WO2018199307A1

WO2018199307A1 - Method for increasing amount of phenolic compound in plant

Info

Publication number: WO2018199307A1
Application number: PCT/JP2018/017259
Authority: WO
Inventors: 敦司岡澤; 藤川　康夫; 智大鶴本
Original assignee: 公立大学法人大阪府立大学; 日亜化学工業株式会社
Priority date: 2017-04-28
Filing date: 2018-04-27
Publication date: 2018-11-01
Also published as: IL269967B2; IL269967B1; IL269967A

Abstract

To provide a method whereby the amount of a phenolic compound such as polyphenol can be effectively/efficiently increased. The present invention provides a method for increasing the amount of a phenolic compound in a plant, said method being characterized by comprising irradiating the plant with ultraviolet light in such a manner that the irradiance of light within a wavelength range of 270-290 nm attains 1500-50000 μmol/m2 and the irradiance of light within a wavelength range of 310-400 nm attains a level corresponding to less than 50% of the irradiance of light within the wavelength range of 270-290 nm, or a method for producing a plant having an increased content of a phenolic compound.

Description

Method for increasing the amount of phenolic compounds in plants

The present invention relates to a method for increasing the amount of a phenolic compound in a plant and a method for producing a plant having an increased content of a phenolic compound.

Phenolic compounds in plants (e.g., polyphenols) have been revealed to have various physiological activities such as antioxidant activity, antibacterial activity, and antihypertensive activity, coupled with the recent increase in health orientation, Much attention has been paid.
Therefore, techniques for increasing the amount of phenolic compounds contained in plants have been developed. In particular, it is thought that plants synthesize flavonoids that have maximum absorption in the ultraviolet region in order to avoid the effects of ultraviolet light contained in sunlight. There is interest in increasing technology.

For example, Patent Document 1 relates to a method for increasing the polyphenol content in a “post-harvest” plant by irradiating ultraviolet light in a specific wavelength region (240 nm or more and 320 nm or less or 300 nm or more and 400 nm or less). In Patent Document 1, an appropriate ultraviolet light irradiation amount is 0.5 J / cm ² or more and 50 J / cm ² or less per day. It describes that the wavelength of the ultraviolet light irradiated to the plant after harvesting should be selected according to the maximum absorption of the target polyphenol.

Patent Document 2 discloses that ascorbic acid and / or polyphenols in a monocotyledonous plant (especially bud leek) are irradiated with ultraviolet light having a wavelength range of 280 to 380 nm and having a peak in the vicinity of a wavelength of 312 nm. It relates to a method for increasing the content. In Patent Document 2, an appropriate ultraviolet light intensity is 0.1 to 1.0 mW · cm ⁻² .

In addition, Non-Patent Document 1 aims at investigating the influence of UVB stress in Arabidopsis thaliana, and narrow-band UV-B (280 to 320 nm; peak wavelength 312 nm; 830 mW / m ² / S) for 1 day or 4 days continuously. As a result, it is described that an increase in anthocyanins and flavonols was observed by ultraviolet light irradiation for one day or longer.

JP 2004-121228 A JP2008-086272

As mentioned above, it is known that exposing a plant to ultraviolet light (UVA or UVB) increases the amount of phenolic compound in the plant. On the other hand, it is also known that ultraviolet light is harmful not only to plants but also to organisms in general. In the prior art, light having a relatively wide wavelength range is used as the ultraviolet light, and the emission spectrum of the UV lamp used in Non-Patent Document 1 is, for example, as shown in FIG. Although it has a peak wavelength at 312 nm, it covers a wide wavelength range.
Therefore, if the wavelength range that truly contributes to the increase of the phenolic compound in the ultraviolet light can be identified, the amount of the phenolic compound can be increased efficiently while reducing the adverse effects of exposure to ultraviolet light in plants. be able to.
Thus, there remains a need for methods that can effectively / effectively increase the amount of phenolic compounds, such as polyphenols, in plants.

As a result of intensive studies on the wavelength and irradiation amount that contribute to the increase in the phenolic compound, the present inventors have found that ultraviolet light in a specific wavelength range shorter than the conventionally known wavelength range is effective for increasing the phenolic compound. On the other hand, it has been found that ultraviolet light that has been used conventionally does not contribute to an increase in the amount of the phenolic compound, but rather includes light in a wavelength range that can cause a decrease in the abundance, and thus the present invention has been completed.

According to the present invention, a plant is irradiated with ultraviolet light in a wavelength range of 270 to 290 nm, the irradiation dose of 1500 to 50000 μmol / m ² , and the irradiation dose of light in the wavelength range of 310 to 400 nm is 270 to 290 nm. There is provided a method for increasing the amount of a phenolic compound in a plant, wherein the irradiation is carried out so as to be less than 50% of the irradiation amount of light in the wavelength region of.
According to the present invention, it is also possible to irradiate the plant with ultraviolet light, the irradiation amount of light in the wavelength region of 270 to 290 nm is 1500 to 50000 μmol / m ² , while the irradiation amount of light in the wavelength region of 310 to 400 nm is There is provided a method for producing a plant having an increased content of a phenolic compound, wherein the irradiation is performed so that the irradiation amount is less than 50% of light irradiation in a wavelength region of 270 to 290 nm.
According to the present invention, it is also possible to emit light in the wavelength range 270 to 290 nm, and the emission amount in the wavelength range 300 nm to 400 nm is less than 50% of the emission amount in the wavelength range 270 to 290 nm. Control that controls the light source so that the amount of emitted light of less than 10% of the amount of emitted light in the wavelength range 270 to 290 nm and the irradiation amount of light in the wavelength range of 270 to 290 nm to the plant is 1500 to 50000 μmol / m ² And a lighting device characterized in that it is used for increasing the amount of phenolic compounds in plants.

According to the method of the present invention, it is possible to avoid adverse effects due to exposure to harmful ultraviolet light exclusively, so that it is possible to efficiently increase the phenolic compound in the plant, and efficiently to the plant rich in phenolic compound Can be produced.

The emission spectrum of the ultraviolet LED used in the example (experiment 1) is shown. In Experiment 1, HPLC chromatogram (500 nm detection) which shows the amount of anthocyanins in the plant (Arabidopsis thaliana) irradiated with ultraviolet light. The peak wavelength of the irradiated ultraviolet light is 280 nm (BD) or 310 nm (FH), and the irradiation time is 15 minutes (B, F), 4 hours (C, G), or 4 days (D, H). It is. A and E are ultraviolet light non-irradiation controls. The emission spectrum of the ultraviolet LED used in the example (experiment 2) is shown. The emission spectra with peak wavelengths of 280 nm and 310 nm are those used in Experiment 1. In the Example (Experiment 2), the HPLC chromatogram (500 nm detection) which shows the amount of phenolic compounds in the plant (Arabidopsis thaliana) irradiated with the ultraviolet light. The peak wavelength of the irradiated ultraviolet light is shown on the left side in the figure. The irradiation time is 0 (ie, UV light non-irradiation control; leftmost column), 15 minutes (second column from the left), 4 hours (third column from the left), or 4 days (rightmost column). The results at peak wavelengths of 280 nm and 310 nm are those of Experiment 1. In the Example (Experiment 3), the HPLC chromatogram (258 nm detection) which shows the amount of phenolic compounds in the plant (Sudachi (fruit skin), pod film (root), chanoki (leaf), and soybean (fruit skin)) irradiated with the ultraviolet light. The peak wavelength of the irradiated ultraviolet light is 280 nm, and the irradiation time is 0 (ie, UV light non-irradiation control; leftmost column), 15 minutes (second column from the left) or 4 hours (third from the left) Column). The rightmost column shows the absorption spectrum of the peak indicated by the arrow. In the Example (experiment 4), the HPLC chromatogram (520 nm detection) which shows the amount of anthocyanins in the grape (fruit peel) irradiated with the ultraviolet light. The peak wavelength of the irradiated ultraviolet light is 280 nm, and the irradiation time is 0 (that is, ultraviolet light non-irradiation control; leftmost column), 15 minutes (second column from the left) or 4 hours (rightmost column). is there. In the Example (Experiment 5), the HPLC chromatogram (500 nm detection) which shows the amount of anthocyanins in the plant (Arabidopsis thaliana) darkened for 12, 24, 48, 72, 92, or 288 hours after ultraviolet irradiation. The peak wavelength of the irradiated ultraviolet light is 280 nm, and the irradiation time is 15 minutes. Biosynthetic pathway of phenolic compounds (anthocyanins) in plants. The emission spectrum of the ultraviolet lamp (CSL-30B, COSMO BIO) used in Non-Patent Document 1 is shown.

From one aspect, the present invention is a method for increasing the amount of a phenolic compound in a plant, wherein the plant is irradiated with ultraviolet light, and the irradiation dose of light in the wavelength region of 270 to 290 nm is 1500 to 50000 μmol / m ² . In addition, the irradiation is performed such that the irradiation amount of light in the wavelength region of 310 nm to 400 nm is less than 50% of the irradiation amount of light in the wavelength region of 270 to 290 nm.
Another aspect of the present invention is a method for producing a plant having an increased content of a phenolic compound, wherein the plant is irradiated with ultraviolet light, and an irradiation amount of light in a wavelength region of 270 to 290 nm is 1500. It is a method, which comprises irradiating ~ 50000μmol / m ^2, and the and so that the irradiation amount of light in the wavelength range of 310 nm ~ 400 nm is less than 50% of the dose of light in the wavelength range of the 270 ~ 290 nm .

In this specification, the numerical range “a to b” (a and b are specific numerical values) means a range including the values “a” and “b” at both ends. In other words, “ab” is synonymous with “a to b”.

In the present invention, as shown in the examples below, ultraviolet light in the wavelength range of about 270 to 290 nm is effective for increasing the amount of phenolic compounds in plants, while ultraviolet light in the wavelength range of about 310 to 400 nm is phenolic. It is based on a new finding that it does not contribute to the increase in the amount of the compound but even has an adverse effect. Therefore, according to the method of the present invention, the specific wavelength effective for increasing the amount of phenolic compound in the plant while reducing the adverse effect of exposure to ultraviolet light (resulting in a decrease in the amount of phenolic compound present). Therefore, the amount of phenolic compounds in the plant can be increased efficiently.

In the present invention, it is preferable to use near-ultraviolet light that is less likely to be absorbed in the atmosphere than far-ultraviolet light, and the irradiation amount of light in the wavelength region of 270 to 290 nm is 1500 to 50000 μmol / m ² , and The plant is irradiated so that the irradiation amount of light in the wavelength region of 310 nm to 400 nm is less than 50% of the irradiation amount of light in the wavelength region of 270 to 290 nm.
If the irradiation dose of ultraviolet light in the wavelength range of 270 to 290 nm is less than 1500 μmol / m ² , it will probably not significantly activate the phenolic compound synthesis system, so a significant increase in the amount of phenolic compounds in the plant Cannot be achieved. On the other hand, when the irradiation dose of light in the wavelength region of 270 to 290 nm exceeds 50000 μmol / m ² , plant damage is increased, so that a plant with an increased amount of phenolic compound cannot be obtained. Preferably, the irradiation dose of light in the wavelength region of 270 to 290 nm is 2000 to 40000 μmol / m ² . By using an irradiation amount in this range, a plant with an increased amount of phenolic compound can be obtained more efficiently.

On the other hand, ultraviolet light in the wavelength range of 310 nm to 400 nm does not contribute to the increase in the amount of phenolic compounds in plants, but rather acts on plant damage. When the amount is 50% or more, it becomes impossible to efficiently obtain a plant in an increased amount of the phenolic compound. From the viewpoint of avoiding adverse effects on plants, the irradiation amount of light in the wavelength region of 310 nm to 400 nm is preferably less than 30% and less than 20% of the irradiation amount of light in the wavelength region of 270 to 290 nm. Is more preferably less than 10%, most preferably less than 5%.
In one embodiment, the irradiation amount of light in the wavelength region of 300 nm to 400 nm is less than 50%, preferably less than 30%, more preferably 20% of the irradiation amount of light in the wavelength region of 270 to 290 nm. Less, more preferably less than 10%. In another embodiment, the irradiation dose of light in the wavelength range of more than 290 nm and less than or equal to 400 nm is less than 50%, preferably less than 30%, more preferably less than 30%. Preferably it is less than 20%, more preferably less than 10%.

Since the maximum absorption wavelength of DNA and RNA is around 260 nm, there is a concern that light having a wavelength of 260 nm or less has a large adverse effect (for example, cell damage) on plants. Therefore, the irradiation amount of light having a wavelength of 200 nm to 260 nm is preferably less than 20%, more preferably less than 10%, and more preferably less than 5% of the irradiation amount of light in the wavelength region of 270 to 290 nm. More preferred is less than 1%.
In one embodiment, the irradiation dose of light in the wavelength region of 200 nm or more and less than 270 nm (preferably 100 nm or more and less than 270 nm, more preferably 10 nm or more and less than 270 nm, more preferably 1 nm or more and less than 270 nm) is 270 to 290 nm. Is less than 20%, preferably less than 10%, more preferably less than 5%, and most preferably less than 1%.

Ultraviolet light having a wavelength range of 270 to 290 nm is irradiated with a photon flux density of, for example, 0.01 to 100 μmol / m ² / s. If it is less than 0.01 μmol / m ² / s, an increase in the phenolic compound in the plant may not be achieved efficiently. If it exceeds 100 μmol / m ² / s, plant damage may be induced early. Ultraviolet light in the wavelength region of 270 to 290 nm is preferably irradiated with a photon flux density of 0.1 to 20 μmol / m ² / s, more preferably 1 to 5 μmol / m ² / s.

The ultraviolet light source is not particularly limited as long as it can irradiate light in the wavelength region of 270 to 290 nm. For example, a commonly used ultraviolet light source such as a UV lamp can be used. As the UV lamp, for example, a xenon lamp using a SrSiO3: Pb phosphor is preferably used. Moreover, you may use what was taken out from sunlight with the optical filter etc. as ultraviolet light.
The light source used is 310 to 400 nm, 300 to 400 nm, 300 to 400 nm, or more than 290 nm to 400 nm or less, and light in the 270 to 290 nm wavelength range. When emitted at 50% or more of the bundle density, the transmittance for light in the wavelength range of 270 to 290 nm is 310 nm to 400 nm, 300 nm to 400 nm, or more than 290 nm to 400 nm or less. You may use together the filter larger than the transmittance | permeability with respect to the light. In addition to light in the wavelength range of 270 to 290 nm, a wavelength range of 200 nm to 260 nm or a wavelength range of less than 270 nm (for example, a wavelength of 200 nm to 270 nm, 100 nm to 270 nm, 10 nm to 270 nm, or 1 nm to 270 nm) Is emitted at 10% or more of the photon flux density of light in the wavelength range of 270 to 290 nm, the transmittance for light in the wavelength range of 270 to 290 nm is 200 nm to 260 nm, respectively. A filter having a larger transmittance than that of light having a wavelength range of less than 270 nm may be used.

From the viewpoint of energy efficiency, light in the wavelength region of 270 to 290 nm is irradiated as light having a main peak wavelength within, for example, 280 ± 5 nm, more preferably within 280 ± 2 nm. It is preferable that the second peak does not exist or even if it exists, its intensity is 1/10 or less of the main peak.
The full width at half maximum of the main peak (within a peak wavelength of 270 to 290 nm) is preferably 5 to 15 nm. The half-width of the main peak is 15 nm or less, so that it does not contribute to the increase in the amount of phenolic compounds in the plant (which may be harmful), while avoiding irradiation of light in the wavelength range to the plant, phenolic in the plant In addition to enabling light irradiation (that is, selective irradiation) in a wavelength range effective for increasing the amount of the compound, energy efficiency is further improved. Light having a main peak half width of less than 5 nm can also be used in the method of the present invention. However, from the viewpoint of cost effectiveness, it is preferable to use light having a main peak half width of 5 nm or more. In one preferred specific embodiment, the ultraviolet light irradiated on the plant is light having a wavelength spectrum having a peak wavelength of 280 ± 5 nm and a half-value width of 5 to 15 nm.

As the ultraviolet light source, a light emitting diode (LED) having a single peak in the emission spectrum is particularly preferable. When using an LED as a light source, it is effective in increasing the amount of phenolic compounds in the plant while avoiding irradiation of the light in the wavelength range that does not contribute to the increase in the amount of phenolic compounds in the plant (which may be harmful). Irradiation of light in the wavelength range (that is, selective irradiation) can be easily realized. The use of LEDs is also preferable from the viewpoint of energy efficiency and economy due to low heat generation, low power consumption and long life. In addition, it becomes easy to control / manage the irradiation dose and / or the photon flux density.
An LED capable of emitting ultraviolet light of 270 to 290 nm can be, for example, one using an AlGaN-based material or an InAlGaN-based material. A specific example of such an LED is Deep UV-LED / model NCSU234BU280 (central wavelength 280 nm; Nichia Corporation).

The amount of irradiation of light in the wavelength range of 270 to 290 nm to the plant is, for example, controlled by turning on and off the light source (for example, when the plant is placed in a closed space) or It can be set to 1500 to 50000 μmol / m ² by controlling the time required for passing (for example, when the plant is moving on the transport device).

In the present specification, the plant is not particularly limited, but herb is preferable. The plant can be, for example, an angiosperm, in particular a dicotyledonous plant. Suitable dicotyledonous plants for the method of the present invention include, for example, Brassicaceae (especially Brassica, Daikon), Solanum (especially Solanum), Barberry (especially Solanum), Camellia (especially Camellia), legumes. Plants of the family (especially soybean genus), citrus (especially mandarin), grapes (especially grapes), roses (especially Dutch strawberrys), asteraceae (especially genus Akinesi), perillas (especially perillas) included. Specific examples of plants that can be used in the present invention include Arabidopsis thaliana, podophyllum, chanoki, soybean, sudachi, grape, cabbage, broccoli, komatsuna, radish, radish, turnip, tomato, eggplant, strawberry, lettuce, and perilla. It is done.

As used herein, a plant can be a plant having a UVR8 photoreceptor.
Although not bound by theory, according to the results of gene expression analysis described later, light in the wavelength region of 270 to 290 nm is transmitted through a UVR8 photoreceptor of a plant via a phenolic compound (for example, phenylpropanoid, flavonoid, It is thought to upregulate the expression of genes of enzymes and transcription factors involved in the biosynthetic pathway of anthocyanins and the like, and as a result, activate biosynthesis of phenolic compounds in plants.

The plant used in the present invention may be in the form of the whole plant including shoots (stems and leaves) and the root system, or may be in the form of a part of the plant such as shoots alone.
When the plant is in the form of the entire plant body, it may be in a cultivated state or in a non-cultivated state (a state in which no nutrient is supplied through the roots). Here, the cultivation may be soil cultivation or hydroponics (for example, hydroponics or solid medium cultivation). Hydroponics can be performed under aseptic conditions.

Cultivation may be performed in a controlled environment. The environmental conditions to be controlled include, for example, the light / dark cycle, temperature, humidity, irradiation amount of natural light and / or artificial light, and carbon dioxide concentration. These conditions are not particularly limited as long as they are suitable for the cultivation / growth of the plant to be used.
The light / dark cycle can be appropriately selected according to the plant to be cultivated and the growth stage (for example, a long-day condition of light period 14-18 hours, or a short-day condition of light period 6-10 hours). As an artificial light source, conventionally used incandescent lamps, fluorescent lamps, white lamps, high-pressure sodium lamps, metal halide lamps, LEDs, and the like can be used. Artificial light is irradiated with a photosynthetic photon flux bundle density that is appropriately set according to the plant to be cultivated and the growth stage. The photosynthetic photon flux density can be, for example, 100 to 500 μmol / m ² / s.
The temperature can be, for example, 20-30 ° C., and the humidity can be, for example, 50-80%.
The carbon dioxide concentration can be, for example, about 1000-1500 ppm.
Fertilizer / liquid fertilizer can be appropriately selected according to the plant to be cultivated. Generally, fertilizer / liquid fertilizer contains nitrogen, phosphorus and potassium.

Plants can be stored under natural light or in the dark, at normal temperatures (eg 15-30 ° C.) or at low temperatures (eg 0-15 ° C.) when not cultivated.

The plant at the time of ultraviolet light irradiation according to the present invention may be in any growth stage. In one embodiment, the plant upon irradiation is in the vegetative growth phase, and in another embodiment, the plant upon irradiation is in a non-cultivated state (ie, after harvest [crop]).
The plant may be a young plant or an adult plant. The seedling can be a sprout. Sprout is also called a germinating plant, and refers to a young plant body after seed germination and before development of the main leaves. The sprout can be obtained, for example, by culturing for 7 to 10 days in a dark place at a temperature of 20 to 25 ° C.
When the plant is in a cultivated state, the irradiation with ultraviolet light according to the present invention may be performed in either the light period or the dark period.

The method of the present invention may further comprise darkening the plant for 12 hours or more after irradiation with light (ultraviolet light) in the wavelength range of 270 to 290 nm. In this specification, “dark placement” means that a plant is in a dark place or a dark room (the level at which the photosynthetic photon flux density does not cause photosynthesis in the plant. More specifically, the photosynthetic photon flux density ≦ 10 μmol / m ² / s. ). The dark period is preferably 24 hours or longer, more preferably 36 hours or longer, more preferably 48 hours or longer, and even more preferably 72 hours or longer. The upper limit of the dark period is not particularly limited as long as the content of the phenolic compound in the plant is increased with respect to the non-irradiated plant, but may be, for example, 300 hours, more specifically, 288 hours (less than). There may be.
Dark storage can be, for example, at room temperature or low temperature.
By darkening a plant for 12 hours or more after irradiation with ultraviolet light, the amount (content) of the phenolic compound in the plant further increases.

In the present specification, the phenolic compound is not particularly limited as long as it can be naturally synthesized in the plant to be used, and may be, for example, phenylpropanoid and polyphenol. Polyphenols include, for example, flavonoids, tannins and lignans. Examples of flavonoids include anthocyanins, flavans (for example, catechin), flavones, isoflavones, and flavonols.
Anthocyanins are glycosides in which sugar chains (for example, glucose, galactose, rhamnose) are bound to anthocyanidins. Common anthocyanidins contained in plants include pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin. Anthocyanins are red-purple to blue-colored pigments widely present in the plant kingdom. They are used as plant colorants (for example in foods) and are also known as antioxidants. It is one of the phenolic compounds suitable for increasing the amount.
For example, as an example of anthocyanins contained in Arabidopsis thaliana, those represented by the following chemical formula can be mentioned.

(The portion surrounded by a line is an anthocyanidin portion, and in this case, a peonidin portion.)

In the present specification, “increased amount of phenolic compound” or “increase in content of phenolic compound” means that the amount of phenolic compound is increased as compared to a plant not irradiated with ultraviolet light according to the present invention. For example, 10% or more, preferably 20% or more, more preferably 50% or more, more preferably 100% or more [result of 15 minutes irradiation in grape (2250 μmol / m ² ) Added in consideration]. “Increased amount of phenolic compound” or “increased content of phenolic compound” includes that a phenolic compound that was not synthesized before irradiation is newly synthesized after irradiation. .
The quantification of the phenolic compound may be performed using any known method, for example, by chromatography. Chromatography includes liquid chromatography (eg, HPLC). The liquid chromatography can be reverse phase chromatography.

By irradiation with ultraviolet light according to the present invention, the amount of the phenolic compound is 1.1 times or more, preferably 1.2 times or more, more preferably 1.5 times or more, more preferably 2 times or more, for example 3 times as compared with the non-irradiated one. Higher plants can be produced [Same as above]. Therefore, according to the method of the present invention, the produced plant can be provided inexpensively as a high value-added food.

The plant produced by the method of the present invention is suitable as a raw material for producing a phenolic compound having an increased content in the plant.
Therefore, the present invention is a method for producing a phenolic compound,
Plants are irradiated with ultraviolet light in the wavelength range of 270 to 290 nm, while the irradiation dose of light in the wavelength range of 310 to 400 nm is 1500 to 50000 μmol / m ² , while the light dose in the wavelength range of 270 to 290 nm is light. The method of irradiating so that it may become less than 50% of the irradiation amount of this invention The process characterized by including the process of acquiring a phenolic compound from this plant is also provided.

The present invention is also a method for producing a phenolic compound comprising:
Plants are irradiated with ultraviolet light in the wavelength range of 270 to 290 nm, while the irradiation dose of light in the wavelength range of 300 to 400 nm is 1500 to 50000 μmol / m ² , while the light dose in the wavelength range of 270 to 290 nm is light. A step of irradiating light having a wavelength in the wavelength range of 200 nm to less than 270 nm to less than 10% of the light dose in the wavelength range of 270 to 290 nm. Also provided is a method comprising the step of obtaining a compound.

The acquisition process of the phenolic compound from a plant can be performed by extraction, for example. The extraction can be performed by any known method, for example by solvent extraction or supercritical extraction.
The solvent used for solvent extraction can be appropriately selected from known solvents. The solvent is, for example, water (room temperature to boiling water), an organic solvent miscible with water, or a mixed solvent thereof (a mixed solvent of water and one or more water-miscible organic solvents, two or more water). It is possible to use a mixed solvent of an organic solvent miscible with the solvent. The water miscible organic solvent can be a polar organic solvent, such as methanol, ethanol, n- or iso-propanol, acetonitrile, acetone, dioxane, ethyl acetate, dimethyl sulfoxide, dimethylformamide, ethylene glycol, tetrahydrofuran. It is done. Water may be used as hot water or boiling water.
Extraction may be performed under heating (for example, 80 to 90 ° C.) and / or under pressure. The extraction may be reflux extraction. The extraction time is not particularly limited and can be appropriately determined from the viewpoint of extraction efficiency.

For the extraction, leaves and / or stems of plants can be used regardless of the growth stage, but preferably the whole plant is used. The plant may be used for extraction as it is, or may be used after pulverization. Prior to extraction or grinding, the plants may be dried and / or frozen.
For example, in order to remove impurities, the extract may be filtered through an appropriate filter or may be subjected to centrifugation.

When the objective phenolic compound is anthocyanin, for example, a polar organic solvent can be used for extraction. An acid may be added to the polar organic solvent. The acid can be, for example, hydrochloric acid, sulfuric acid, formic acid, acetic acid, phosphoric acid, trichloroacetic acid, trifluoroacetic acid or perchloric acid. The acid may be included in the organic solvent in a weight ratio of, for example, 0.1 to 10%, preferably 1 to 3%. Specific examples of the extraction solvent include trifluoroacetic acid, formic acid or a mixed solvent of acetic acid / methanol, a mixed solvent of acetone / methanol / formic acid or acetic acid, and a mixed solvent of hydrochloric acid / methanol.

You may refine | purify a phenolic compound from the obtained extract.
Purification can be performed, for example, by chromatography. Chromatography can be, for example, column chromatography (particularly HPLC), preferably in reverse phase mode.
The column used for column chromatography can be appropriately selected from known ones according to the separation mode. For reverse phase chromatography, an octadecylated silica gel (ODS) column (also referred to as a C18 column) is generally used, but the present invention is not limited to this. For example, a C30 column can also be used.

In chromatography, water, a polar organic solvent, or a mixed solvent thereof (a mixed solvent of water and one or more polar organic solvents, a mixed solvent of two or more polar organic solvents) can be used as an eluent. The polar organic solvent is as described above. For example, 0.01 to 10 M of an acid such as trifluoroacetic acid, formic acid, acetic acid, phosphoric acid, trichloroacetic acid may be added to the eluent.
In chromatography, a gradient method can be employed. In this case, a mixed solvent of a polar solvent and water (for example, a mixing ratio of 0: 100 to 10:90) optionally containing 0.01 to 10 M acid is used as the eluent A. Using a mixed polar solvent containing 10 M acid (for example, a mixing ratio of 50:50) or a mixed solvent of a polar solvent and water (for example, a mixing ratio of 50:50 to 100: 0), the gradient is increased, for example, for 30 to 60 minutes. , A: B = between 100: 0 and 0: 100.
The flow rate is not particularly limited and can be, for example, 0.2 to 2 ml / min.
The detection of the phenolic compound can be performed, for example, by measuring the absorbance at 250 to 300 nm. Detection of anthocyanins can be performed by measuring absorbance at 500 to 550 nm.

The phenolic compound produced by the method of the present invention can be used as a raw material for functional foods (food for specified health use or functional nutrition food), pharmaceuticals and other industrial products. Therefore, according to the method of the present invention, the produced phenolic compound can be provided at low cost as a raw material for functional foods, pharmaceuticals and other industrial products.
In one embodiment, the plant is Arabidopsis. Since Arabidopsis thaliana grows in a relatively short period of time and is easy to grow, it is suitable for the production of phenolic compounds (for example, anthocyanins) by the method of the present invention.

The present invention can also emit light in the wavelength region of 270 to 290 nm, and the emission amount in the wavelength region of 300 nm to 400 nm is less than 50% of the emission amount of the wavelength region of 270 to 290 nm, and the emission in the wavelength region of 200 nm to less than 270 nm. A light source whose amount is less than 10% of the light emission amount in the wavelength range 270 to 290 nm, and a control unit that controls the light source so that the irradiation amount of light in the wavelength range 270 to 290 nm to the plant is 1500 to 50000 μmol / m ² It is related with the illuminating device characterized by using to increase the amount of phenolic compounds in a plant.
The lighting device of the present invention is the above-described method for increasing the amount of the phenolic compound in the plant according to the present invention, the method for producing the plant having an increased content of the phenolic compound, and the method for producing the phenolic compound (collectively, Useful in “methods of the invention”).

The light source is similar to that described above with respect to the method of the present invention.
The light source preferably has a wavelength of light of 270 to 290 nm in the wavelength region of 200 nm to less than 270 nm (more preferably 100 nm to less than 270 nm, more preferably 10 nm to less than 270 nm, and even more preferably 1 nm to less than 270 nm). The light source is less than 5%, more preferably less than 1% of the light irradiation amount of the area.
The light source preferably also has an irradiation amount of light in the wavelength range of 300 to 400 nm of less than 30%, more preferably less than 20%, and even more preferably less than 10% of the irradiation amount of light in the wavelength range of 270 to 290 nm. Is a light source. In one more specific embodiment, the light source has an irradiation dose of light in a wavelength range of more than 290 nm and less than or equal to 400 nm less than 50% of an irradiation dose of light in the wavelength range of 270 to 290 nm, preferably Less than 30%, more preferably less than 20%, more preferably less than 10%.
From the above viewpoint, preferred specific examples of the light source include an LED or a xenon lamp (for example, one using a SrSiO3: Pb phosphor) (with a necessary filter (see above)). preferable.
The lighting device of the present invention may have only one light source or a plurality of light sources.

The control unit controls the timing of turning on and off the light source and / or dimming. The control unit can be, for example, a timer and / or a pulse width modulation circuit.

The lighting device of the present invention may be installed in a closed space for growing and / or storing plants. The closed space is not limited in shape and size as long as plants can be grown and / or stored, and may be, for example, a plant cultivation facility, a plant factory, a warehouse, a container, a refrigerator, or the like.
Within the enclosed space, the lighting device can be arranged above and / or laterally and / or below the plant.
An air conditioner that controls the internal temperature and / or humidity to a predetermined value may be installed in the closed space.

In one preferred embodiment, the photosynthetic photon flux density in the closed space is 10 μmol / mm for 12 hours or more after the dose of light in the wavelength region 270 to 290 nm from the illumination device reaches 1500 to 50000 μmol / m ^2. m ² / s or less (ie, “dark” or “dark room” state). The dark state is preferably continued for 24 hours or longer, more preferably 36 hours or longer, more preferably 48 hours or longer, and further preferably 72 hours or longer. The upper limit of the duration of the dark state is not particularly limited as long as the content of the phenolic compound in the plant is increased with respect to the non-irradiated plant, but can be, for example, 300 hours, for example, 288 hours (less than) Also good.

(Experiment 1)
Arabidopsis thaliana was aseptically grown on MS (Murashige-Skoog) agar. Specifically, sterilized seeds are sown on MS agar medium, and in a plant incubator (BMS-PS08RGB2, biomedical science) at a temperature of 22-23 ° C for long days (light period 16 hours / dark period) 8 hours; under a photosynthetic effective photon flux density of 200 μmol / m ² / s), the seedlings were grown for 14 days.
After growing for 14 days, using Arabidopsis thaliana with LED, peak wavelength 280nm (half width 10nm; Deep UV-LED / model: NCSU234BU280) or peak wavelength 310nm (half width 10nm; Deep UV-LED / model: NCSU234BU310) Ultraviolet light was irradiated for 15 minutes, 4 hours or 4 days continuously at an illuminance with a photon number of 2.5 μmol / m ² / s (accumulated photon number: 2250, 36000 and 864000 μmol / m ² ). The emission spectrum of the LED used is shown in FIG.
Then, it left still in the dark until the measurement of anthocyanin. As a control, one that was allowed to stand for 24 hours in the dark without being irradiated with ultraviolet light was used.

Twenty-four hours after irradiation with ultraviolet light, Arabidopsis thaliana (dry weight about 30 mg) was freeze-ground and subjected to solvent extraction using 80% methanol.
The amount of anthocyanins in the obtained extract was analyzed by high performance liquid chromatography (LC-10, Shimadzu Corporation) under the following conditions.
HPLC conditions / column: ODS column (Kinetex 5u C18 100 A, Phenomenex)
・ Column temperature: 40 ℃
・ Flow rate: 2 ml / min ・ Injection volume: 10 μl
-Mobile phase: eluent A 0.1% formic acid aqueous solution eluent B 0.1% formic acid acetonitrile solution linear gradient B: 1% to 99% over 49 minutes
・ Detection: 210 ~ 750nm

The results are shown in FIG.
In Arabidopsis thaliana irradiated with ultraviolet light having a peak wavelength of 280 nm for 15 minutes or 4 hours, the amount of anthocyanin (peak height) increased to 3 times or more (B) or nearly 2 times (C) of the control (A), respectively. Anthocyanins could not be detected in plants after 4 days of irradiation (D).
On the other hand, in Arabidopsis thaliana irradiated with ultraviolet light having a peak wavelength of 310 nm for 15 minutes, 4 hours, or 4 days, the amount of anthocyanins was decreased with respect to the control (E) (F to H).

From these results, it is clear that irradiation with ultraviolet light near 280 nm is effective for increasing the amount of anthocyanins in Arabidopsis, while irradiation with ultraviolet light near 310 nm does not contribute, but rather has some negative effect that causes a decrease in the amount of anthocyanins. It was shown that.

(Experiment 2)
The peak wavelength of the LED used is 270 nm (half-width 10 nm; Deep UV-LED / model: NCU234BU270) or 300 nm (half-width 10 nm; Deep UV-LED / model: NCU234BU300), and the irradiation time is 15 for the peak wavelength 270 nm. The same experiment as in Experiment 1 was performed except that the time was 1 minute (integrated photon number: 2250 μmol / m ² ), and the peak wavelength of 300 nm was 15 minutes or 4 hours (integrated photon number: 2250 and 36000 μmol / m ^2, respectively). The emission spectrum of the LED used is shown in FIG. In FIG. 3, emission spectra with peak wavelengths of 280 nm and 310 nm are also shown for comparison.

The results are shown in FIG. In addition, the figure also showed the result of Experiment 1 for comparison.
In Arabidopsis thaliana irradiated with ultraviolet light with a peak wavelength of 270 nm for 15 minutes or with ultraviolet light with a peak wavelength of 300 nm for 15 minutes or 4 hours, the amount of anthocyanin (peak height) was decreased relative to the control.
These results indicate that irradiation with UV light below 270 nm and UV light near 300 nm do not contribute to an increase in the amount of anthocyanins in Arabidopsis, but rather have some negative effect that causes a decrease in the amount of anthocyanins. It was.

The wavelength component ratio of the LED used in

Experiments

1 and 2 is shown.

(Experiment 3)
Collected or purchased sudachi (pepper), pod film (root), tea tree (leaf), and soybean (pepper) using LED (Deep UV-LED / model: NCSU234BU280), peak wavelength 280nm (half width 10nm) ultraviolet Light was continuously irradiated for 15 minutes at an illuminance of 5 μmol / m ² / s of photon number (integrated photon number: 4500 μmol / m ² ). The leaves and stems were irradiated with ultraviolet light in a state where they were lightly immersed in pure water to prevent drying and aging.
Then, it left still in the dark until the measurement of a phenolic compound. As a control, one that was allowed to stand for 24 hours in the dark without being irradiated with ultraviolet light was used. In addition, about the leaf and stem, in order to prevent dryness and aging, it was left in the state where it was lightly immersed in pure water.

24 hours after the irradiation with ultraviolet light, each plant part (dry weight about 30 mg) was freeze-ground and subjected to solvent extraction using 80% methanol.
The amount of phenolic compound in the resulting extract was analyzed by high performance liquid chromatography (Prominence, Shimadzu Corporation) under the following conditions.
HPLC conditions / column: ODS column (Triart C18 (150 × 4.6mm, S-5μm), YMC)
・ Column temperature: 40 ℃
・ Flow rate: 1 ml / min ・ Injection volume: 10 μl
Mobile phase: Eluent A 0.1% formic acid aqueous solution Eluent B 0.1% formic acid acetonitrile solution Linear gradient B: 30% for 1% to 100%
・ Detection: 190-800nm

The results are shown in FIG.
Sudachi (peel), pod film (root), chanoki (leaf), and soybean (peel) irradiated with ultraviolet light with a peak wavelength of 280 nm for 15 minutes or 4 hours have absorption at around 250 to 300 nm, which is characteristic of phenolic compounds An increase in peak was confirmed.
From this result, it was shown that the irradiation of ultraviolet light around 280 nm is effective for increasing phenolic compounds in sudachi, podophyllum, tea tree and soybean.

(Experiment 4)
Using the LED (Deep UV-LED / model: NCSU234BU280), UV light with a peak wavelength of 280 nm (half width 10 nm) is applied to each purchased fruit (fruit skin) with a photon number of 2.5 μmol / m ² / s. Irradiation was continued for 15 minutes or 4 hours at an illuminance (integrated photon number: 2250 or 36000 μmol / m ^2, respectively). Then, it left still in the dark until the measurement of a phenolic compound. As a control, one that was allowed to stand for 24 hours in the dark without being irradiated with ultraviolet light was used.
After 24 hours of ultraviolet light irradiation, the peel (dry weight about 40-100 mg) was freeze-ground and subjected to solvent extraction using 80% methanol. The amount of phenolic compound in the obtained extract was analyzed by high performance liquid chromatography (Prominence, Shimadzu Corporation) under the same conditions as in Experiment 3.

The results are shown in FIG.
In grapes (fruit skin) irradiated with ultraviolet light with a peak wavelength of 280 nm for 15 minutes or 4 hours, an increase in the total area of the peak having an absorption around 520 nm, which is characteristic of anthocyanins, was confirmed (1.2 to 1.9 times increase).
From this result, it was shown that irradiation with ultraviolet light near 280 nm is effective for further increase of anthocyanins even in grapes having a high anthocyanin content in a non-irradiated state.

(Experiment 5)
According to the method described in Experiment 1, Arabidopsis thaliana was irradiated with ultraviolet light having a peak wavelength of 280 nm continuously for 15 minutes (integrated photon number: 2250 μmol / m ² ).
Then, it was left still in the dark for 12, 24, 48, 72, 96 or 288 hours until the measurement of anthocyanin.
After darkening, Arabidopsis thaliana (dry weight about 30 mg) was freeze-ground and subjected to solvent extraction using 80% methanol.
The amount of anthocyanin in the obtained extract was analyzed according to the method described in Experiment 1.

The results are shown in FIG. The multiples shown in the figure (for example, “× 0.4” or “× 4.0”) are relative to the amount (reference value) when darkened for 24 hours after irradiation with ultraviolet light. It should be noted that the amount of anthocyanin increased by a factor of 3 with respect to the non-irradiated control when darkened for 24 hours after UV irradiation [see FIG. 1]. That is, “0.4 times” and “4 times” with respect to dark storage for 24 hours after ultraviolet light irradiation mean “1.2 times” and “12 times” for the non-irradiation control, respectively.
From this result, it was shown that the amount of the phenolic compound in the ultraviolet irradiation plant was further increased when left for 24 hours or longer after the ultraviolet irradiation.

(Experiment 6)
Preparation of total RNA for gene expression analysis Total RNA is RNeasy mini kit (Qiagen) immediately after irradiation from Arabidopsis thaliana irradiated for 45 minutes with UV light of peak wavelength 280nm (half width 10nm; Deep UV-LED / model: NCSU234BU280) And prepared according to the instruction manual.

Expression analysis RNA-seq analysis was performed on the prepared total RNA (Takara Bio Inc.). For sequence analysis, HiSeq2500 system (Illumina) was used and TAIR10.37 was used as reference sequence data.

Table 2 shows genes whose expression levels (FPKM: Fragments Per Kilobase of exon per million mapped reads) are more than twice.

In the table, a gene marked with “*” indicates a gene of an enzyme involved in the biosynthesis pathway of a phenol compound (FIG. 8).

The increase of anthocyanins in plants irradiated with ultraviolet light near 280 nm is caused by UV-B photoreceptor-mediated signal transduction via UVR8 activation (monomerization). It is speculated that this was caused by stimulating (Table 2) and, as a result, increasing the expression of chalcone synthase, which is one of the rate limiting factors of the shikimate pathway. Therefore, irradiation with ultraviolet light near 280 nm increases not only the amount of anthocyanins but also the amount of phenolic compounds synthesized in general via the shikimic acid pathway (

Experiments

3 and 4, FIGS. 5 and 6). .
Furthermore, the photoreceptor UVR8, UV-B photoreceptor-mediated signal transduction system and shikimate pathway exist not only in Arabidopsis but also in the plant kingdom. The amount is thought to increase.

Claims

Plants are irradiated with ultraviolet light in the wavelength range of 270 to 290 nm, and the irradiation dose of light in the wavelength range of 270 to 290 nm is 1500 to 50000 μmol / m 2 and the irradiation dose of light in the wavelength range of 310 to 400 nm is A method for increasing the amount of a phenolic compound in a plant, wherein the irradiation is performed so that the irradiation amount is less than 50%.
The method according to claim 1, wherein the irradiation amount of light in the wavelength region of 310 nm to 400 nm is less than 10% of the irradiation amount of light in the wavelength region of 270 to 290 nm.
The method according to claim 1, wherein the irradiation amount of light in the wavelength region of 300 to 400 nm is less than 50% of the irradiation amount of light in the wavelength region of 270 to 290 nm.
The method according to any one of claims 1 to 3, wherein the light in the wavelength region of 270 to 290 nm is irradiated at a photon flux density of 1 to 5 µmol / m 2 / s.
The method according to any one of claims 1 to 4, wherein an irradiation amount of light in a wavelength region of 200 nm to 260 nm is less than 20% of an irradiation amount of light in the wavelength region of 270 to 290 nm.
The method according to any one of claims 1 to 5, wherein an irradiation amount of light in a wavelength region of 200 nm or more and less than 270 nm is less than 10% of an irradiation amount of light in the wavelength region of 270 to 290 nm.
The method according to any one of claims 1 to 6, wherein the ultraviolet light is light having a wavelength spectrum having a peak wavelength of 280 ± 5 nm and a half-value width of 5 to 15 nm.
The method according to any one of claims 1 to 7, wherein the ultraviolet light is light having an LED as a light source.
The method according to any one of claims 1 to 8, wherein the plant is irradiated with ultraviolet light in a dark place.
The method according to any one of claims 1 to 9, further comprising darkening the plant for 12 hours or longer after the irradiation with ultraviolet light.
The method according to claim 10, wherein the plant is darkened for 48 hours or more and less than 288 hours.
The method according to any one of claims 1 to 11, wherein the plant is a cruciferous, barberry, camellia, legume, citrus or grape plant.
The plant according to any one of claims 1 to 12, wherein the plant is a plant selected from Arabidopsis thaliana, podophyllum, chanoki, soybean, sudachi, grape, cabbage, broccoli, komatsuna, chingensai, radish, turnip, tomato, eggplant, strawberry, lettuce and perilla. The method according to claim 1.
The method according to any one of claims 1 to 13, wherein the phenolic compound is polyphenol.
15. The method of claim 14, wherein the polyphenol is a flavonoid.
The method of claim 15, wherein the flavonoid is an anthocyanin.
Plants are irradiated with ultraviolet light in the wavelength range of 270 to 290 nm, while the irradiation dose of light in the wavelength range of 310 to 400 nm is 1500 to 50000 μmol / m 2 , while the light dose in the wavelength range of 270 to 290 nm is light. A method for producing a plant having an increased content of a phenolic compound, wherein the irradiation is performed so that the amount of irradiation is less than 50% of the amount of irradiation.
Plants are irradiated with ultraviolet light in the wavelength range of 270 to 290 nm, while the irradiation dose of light in the wavelength range of 300 to 400 nm is 1500 to 50000 μmol / m 2 , while the light dose in the wavelength range of 270 to 290 nm is light. Irradiation is performed so that the irradiation amount of light in a wavelength region of less than 50% and less than 200 nm and less than 270 nm is less than 10% of the irradiation amount of light in the wavelength region of 270 to 290 nm. A method for producing a plant having an increased content of a sex compound.
The method according to claim 17 or 18, wherein the plant is in a sprout state.
The method according to claim 17 or 18, wherein the plant is in a non-cultivated state.
It can emit light in the wavelength range 270 to 290 nm, and the emission amount in the wavelength range 300 nm to 400 nm is less than 50% of the emission amount in the wavelength range 270 to 290 nm, and the emission amount in the wavelength range 200 nm to less than 270 nm is the wavelength range 270. A light source that is less than 10% of the emitted light of ~ 290 nm;
Illumination characterized by having a control unit that controls the light source so that the irradiation amount of light in the wavelength range of 270 to 290 nm to the plant is 1500 to 50000 μmol / m 2, and is used to increase the amount of phenolic compounds in the plant apparatus.