid nitrogen, and stored at 85 ° C. lmL of 2% (v Zv) DMSO in methanol was placed in each sample and ground using a MM200 (registered trademark, Retch GmbH & Co., Germany) at 30 Hz for 6 minutes. Next, centrifugation was performed at 4000 rpm (1467xg) and 4 ° C for 15 minutes (Kubota (registered trademark), Kubota Corporation). The extract (0.5 mL) was filtered through a syringe filter (Dismic-13HP, Advantech, Toyo Roshi Kaisha Co., Ltd.) and diluted with 0.5 mL of 2% (vZv) DMSO in methanol. . Half of each aliquot was used for analysis of hypericin and pseudohypericin and hyperforin.

[0031] For the analysis of hyperforin, 3.5 mL of a 2% (vZv) DMSO methanol solution was prepared, and then the extraction was performed in a dark room at room temperature.

Samples for analysis of hypericin and pseudohypericin are placed in a transparent glass vial and irradiated with a light source of 155 ± 5 / ζ πιο1π ² 5 ^_ 1ΡΡΡ (100W tungsten lamp, Toshiba) for 40 minutes. form) was converted to hypericin and pseudohypericin.

A sample of 20 μ1 extract was injected into a Phenomenex Hypersil C18 column (3.m, 4.6 mm × 100 mm) and placed in an HPLC system. The HPLC system consists of an SCL-10A system controller, a SIL-10A autoinjector, and a CTO-10A column oven (Shimadzu Corporation). For isocratic separation of analytes using 0.1 Imol L ^-1 triethylammonium acetate and acetonitrile (33:67, vZv) as mobile phase, flow rates for neutral and pseudohypericin and hyperforin, 0. 5mLmin ^{_ 1} and 1.2 were carried out with the OmLmin.

[0032] Hyperforin analysis was performed at 270 nm, and hypericin and pseudohypericin analysis at 588 nm, respectively, using an SPD-M10AV photodiode array detector. The standard curve was created using standard concentrations of pseudohybelicin (0.5, 2.5, 5 , 25 and 50 / z GML), standard concentrations (0.5 of Nono Iperishin, 2.5, 5, 25 and 50 GML ^_1) and standard concentrations of hyperforin (0.5, 2.5, 5, 25 and 50 GML ^_1) was performed using (both r ^2> 0. 99).

Quantification was performed by comparing peak areas (RT for 5.8 min, 3.5 min and 7.8 min for each) with a standard curve.

The concentration of secondary metabolites represents the dry weight of the MGG ^_1 leaf was their amount is determined by multiplying the dry weight of the total of the plant to the concentration.

[0033] 1.3 Statistical analysis

The test was performed in 10 iterations with a fully random design for 3 x 3 variables. The test was conducted twice. Statistical significance was determined by one-way analysis of variance (ANOVA) using the Sigma Stat program (Sigma (Static) V2.03, SPSS Inc-) of Windows (registered trademark). The difference in values was evaluated by the Student-Newman-Keuls test with P≤0.05.

[0034] 2. Results

Green and dry weights of Hypericum perforatum increased with increasing photosynthetic photon flux density (PPF) and Z or CO concentrations (Figures 2-4). On day 45 after transplantation, HH

2

The fresh weight and the dry weight were the highest in the treated area, 29 and 30 times that of the control area (FC), respectively. Raw weight and dry weight increased with increasing CO concentration in the case of PPF force S300 or 600 μmolm " ² s ¹ .

2

The number of stem nodes increased with increasing PPF and Z or CO concentrations (Figure 5). Stem

2

The number of nodes was the highest in the HH-treated group, about four times that in the control group.

[0035] The net photosynthetic rate (Pn) in leaves increases with increasing PPF and Z or CO concentrations.

2

(Fig. 6A), the largest in the HH treatment section. When PPF is low (100 / ζ πιο1π ² 5 ^_ , the effect of CO concentration on Pn is small force. When PPF is large (300 or 60

2

0 / ζ πιο1π ² 3 ^{_ 1} ), Pn markedly increased with increasing CO concentration. Smell in the field

2

The Pn of the plant grown in this way was less potent than the plant under controlled growth conditions (Fig. 6B). Chlorophyll a concentration was unaffected by differences in growth conditions. The concentration of chlorophyll b was the highest in the HH treatment area (FIGS. 7A and 7B). CO concentration is 1500 In the case of ^ mol mol ^_1 , the chlorophyll a / b ratio decreased with increasing PPF (Figure 7C).

) o

[0036] Hypericin and pseudohypericin concentrations (mgZplant) in leaf tissue increased with increasing PPF and CO concentrations (Figures 8A and 8B). Hypericin and

2

The concentration of pseudohypericin was highly correlated with the rate of pure photosynthesis (FIGS. 9A and 9B). That is, the concentration was 0.82 and 0.79 for R ² as a quadratic function of Pn, respectively. The concentrations of hypericin and pseudohypericin were the highest in the HH-treated group, 30 and 41 times stronger than the control group, respectively (mg g ^_1 DM).

[0037] The total hypericin concentration, which is the sum of the concentrations of rhino, ipericin and pseudonoypericin, correlates with the PPF and CO concentrations, and is an approximated curve (Figure 10A and 10B)

2

R ² = l) was obtained.

In the controlled plots, hyperforin concentration increased with increasing PPF and reached a maximum at 300 or 600 / ζ πιο1π ² 5 ^{_ 1} (Figure 8C). Overall, the concentration of hyperforin in the MH treatment group was the highest, 45 times that of the control group.

[Example 2]

Increased production of secondary metabolites in Glycyrrhiza uralensis (eg UV effects) 1. Materials and methods

1. 1 Plant, treatment and growth conditions

'Plant before transplanting ········ Glycyrrhiza uralensis seedlings with 10 days of age (with 4 unfolded leaves)

Glycyrrhiza uralensis (Fisch.) Seeds were soaked in concentrated sulfuric acid for 20 minutes, washed several times with tap water, and immediately sown in a multi-tray containing wet mixed soil (Yanmar Agricultural Machinery Co., Ltd.). After 4-5 days of germination, the tray was stored in artificial light (100 μmolm ” ² s ^{_ 1} PPF (photosynthetic effective photon flux)).

Day length: 16L8D (16 hours light period 8 hours dark period)

Temperature: 28 ° C and 26 ° C in each of light and dark periods

[0039] 'After transplanting · · ·' Select seedlings randomly and transplant them to a hydroponic system (with deep flow technique (DFT)) or plastic pot (15cm in diameter, 19cm in height). Leave in a room with environmental condition control equipment. (1) Transplanted in hydroponic system · · · Styrofoam 'sheet hole (supporting 5cm height) placed on plastic tray (length 54cm x width 39cm x depth 8cm, including nutrient solution) With a sponge for).

Planting density '··· 12 strains per train (38 strains Zm ² )

Number of trays per process · '· 3

(2) Transplanted into a plastic pot

Soil: Wet mixed soil (Yanmar Agricultural Machinery Co., Ltd.). 550g per pot.

Number of pots per process ... 30

Planting density ··· 52 strains Zm ²

[0040] Luminous intensity (PPF): 300 μmolm " ² s ^_1

Light wavelength: as shown in Table 2 below

[Table 2]

Spectral characteristics of each treatment

* Measurement of each data was performed at photosynthetic photon flux density (PPF) ΙΟθ ίπιοΙπΓ ² s- ¹ .

. Blue, green, and red mean that the main components are blue, green, and red (40-81%), respectively, and do not mean each single color.

[0041] (3) Growth conditions common to those transplanted into hydroponic systems and those transplanted into plastic pots

Day length: 16L8D (16 hours light period 8 hours dark period)

Temperature: 28 ° C and 26 ° C in each of light and dark periods

CO concentration: 1000 μmol mol " ¹

2

Luminous intensity: 100 molm " ² s ^_1 PPF for 7 days

Relative humidity: 65—70%

Fertilization: Hyponex (registered trademark, N: P: K = 6: 6: 6, Noyponex Inc.) was used as a regular titer. ΡΗ of nutrient solution

Application of nutrients · · 'Those transplanted in plastic pots were performed every 3 days. For those transplanted to the hydroponic system, replace them with new ones every 10 days, and keep the amount of nutrient solution at a frequency of once every two to three days to keep the amount of nutrient solution constant. Replenished.

[0042] Test period · · · 3 months transplanted to hydroponic system and 6 months transplanted to plastic pot.

Sample collection '·' For those transplanted into hydroponic systems, 1, 3 and 6 months after transplanting and 6 months for those transplanted into plastic pots.

[0043] (4) UV—irradiation of sputum

Growth conditions after transplanting '· · Transplanted into a plastic pot (15cm in diameter, 19cm in height). Leave in indoor experimental equipment.

Number of cultivated strains · '· 60

Other growth and nutritional conditions '·' Same as above for transplanted plastic pots where applicable

1 ^ -irradiation '· · 3 months after transplantation, the following treatments were performed.

i) High UV—B irradiation for a short period (1.13 W m ^_2 , 3 days), 300 molm ^_2 s ^_1 PPF ii) Low UV—B irradiation for a long period (0.43 W m ^_2 , 15 days), 300 μ molm " ² s ^_1 PPF iii) Control (no UV—B irradiation), 300 μmolm" ² s ^_1 PPF

The net photosynthetic rate (Pn) was also measured before and after each UV-B irradiation.

[0044] 1.2 Extraction and determination of glycyrrhizic acid

Extraction and isolation of glycyrrhizic acid was performed by a modified method described in Sato et al. (2004), Plant Sci. 166, 333-340. The outline is as follows.

Root yarn and weave (fresh weight 0.4-0.45g FM) was placed in a yellow Eppendorf tube (20mL), immediately frozen in liquid nitrogen and stored at -85 ° C.

lmL of 80% (vZv) ethanol was added to each sample and ground using a MM200 (registered trademark, Retch Gmblt & Co., Germany) at 30 Hz for 6 minutes. Next at 10, OOOrpm, Centrifugation was performed for 10 minutes (Kubota (registered trademark), Kubota Corporation). The extract was filtered through a 0.2 m syringe filter (Dismic-13HP, Advantech, Toyo Roshi Kaisha, Ltd.), and 300 μL of the aliquot was placed in a plastic HPLC autosampler vial and a 20 L sample. The glycyrrhizic acid was quantified from the peak area by HPLC.

[0045] Glycyrrhizic acid was quantified by HPLC system consisting of SCL-10A system controller, SIL-10A autoinjector, and CTO-10A column oven (Shimadzu Corporation), and SPD-M10AV photodiode array detector at 254 nm. Was used. Separation of glycyrrhizin was performed by injection onto a Phenomenex Hypersil C18 column (3. m, 4.6 mm x 100 mm). A lysocratic separation of the analyte was carried out using 0.1 mol L- ¹ sodium diphosphate and acetonitrile (65:35, vZv) as the mobile phase and a flow rate of 1. OmLmin- ¹ . A standard curve was prepared using a standard product of glycyrrhizin (Wako Pure Chemical Industries, Ltd.) (r ² > 0.99), and the amount of glycyrrhizin was determined using the standard curve.

[0046] 1.3 Statistical analysis

The test was conducted twice. Statistical significance was determined by one-way analysis of variance (ANOVA) using the Sigma Stat program (Sigma® Stat ™ V2.03, SPSS Inc. from Windows®). Differences in mean values were made by Tukey test with P≤0.05.

[0047] 2.Result

Those transplanted into plastic pots grew more vigorously and had better morphological appearance than those transplanted into hydroponic systems (Figure l la, l lb).

Three months after transplantation, the leaves and stems transplanted to the hydroponic system did not differ between the red and blue light-irradiated groups and the control (white light-irradiated group) (Figures 13a to 13d). ). On the other hand, in the fresh weight and dry weight of the roots, the red light irradiation group and the blue light irradiation group were smaller than the control (FIGS. 13e and 13f).

[0048] In those transplanted to plastic pots, the growth of the red light irradiation group was superior to that of the blue light irradiation group or the control. The fresh weight and dry weight of leaves and stems 3 months after transplantation were 1.4 times, 1.8 times, and 2 times greater in the red light-irradiated area than in the blue light-irradiated area, respectively (Figs. 14a to 14d) . The control group is a red light irradiation group and a blue light irradiation group. It was located in the middle.

The fresh weight and dry weight of roots at 3 months and 6 months after transplantation were 1.19 times, 1.3 times and 2 times, 1.5 times as large in the red light irradiation region than in the blue light irradiation region, respectively. (Figs. 15a and 15b).

[0049] In the case of transplanted to a plastic pot, the concentration of glycyrrhizic acid (amount per g of raw root weight) was measured 1 month, 3 months, and 6 months after transplantation. As a result, the concentration of glycyrrhizic acid increased markedly after 1 to 3 months after transplantation, but the roots became thick (Fig. 12), but hardly increased (Fig. 16b).

Regarding the effect of irradiation light on the concentration of glycyrrhizic acid, the concentration of glycyrrhizic acid in the red light irradiation group was the highest in the control group, followed by the lowest in the blue light irradiation group.

[0050] In the case of transplanted to a hydroponic system, regarding the effect of irradiation light on the concentration of glycyrrhizic acid (3 months after transplantation), the concentration of glycyrrhizic acid in the red light irradiation group was the highest in the control group and in the blue group. There was no significant difference in the light irradiation zone (Fig. 6A). When the concentration of glycyrrhizic acid at 3 months after transplantation was compared with that transplanted into plastic pots and those transplanted into hydroponic systems, those transplanted into plastic pots were 1.6 times stronger (Fig. 1 6a, 16b).

[0051] When UV-B was irradiated, the concentration of glycyrrhizic acid increased. In other words, the concentration of glycyrrhizic acid was about 1.5 times greater than that of the control group in both the group subjected to high UV-B irradiation for a short period and the group subjected to low UV-B irradiation for a long period of time (Fig. 17). . In the comparison of the UV-B irradiation sections, the section where low UV-B irradiation was performed for a long time showed a higher concentration of darlicyllithic acid.

The net photosynthetic rate during each UV-B irradiation period was smaller than that before and after that period (Figs. 18 and 19).

Industrial applicability

[0052] As is apparent from the above examples, according to the present invention, the secondary metabolite of a plant can be produced much more rapidly and in a large amount as compared with the conventional method. Accordingly, the present invention greatly contributes to the development of the pharmaceutical industry, food industry and other related industries.

Claims

The scope of the claims

[I] A method for increasing the production of secondary metabolites in a plant, wherein at least one of the elements affecting the growth of the plant is controlled on the plant body that has passed the seedling stage. Said method.

[2] The method according to claim 1, wherein the method is performed indoors.

[3] The factor affecting plant growth is one or more of photosynthetic photon flux density, carbon dioxide concentration, light wavelength, light period, temperature, humidity, and nutrients. The method described in 1.

[4] The method according to claim 3, wherein the factors affecting plant growth are photosynthetic photon flux density and carbon dioxide concentration.

[5] Photosynthetic photon flux density is 50 ~: LOOO μmolm ^_2 s ^_1 and diacid carbon concentration is 300

To 2000 is μ mol mol ^_1, The method of claim 4.

6. The method according to claim 1, further comprising applying stress to the plant.

[7] The method according to claim 5, wherein the stress is UV-B irradiation.

[8] The method according to any one of claims 1 to 7, wherein the plant is a Hypericaceae plant.

[9] The method of claim 8, which is Hypericaceae plant power.

[10] The method according to claim 8 or 9, wherein the secondary metabolite is hypericin or a derivative thereof.

[II] The method according to any one of claims 1 to 10, wherein the plant is a plant belonging to the genus Glycyrrhiza.

[12] The method according to claim 11, wherein the plant is Glycyrrhiza uralensis.

[13] The method according to claim 11 or 12, wherein the secondary metabolite is glycyrrhizic acid or a derivative thereof.