JP7133812B2 - How to treat Madagascar periwinkle to increase vinblastine - Google Patents

Description

The present invention relates to a method of treating Madagascar periwinkle to increase the amount of vinblastine.

Vinblastine contained in Catharanthus roseus (particularly its leaves) is used as an antineoplastic or anticancer agent.
However, since the amount of vinblastine naturally contained in Madagascar periwinkle is extremely small (on the order of 0.01 wt% per dry weight), obtaining it from Madagascar periwinkle (by extraction and purification) requires a large amount of Madagascar periwinkle, resulting in high costs. there is
Madagascar periwinkle contains more vindoline and catharanthine, the synthetic precursors of vinblastine, than vinblastine. Therefore, a method of synthesizing vinblastine using these precursors extracted from Madagascar periwinkle (semi-synthetic method) has been devised, and a technique for increasing the yield of vinblastine by irradiating with ultraviolet (UV) light during synthesis has also been proposed. (Patent Documents 1 and 2).

For example, US Pat. No. 6,200,001 discloses a method for efficiently producing vinblastine by irradiating light within the UV/visible spectrum (preferably light with a wavelength greater than 254 nm, especially greater than 400 nm) during the reaction of cataratine and vindoline. is described. The light used in the examples of this document is light with wavelengths λ>400 nm, light with λ>345 nm and light with λ>360 nm.
Patent document 2 increases the yield of vinblastine produced by reacting vindoline and catharanthine in an acidic aqueous solution under UV light irradiation with a wavelength of 180 to 400 nm (mainly 254, 265, 297 and 366 nm). It states that it is possible.

On the other hand, it is known that vinblastine content can be increased by irradiation with near-ultraviolet (UVA) light such as an ultraviolet lamp in plants or cultures of Madagascar periwinkle (Patent Document 3, Non-Patent Documents 1 and 2). .
For example, in Patent Document 3, under the irradiation of light in the near-ultraviolet region of 400 nm or less, or under the conditions in which the light used in normal culture is supplemented with the light in the near-ultraviolet region, the foliar organs of Catharanthus periwinkle are cultured, and the alkaloid content is high. An organ culture method of Catharanthus roseus to obtain strains is described.
Non-Patent Document 1 describes that vinblastine levels in multiple shoot cultures increase under irradiation with near-ultraviolet light with a peak wavelength of 370 nm.
Non-Patent Document 2 describes that UVA light (light in the wavelength range of 290 nm to 380 nm among artificial light with a peak wavelength of 370 nm and natural light) stimulates the synthesis of dimeric indole alkaloids in whole plants of Madagascar periwinkle. there is

International Publication No. WO99/62912 International Publication No. WO89/12056 JP-A-01-252229

J. Ferment. Bioeng., 74(4): 222-225, 1992 Planta Med., 59(1): 46-50, 1993

As described above, it is known that UVA light irradiation increases the amount of vinblastine in Madagascar periwinkle. However, in the prior art as described above, light with a wide range of wavelengths is used as ultraviolet light. For example, the main peak wavelength mentioned in Patent Document 2 is presumed to correspond to the peak wavelength in the ultraviolet light region of the mercury lamp used (having a wide wavelength band spectrum).
Therefore, if it is possible to identify a wavelength range of ultraviolet light that truly contributes to increasing the amount of vinblastine in Madagascar periwinkle, vinblastine in Madagascar periwinkle can be efficiently and / or dramatically increased by using light of the identified wavelength. The dosage can be increased, and as a result, vinblastine and its derivatives can be provided at relatively low cost.
Thus, there is still a need for a method that can efficiently increase the amount of vinblastine in Madagascar periwinkle.

The present inventors have extensively studied the wavelengths that contribute to increasing the amount of vinblastine in Madagascar periwinkle. The present invention was completed by discovering that it hardly contributes to

According to the present invention, the whole of Madagascar periwinkle or at least a portion including leaves is irradiated with light in a wavelength range of 370 nm or more and 400 nm or less in an amount effective to increase the amount of vinblastine in the Madagascar periwinkle, A method of treating Madagascar periwinkle, wherein the irradiation amount of light in the wavelength range of 200 nm or more and less than 370 nm irradiated to the Madagascar periwinkle is less than 20% of the irradiation amount of light in the wavelength range of 370 nm or more and 400 nm or less. provided.
According to the present invention, there is also provided a method for obtaining vinblastine, which comprises extracting and/or purifying vinblastine from Madagascar periwinkle treated by the above treatment method.

Further, according to the present invention, it is possible to mainly emit light in a wavelength range of 370 nm or more and 400 nm or less, and the radiation amount of light in the wavelength range of 200 nm or more and less than 370 nm is the light emission in the wavelength range of 370 nm or more and 400 nm or less. A light source that is less than 20% of the amount of light; the illuminance of light in the wavelength range of 370 nm to 400 nm for Madagascar periwinkle is 0.5 W/m ² or more and 500 W/m ² or less, or the irradiation amount of the light is 150 kJ/m ² or more and a controller for controlling the light source so that the light source is 150000 kJ/m ² or less, and is used to increase the amount of vinblastine in Madagascar periwinkle.

According to the present invention, there is further provided a container for containing the whole plant of Madagascar periwinkle or at least a portion including leaves, and a first device for irradiating the Madagascar periwinkle in the container with light in a wavelength range of 370 nm or more and 400 nm or less. A light source that emits light in the wavelength range of 370 nm or more and 400 nm or less, and the amount of light emitted in the wavelength range of 200 nm or more and less than 370 nm is less than 20% of the light emission amount of the wavelength range of 370 nm or more and 400 nm or less. The first light source and the illuminance of light in a wavelength range of 370 nm to 400 nm for the capsicum in the container is 0.5 W/m ² or more and 500 W/m ² or less, or the irradiation amount of the light is 150 kJ/m ² or more. a first control unit for controlling the first light source so that the intensity is 150,000 kJ/m ² or less; a second light source for irradiating the periwinkle in the container with light containing red light; and the container. and a second control unit for controlling the second light source so that the photosynthetically active radiant flux density (PPFD) for Catharanthus roseus in the plant is 100 μmol/m ² /s or more and 400 μmol/m ² /s or less. A device for increasing the amount of vinblastine in Madagascar periwinkle is provided.

According to the present invention, it is possible to irradiate light in a specific wavelength range, which is remarkably effective in increasing the amount of vinblastine synthesized in Madagascar periwinkle, at a relatively high irradiation dose, while tissue by exposure to harmful shorter wavelength light exclusively Since the adverse effect on the periwinkle can be avoided, the amount of vinblastine in Madagascar periwinkle can be increased efficiently and/or dramatically.

The emission spectra of LEDs used in Examples (Experiments 1 to 3) are shown. The amount of vinblastine in Madagascar periwinkle (leaf discs) irradiated with LED light of various wavelengths (5 W/m ² ) for 5 days is shown. Number of samples: n=1-2. "ND" indicates that the vinblastine concentration was below the limit of detection. The amount of vinblastine in Madagascar periwinkle (leaf disc) irradiated with LED light (peak wavelength: 385 nm) at various illuminances for 5 days is shown. "Cont." indicates that only the white fluorescent lamp was used. Vinblastine content in Madagascar periwinkle (leaf discs) irradiated with LED light (peak wavelength: 385 nm; 50 W/m ² ) for various periods. Vinblastine synthesis rate in Madagascar periwinkle (leaf disc) irradiated with LED light (peak wavelength: 385 nm; 50 W/m ² ) for various periods. The emission spectra of LEDs and LDs used in Examples (Experiments 4 and 5) are shown. The amount of vinblastine in Madagascar periwinkle (leaf discs) irradiated with LED light (50 W/m ² ) with a peak wavelength of 375 nm or 396 nm for 5 days is shown. The amount of vinblastine in Madagascar periwinkle (leaf disc) irradiated with LED light (50 W/m ² ) with a peak wavelength of 375 nm or LD light (50 W/m ² ) with a peak wavelength of 376 nm for 5 days is shown. The amounts of vinblastine (A) and vincristine (B) in Madagascar periwinkle (leaf disc) used in Experiment 3 are shown.

From a certain point of view, the present invention is a method for treating Madagascar periwinkle, in which the whole or at least a part including leaves of Madagascar periwinkle is irradiated with light in a wavelength range of 370 nm or more and 400 nm or less to increase the amount of vinblastine in the Madagascar periwinkle. While irradiating in an effective amount, the irradiation amount of light in the wavelength range of 200 nm or more and less than 370 nm irradiated to the Madagascar periwinkle during the irradiation is less than 20% of the irradiation amount of light in the wavelength range of 370 nm or more and 400 nm or less. To provide a method characterized by:
In other words, the present invention is a method for increasing the amount of vinblastine in Madagascar periwinkle, wherein Madagascar periwinkle is irradiated with light in the wavelength range of 370 nm or more and 400 nm or less, while the 200 nm or more irradiated to the Madagascar periwinkle during the irradiation. The method is characterized in that the irradiation amount of light in the wavelength range of less than 370 nm is less than 20% of the irradiation amount of light in the wavelength range of 370 nm or more and 400 nm or less.
From another aspect, the present invention provides a method for producing Madagascar periwinkle with an increased vinblastine content, wherein Madagascar periwinkle is irradiated with light in a wavelength range of 370 nm or more and 400 nm or less, and during the irradiation, Provided is a method characterized in that the dose of light in the wavelength region of 200 nm or more and less than 370 nm with which Catharanthus roseus is irradiated is less than 20% of the dose of light in the wavelength region of 370 nm or more and 400 nm or less.

In the present invention, as shown by the examples described later, light in the wavelength range of about 370 nm to about 400 nm is remarkably effective in increasing the amount of vinblastine in Madagascar periwinkle, while ultraviolet light with a wavelength of less than 370 nm and light with a wavelength of more than 400 nm It is based on the new knowledge that it does not contribute to the increase of vinblastine in Catharanthus roseus. Considering that short-wavelength light, such as light with a wavelength of less than 370 nm, can damage plant tissue (thus reducing the yield of vinblastine), according to the present invention, a method effective for increasing the amount of vinblastine in Madagascar periwinkle It becomes possible to effectively/efficiently irradiate light of a specific wavelength/wavelength range, and as a result, the amount of vinblastine in Madagascar periwinkle can be efficiently and/or dramatically increased.

In the present invention, Catharanthus roseus has a specific wavelength range effective for increasing the amount of vinblastine, that is, 370 nm or more and 400 nm or less, more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less. Irradiate with light.
The illuminance of light in the wavelength region of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less) should be an amount effective to increase the amount of vinblastine in the irradiated Madagascar periwinkle. However, for example, 0.2 W/m ² or more and 500 W/m ² or less, preferably 0.5 W/m ² or more and 500 W/m ² or less, more preferably 1 W/m ² or more and 500 W/m ² or less, more preferably 1 W /m ² or more and 300 W/m ² or less, more preferably 3 W/m ² or more and 200 W/m ² or less, more preferably 5 W/m ² or more and 150 W/m ² or less, more preferably 10 W/m ² or more and 150 W/m ² or more 20 W/m ² or more and 100 W/m ² or less, more preferably 25 W/m ² or more and 80 W/m ² or less, and more preferably 25 W/m ² or more and 50 W/m ² or less. If it is less than 0.2 W/m ² or more than 500 W/m ² , it may not be possible to efficiently increase the amount of vinblastine in Madagascar periwinkle.

The irradiation time of light in the wavelength region of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less) is an effective time for increasing the amount of vinblastine in the irradiated Madagascar periwinkle. For example, 1 hour to 480 hours, preferably 1 hour to 320 hours, more preferably 1 hour to 168 hours, more preferably 24 hours to 168 hours, more preferably 36 hours to 160 hours. less than an hour. If it is less than 1 hour or more than 480 hours, it may not be possible to effectively increase the amount of vinblastine in Madagascar periwinkle.
The light in the wavelength region of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less) may be continuous light or intermittent light (for example, pulsed light). There may be.

For example, an effective dose-boosting amount of vinblastine in Madagascar periwinkle may have a lower limit of irradiation dose of 150, 300, 600, 1200, 2000, 2500, 3000, 4500, 6000, 7000 or 8000 kJ/m ² and an upper limit of can be a dose of 150000, 90000, 60000, 45000, 30000, 25000, 20000 or 15000 kJ/ ^m2 . If the irradiation dose is less than 50 kJ/m ² or more than 300000 kJ/m ² , it may not be possible to effectively increase the amount of vinblastine in Madagascar periwinkle. Specifically, the effective amount for increasing the amount of vinblastine in Madagascar periwinkle is 150 kJ/m ² or more and 150000 kJ/m ² or less, preferably 150 kJ/m ² or more and 90000 kJ/m ² or less, more preferably 150 kJ/m ² or more and 60,000 kJ/m ² or less, more preferably 300 kJ/m ² or more and 60,000 kJ/m ² or less, more preferably 600 kJ/m ² or more and 60,000 kJ/m ² or less, more preferably 1200 kJ/ ^m2 or more and 60000 kJ/ ^m2 or less, more preferably 1200 kJ/ ^m2 or more and 45000 kJ/ ^m2 or less, more preferably 2000 kJ/ ^m2 or more and 45000 kJ/ ^m2 or less, more preferably 2000 kJ/m2 ² or more and 30000 kJ/ ^m2 or less, more preferably 2500 kJ/ ^m2 or more and 30000 kJ/ ^m2 or less, more preferably 2500 kJ/ ^m2 or more and 25000 kJ/ ^m2 or less, more preferably 3000 kJ/ ^m2 or more and 25000 kJ/ ^m2 or less, More preferably 4500 kJ/m ² or more and 25000 kJ/m ² or less, more preferably 6000 kJ/m ² or more and 25000 kJ/m ² or less, more preferably 7000 kJ/m ² or more and 25000 kJ/m ² or less.

Madagascar periwinkle may be irradiated with light in a specific wavelength range of 370 nm or more and 400 nm or less alone, or may be irradiated as light containing light in a wavelength/wavelength range other than the specific wavelength range. When the light irradiated to Madagascar periwinkle contains light in the wavelength range of 200 nm or more and less than 370 nm, the irradiation amount of light in the wavelength range of 200 nm or more and less than 370 nm is 370 nm or more and 400 nm or less (more specifically, 375 nm or more and 396 nm or less, more Specifically, less than 20%, preferably less than 10%, more preferably less than 5%, preferably less than 2%, preferably less than 1% of the irradiation amount of light in the wavelength range of 375 nm or more and 395 nm or less) do. While irradiating Catharanthus roseus with light of a wavelength/wavelength range that is effective for increasing vinblastine dosage, irradiation doses of light of shorter wavelengths/wavelength ranges that do not contribute to increasing vinblastine dosage and may adversely affect plant tissues (e.g., lead to death) can efficiently increase the amount of vinblastine in Madagascar periwinkle from the viewpoint of yield/productivity (in other words, obtain a large number of Madagascar periwinkle with increased vinblastine content).

On the other hand, light with a wavelength around 405 nm contributes almost no increase in vinblastine, even though it has a wavelength close to the specific wavelength range. Therefore, from the viewpoint of energy efficiency, the light in the wavelength range of 370 nm or more and 400 nm or less is preferably light from a light source that mainly emits light in the wavelength range of 370 nm or more and 400 nm or less.
As used herein, the term “light source that mainly emits light in the wavelength range of 370 nm or more and 400 nm or less” means that the amount of emitted light in the wavelength range of 370 nm or more and 400 nm or less is equal to the amount of light emitted in the entire wavelength range. A light source that emits 50% or more, more specifically 60% or more, more specifically 70% or more, more specifically 80% or more. The radiation amount of light in a wavelength range of 200 nm or more and less than 370 nm from a light source that mainly emits light in a wavelength range of 370 nm or more and 400 nm or less is preferably 370 nm or more and 400 nm or less (more specifically, 375 nm or more and 396 nm or less, more specifically Specifically, it is less than 20%, more preferably less than 10%, more preferably less than 5%, more preferably less than 2%, more preferably 1% of the irradiation amount of light in the wavelength range of 375 nm or more and 395 nm or less) is less than The radiation amount of light in the wavelength range of more than 400 nm and less than 430 nm from a light source that mainly emits light in the wavelength range of 370 nm or more and 400 nm or less is 370 nm or more and 400 nm or less (more specifically, 375 nm or more and 396 nm or less, more specifically is more preferably less than 40% of the radiation amount of light in the wavelength range of 375 nm or more and 395 nm or less) from the viewpoint of energy efficiency, more preferably less than 30%, more preferably less than 20%, more preferably 10 %.
The light source that mainly emits light in the wavelength range of 370 nm or more and 400 nm or less is preferably a light source that has a single peak in the wavelength range of 370 nm or more and 400 nm or less.

The light in the wavelength range of 370 nm or more and 400 nm or less includes at least red light (more specifically, light in the wavelength range of 600 nm or more and 700 nm or less) (for example, monochromatic red light or white light). Madagascar periwinkle may be irradiated with a combination of Since red light promotes the synthesis of vindoline and catharanthine, which are precursors for synthesizing vinblastine, the amount of vinblastine in Madagascar periwinkle can be further increased by irradiating Madagascar periwinkle in addition to light in the wavelength range of 370 nm or more and 400 nm or less. . In a specific embodiment, the light in the wavelength range of 370 nm or more and 400 nm or less is irradiated to Madagascar periwinkle at the same time as the light comprising at least red light.
The light comprising at least red light is, for example, 50 μmol/m ² /s or more and 500 μmol/m ² /s or less, more specifically 100 μmol/m ² /s or more and 400 μmol/m ² /s or less of photosynthetically active radiation. Catharanthus roseus can be irradiated at the flux density (PPFD).

When the light in the wavelength range of 370 nm or more and 400 nm or less is irradiated to Madagascar periwinkle together with the light containing at least red light, the light in the wavelength range of 370 nm or more and 400 nm or less emits light containing at least red light. may be emitted from the same light source as , or may be emitted from a different light source.
In a specific embodiment, the light in the wavelength range of 370 nm or more and 400 nm or less is irradiated to Madagascar periwinkle from a light source (first light source) that mainly emits light in the wavelength range of 370 nm or more and 400 nm or less, and at least red light is emitted. The comprising light is applied to the Madagascar periwinkle from a light source emitting primarily red light and/or a white light source (second light source or second and third light sources). As used herein, the term “light source that mainly emits red light” means that the amount of emitted red light is 50% or more, more specifically 60%, of the amount of emitted light in the entire wavelength range. Above, more specifically 70% or more, and more specifically 80% or more.

The light source that can be used to irradiate the Catharanthus roseus with light in the wavelength range of 370 nm or more and 400 nm or less is not particularly limited as long as it can irradiate the light in the wavelength range. For example, commonly used ultraviolet light such as a UV lamp A light source can be used. As the UV lamp, it is preferable to use, for example, a metal halide lamp or a high-pressure mercury lamp. Alternatively, light extracted from sunlight through an optical filter or the like may be used.
The light source used emits light in the wavelength range of 200 nm to less than 370 nm together with light in the wavelength range of 370 nm to 400 nm, and the amount of radiation is 20% or more of the radiation amount of light in the wavelength range of 370 nm to 400 nm. In some cases, a filter having a higher transmittance for light in the wavelength range of 370 nm or more and 400 nm or less than that for light in the wavelength range of 200 nm or more and less than 370 nm may be used together.

From the viewpoint of energy efficiency, the light in the wavelength range of 370 nm or more and 400 nm or less is irradiated as light having a main peak wavelength, for example, within 385 ± 10 nm, more preferably within 385 ± 5 nm, more preferably within 385 ± 2 nm. be. Preferably, the second peak does not exist (that is, a single peak), or if it does exist, its intensity is 1/10 or less of that of the main peak.
The half width of the main peak (having a wavelength of 370 nm or more and 400 nm or less) is preferably 0.5 nm or more and 15 nm or less. The half-value width of the main peak of 15 nm or less reduces the amount of vinblastine in Madagascar periwinkle while avoiding irradiation of the Madagascar periwinkle with light in a shorter wavelength region that does not contribute to increasing the amount of vinblastine in Madagascar periwinkle (which can be exclusively harmful). In addition to being able to irradiate light in a wavelength range that is remarkably effective for increasing the amount (that is, selective irradiation), energy efficiency is further improved. Although light with a main peak half-value width of less than 0.5 nm can also be used in the present invention, it is currently preferred to use light with a main peak half-value width of 0.5 nm or more from the viewpoint of cost effectiveness. In a preferred specific embodiment, the light with which Madagascar periwinkle is irradiated is light having a wavelength spectrum with a peak wavelength of 385±5 nm and a half width of 0.5 nm or more and 15 nm or less.

Light emitting diodes (LEDs) and laser diodes (LDs) having a single peak in the emission spectrum are particularly preferable as the light source for irradiating the Madagascar periwinkle with light in the wavelength range of 370 nm or more and 400 nm or less. When using an LED or LD as a light source, it is effective to increase the amount of vinblastine in Madagascar periwinkle while avoiding irradiation of the Madagascar periwinkle with light in the shorter wavelength range that does not contribute to increasing the amount of vinblastine in Madagascar periwinkle (which can be exclusively harmful). It becomes possible to easily irradiate light in the wavelength range. The use of LEDs or LDs is also preferable from the viewpoint of energy efficiency and economy due to their energy intensity, low heat generation, low power consumption, and long life. In addition, it becomes easier to control/manage the dose.
An LED capable of emitting light in a wavelength range of 370 nm or more and 400 nm or less can be one using, for example, InGaN-based material or AlInGaN-based material. A specific example of such an LED is type: NVSU233BU385 (center wavelength 385 nm; Nichia Corporation). Further, as a specific example of the LD, type: NDU4116 (center wavelength 375 nm; Nichia Corporation) can be mentioned.

The light source that mainly emits red light is not particularly limited as long as it can irradiate light in the relevant wavelength range, for example, red fluorescent lamp, red lamp, high pressure sodium lamp, red LED etc. A red light source can be used.
Any white light source used for plant cultivation, such as an incandescent lamp, a white fluorescent lamp, a white lamp, a white metal halide lamp, and sunlight can be used as the white light source.

Catharanthus roseus as used herein is not particularly limited as long as it belongs to the Catharanthus roseus species of the family Apocynaceae, and may be any variety (original species, variety, cultivated/horticultural variety).
Madagascar periwinkle may be genetically engineered, for example, genes involved in the vinblastine synthesis system, proteins involved in the transport of catalatin, the synthetic precursor of vinblastine, or photoreceptors sensitive to the UVA region. may

Catharanthus roseus may be in the form of the whole plant body, or may be in the form of parts of the plant body such as shoots or leaves, when irradiated with light in the wavelength range of 370 nm or more and 400 nm or less. It may be in the form of a product (eg, leaf-stem organ culture, callus culture). Madagascar periwinkle is preferably a plant having at least leaves from the viewpoint of yield.
When the Madagascar periwinkle is in the form of the whole plant when irradiated with light in the wavelength range of 370 nm or more and 400 nm or less, it may be in a cultivated state or in a non-cultivated state (a state in which nutrients are not supplied through the roots). good. Here, the cultivation may be soil cultivation or hydroponic cultivation (for example, hydroponics or solid medium cultivation). Hydroponic cultivation is preferable in that it can be performed under sterile conditions.

Madagascar periwinkle when irradiated with light in the wavelength range of 370 nm or more and 400 nm or less may be harvested plants or parts thereof as long as they are in a viable state. As used herein, the phrase "in a viable state" with respect to Madagascar periwinkle refers to a state in which the vinblastine synthesis system is functioning.
The plant parts of Madagascar periwinkle (in particular, the parts containing leaves) may be cut. Any cutting method can be used, but it is preferable that the cutting surface is sharp, and it is more preferable to cut so that the cutting area is large. According to this embodiment, it is possible to efficiently irradiate and/or mass-process light of a wavelength that is remarkably effective for increasing the amount of vinblastine (to leaf tissue containing a relatively large amount of vinblastine), thereby increasing the amount of vinblastine more efficiently. can be achieved effectively.
When irradiation of light in the wavelength range of 370 nm or more and 400 nm or less according to the present invention is performed on a non-cultivated plant body of Madagascar periwinkle or a part thereof (preferably a cut part), it prevents aging and / or removes light from the light source. From the viewpoint of reducing heat reception, the plant body or part is preferably placed in water or sprayed with water. The water can be any water, but pure water is preferred. Additives to water include alcohol for sterilization purposes, surfactants to improve landing, acids such as vinegar and citric acid for antibacterial/bacteriostatic purposes, and sugars, vitamins, and inorganic salts as energy sources. , plant aging hormone inhibitors and the like may be added.

The Madagascar periwinkle at the time of irradiation with light in the wavelength region of 370 nm or more and 400 nm or less according to the present invention may be at any stage of growth, but from the viewpoint of yield, it is preferably a mature plant or a part thereof.
When Madagascar periwinkle is cultivated or cultured, irradiation with light in the wavelength range of 370 nm or more and 400 nm or less according to the present invention may be performed in either the light period or the dark period, but it is necessary to supply a synthetic precursor that contributes to increasing the amount of vinblastine. From this point of view, it is preferable to perform it in the light period.

Cultivation or culture of Madagascar periwinkle may be open field cultivation or cultivation or culture under a controlled environment. Environmental conditions to be controlled include, for example, light-dark cycle, temperature, humidity, natural and/or artificial light exposure, and carbon dioxide concentration. These conditions are not particularly limited as long as they are suitable for cultivation/growth of Madagascar periwinkle to be used.
The light-dark cycle can be appropriately selected according to the Madagascar periwinkle to be cultivated and the stage of growth (for example, long-day conditions with a light period of 14-18 hours, or short-day conditions with a light period of 6-10 hours, for example). As a light source for artificial light, conventionally used incandescent lamps, fluorescent lamps, white lamps, high-pressure sodium lamps, metal halide lamps, LEDs, and the like can be used. The artificial light is applied by PPFD, which is appropriately set according to the Madagascar periwinkle to be cultivated, the stage of growth, and the like. PPFD can be, for example, 50 μmol/m ² /s or more and 500 μmol/m ² /s or less, more specifically 100 μmol/m ² /s or more and 400 μmol/m ² /s or less.
The temperature can be eg 20-30° C. and the humidity can be eg 50-80%.
Carbon dioxide concentration can be, for example, about 1000-1500 ppm.
Fertilizer/liquid fertilizer can be appropriately selected according to the Madagascar periwinkle to be cultivated. Fertilizers/liquids generally contain nitrogen, phosphorus and potassium.

As used herein, "increased amount of vinblastine" or "increased content (of) vinblastine" or "increased amount of vinblastine" refers to 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more Specifically, the amount of vinblastine is increased, for example, twice or more the amount of vinblastine in non-irradiated Madagascar periwinkle, preferably at least 2 times the amount of vinblastine in non-irradiated Madagascar periwinkle. 3 times or more, more preferably 4 times or more, more preferably 5 times or more, more preferably 10 times or more, more preferably 20 times or more, more preferably 30 times or more, more preferably 40 times or more, more preferably 50 times This means that it has more than doubled.
Quantitation of vinblastine may be performed using any known method, for example by chromatography. Chromatography includes liquid chromatography (eg, HPLC). Liquid chromatography can be reverse phase chromatography.

From another point of view, irradiation with light in the wavelength region of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less) according to the present invention, compared with the non-irradiated one. , the amount of vinblastine is 2 times or more, for example 3 times or more, more preferably 4 times or more, more preferably 5 times or more, preferably 10 times or more, more preferably 20 times or more, more preferably 30 times or more, more preferably Catharanthus roseus that is 40 times or more, more preferably 50 times or more can be produced. Therefore, from this point of view, the present invention irradiates Catharanthus roseus with light in a wavelength range of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less). The amount of light in the wavelength range of 200 nm or more and less than 370 nm that is irradiated to the Madagascar periwinkle during irradiation is reduced to the above 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less). Provided is a method for producing Madagascar periwinkle with increased vinblastine content, characterized in that the irradiation dose of light in the wavelength range is less than 20%.
According to the method for producing Madagascar periwinkle of the present invention, the extraction efficiency of vinblastine from Madagascar periwinkle is dramatically increased. As a result, vinblastine can also be provided at low cost.
With respect to the method for producing Madagascar periwinkle of the present invention, all of the matters described above for the method of treating Madagascar periwinkle of the present invention (that is, the method for increasing the amount of vinblastine) are all applicable.

Madagascar periwinkle produced by the above method of the present invention is suitable as a raw material for producing vinblastine or a salt thereof.
Accordingly, the present invention provides a method for producing vinblastine or a salt thereof, comprising:
A step of irradiating the Madagascar periwinkle with light in a wavelength range of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less), wherein the Madagascar periwinkle is irradiated during the irradiation. The irradiation amount of light in the wavelength range of 200 nm or more and less than 370 nm is less than the irradiation amount of light in the wavelength range of 370 nm or more and 400 nm or less (more specifically, 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less). A process comprising obtaining vinblastine or a salt thereof from said Madagascar periwinkle which is less than 20%.

Madagascar periwinkle is irradiated with light in a wavelength range of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less), while the Madagascar periwinkle is irradiated during the irradiation. The irradiation amount of light in the wavelength range of 200 nm or more and less than 370 nm is 20% of the irradiation amount of light in the wavelength range of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less). By making it less than, as described above, Madagascar periwinkle with an increased vinblastine content can be efficiently produced/obtained.
With respect to the Madagascar periwinkle production process of the present invention, all of the above statements regarding the method of increasing the amount of vinblastine of the present invention apply.

The salt of vinblastine may be, for example, hydrochloride, sulfate, acetate, tartrate or citrate, preferably sulfate.
The step of obtaining vinblastine or a salt thereof from Madagascar periwinkle can be performed, for example, by extraction. 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 organic solvents. Organic solvents are, for example, methanol, ethanol, n- or iso-propanol, butanol, acetonitrile, acetone, dioxane, ethyl acetate, dimethylsulfoxide, dimethylformamide, ethylene glycol, tetrahydrofuran, dichloromethane, trichloromethane, tetrachloromethane, chloroform, Examples include trichlorethylene, hexane, toluene, and benzene. An acid may be added to the organic solvent. Acids 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 contained in the organic solvent in a weight ratio of eg 0.1-10%, preferably 1-3%.
Extraction may be performed at elevated temperature (eg, 80-90° C.) and/or under pressure. The extraction may be reflux extraction. The extraction time is not particularly limited and can be determined appropriately from the viewpoint of extraction efficiency.

For extraction, the whole plant of Madagascar periwinkle can be used, but a part of the plant including at least the leaves may also be used. Since vinblastine is abundantly contained in leaves, it is preferable to use leaves for extraction from the viewpoint of efficiency. The plant body (whole or part) may be used for extraction as it is, or may be used after pulverization. The plant may be dried and/or frozen prior to extraction or grinding. Drying may be performed by any method, such as hot air drying, normal temperature drying, reduced pressure drying, or freeze drying.
The extract may be filtered through an appropriate filter or subjected to centrifugation, for example, to remove contaminants.
A specific extraction method for vinblastine is described, for example, in US Pat. No. 4,749,787.

Vinblastine may be purified from the resulting extract.
Purification can be performed, for example, by chromatography. Chromatography can be, for example, column chromatography (especially HPLC), preferably in reversed-phase mode.
The column used for column chromatography can be appropriately selected from known columns according to the separation mode. For reversed-phase chromatography, octadecylated silica gel (ODS) columns (also called C18 columns) can generally be used, but are not limited thereto.

In chromatography (e.g., gradient method), 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) is used as an eluent. can be used. Polar organic solvents include, for example, methanol, ethanol, n- or iso-propanol, acetonitrile, acetone, dioxane, ethyl acetate, dimethylsulfoxide, dimethylformamide, ethylene glycol, tetrahydrofuran. Acids such as trifluoroacetic acid, formic acid, acetic acid, phosphoric acid and trichloroacetic acid may be added to the eluent, for example 0.01-10M.
Although the flow rate is not particularly limited, it can be, for example, 0.1-2 ml/min.
Vinblastine can be detected, for example, by measuring absorbance at 250-300 nm.
A specific method for isolating vinblastine from Madagascar periwinkle is described, for example, in US Pat. No. 4,749,787.

Vinblastine or a salt thereof produced by the above method of the present invention can be used as a raw material for pharmaceuticals and other industrial products. Therefore, according to the present invention, the produced vinblastine can be inexpensively provided as a raw material for pharmaceuticals and other industrial products.
Vinblastine or a salt thereof produced by the above method of the present invention is also suitable as a raw material for (semi)synthesizing vinblastine derivatives such as vincristine and vindesine, or salts thereof.

(１)
で表されるビンブラスチン誘導体又はその塩の製造方法であって、
ニチニチソウに、370nm以上400nm以下(より具体的には375nm以上396nm以下、より具体的には375nm以上395nm以下)の波長域の光を照射する工程であって、該照射の間に該ニチニチソウに照射される200nm以上370nm未満の波長域の光の照射量が前記370nm以上400nm以下(より具体的には375nm以上396nm以下、より具体的には375nm以上395nm以下)の波長域の光の照射量の20％未満である工程、
該ニチニチソウからビンブラスチン又はその塩を取得する工程、及び
該ビンブラスチン又はその塩を化学的に修飾して前記ビンブラスチン誘導体又はその塩を合成する工程
を含むことを特徴とする方法も提供する。 Accordingly, the present invention provides the following formula (1):

(1)
A method for producing a vinblastine derivative or a salt thereof represented by
A step of irradiating the Madagascar periwinkle with light in a wavelength range of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less), wherein the Madagascar periwinkle is irradiated during the irradiation. The irradiation amount of light in the wavelength range of 200 nm or more and less than 370 nm is less than the irradiation amount of light in the wavelength range of 370 nm or more and 400 nm or less (more specifically, 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less). a process that is less than 20%,
A method is also provided, comprising the steps of: obtaining vinblastine or a salt thereof from Madagascar periwinkle; and chemically modifying the vinblastine or salt thereof to synthesize the vinblastine derivative or salt thereof.

In formula (1),
n Rs are each independently halogen (preferably halogen selected from fluorine, chlorine, bromine and iodine), C1-C4 (preferably C1-C3, more preferably C1-C2) alkyl, alkenyl, alkynyl, alkyloxycarbonyl or alkylthio, aryl (preferably substituted or unsubstituted phenyl), cyano, nitro, or formyl;
n is an integer of 0 to 4, preferably an integer of 0 to 3, more preferably an integer of 0 to 2, more preferably an integer of 0 or 1;
R ¹ and R ³ are each independently C1-C4 (preferably C1-C3, more preferably C1-C2) alkyloxycarbonyl, aminocarbonyl, methylaminocarbonyl or hydrazinocarbonyl, preferably CH ₃ OCO- or _NH2CO- ;
R ² is hydrogen, methyl or formyl, preferably methyl or formyl,
R ⁴ is hydrogen or C1-C4 (preferably C1-C3, more preferably C1-C2) alkylcarbonyl, preferably hydrogen or CH ₃ CO-.

A specific example of the vinblastine derivative represented by formula (1) may be vincristine or vindesine, or a salt thereof.
The salt of the vinblastine derivative is not particularly limited as long as it is a pharmaceutically acceptable salt, and may be, for example, hydrochloride, sulfate, acetate, tartrate or citric acid, preferably sulfate.

Regarding the step of obtaining vinblastine or a salt thereof from Madagascar periwinkle in the method for producing a vinblastine derivative or a salt thereof of the present invention, all of the matters described above for the method of producing vinblastine or a salt thereof of the present invention apply.
Chemical modification can be, for example, deacetylation, oxidation, substitution of an ester or carboxyl group with an amide group, and/or transesterification. Specific methods of chemical modification are described, for example, in Molecules 17, 5893-5914, 2012 and US Pat. No. 4,203,898.
All of the matters described above regarding the method for increasing the amount of vinblastine of the present invention apply to the Catharanthus roseus production step in the method for producing a vinblastine derivative or salt thereof of the present invention.

The present invention also provides
It can mainly emit light in the wavelength range of 370 nm or more and 400 nm or less (more specifically, 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less), and the amount of light emitted in the wavelength range of 200 nm or more and less than 370 nm. is less than 20% of the radiation amount of light in the wavelength range of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less);
The illuminance of light in the wavelength range of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less) for Catharanthus roseus is 0.5 W/m ² or more and 500 W/m ² or less, or and a controller for controlling the light source so that the irradiation amount of the light is 150 kJ/m ² or more and 150000 kJ/m ² or less, and is used to increase the amount of vinblastine in Catharanthus roseus.
The lighting device of the present invention includes the method for treating Madagascar periwinkle according to the present invention (or the method for increasing the amount of vinblastine in Madagascar periwinkle according to the present invention), the method for producing Madagascar periwinkle with increased vinblastine content, and the production of vinblastine or a salt thereof. It is suitable for use in a method of production and a method of producing a vinblastine derivative or a salt thereof (hereinafter collectively referred to as "the method of the present invention").

For a light source that mainly emits light in the wavelength range of 370 nm or more and 400 nm or less (more specifically 375 nm or more and 396 nm or less, more specifically 375 nm or more and 395 nm or less), all of the matters described above with respect to the method of the present invention apply. .
This light source emits light in a wavelength range of 200 nm or more and less than 370 nm (preferably 100 nm or more and less than 370 nm, more preferably 10 nm or more and less than 370 nm, still more preferably 1 nm or more and less than 370 nm). is less than 20%, preferably less than 10%, more preferably less than 5%, more preferably 2% less than, more preferably less than 1%.
In some embodiments, from the viewpoint of energy efficiency, the light source preferably emits light in a wavelength range of more than 400 nm and less than or equal to 430 nm. Below, more specifically, less than 40%, more preferably less than 30%, preferably less than 20%, more preferably less than 10% of the light in the wavelength range of 375 nm or more and 395 nm or less. . In a more specific embodiment, this light source is a light source having a single peak in the wavelength range of 370 nm or more and 400 nm or less.
Suitable specific examples of light sources that mainly emit light in the wavelength range of 370 nm or more and 400 nm or less include LEDs, LDs, metal halide lamps (with necessary filters (see above)), high-pressure mercury lamps, etc. LED or LD is more preferable, and LED or LD having a peak wavelength of 375 nm or more and 396 nm or less, more specifically, a wavelength range of 375 nm or more and 395 nm or less is more preferable. LEDs or LDs with a wavelength spectrum are more preferred.
The illumination device of the present invention may have only one light source that mainly emits light in the wavelength range of 370 nm or more and 400 nm or less, or may have a plurality of light sources. In the latter case (with more than one), the light sources may be provided as LDs, arrays of LEDs or clusters of LEDs.

The control unit controls the illuminance of light in the wavelength range of 370 nm to 400 nm (more specifically, 375 nm to 396 nm, more specifically 375 nm to 395 nm) for Madagascar periwinkle so that the illuminance is 0.5 W/m ² to 500 W/m ² . (more preferably 0.5 W/m ² or more and 100 W/m ² or less, more preferably 0.5 W/m ² or more and 50 W/m ² or less), or the light irradiation amount is 150 kJ/m ² or more and 150000 kJ/m ² The light source is controlled so as to achieve the following (more preferably 1200 kJ/m ² or more and 60000 kJ/m ² or less, more preferably 2000 kJ/m ² or more and 30000 kJ/m ² or less). More specifically, the controller may control the dimming of the light source, and additionally control the timing of turning on and off the light source. The control unit can be, for example, a pulse width modulation circuit or a pulse width modulation circuit and a timer, and can be configured with, for example, a microcomputer, relays and/or switching elements. For example, the timer can set the lighting time of the light source to 8 to 16 hours or 1 hour or more, for example 1 hour or more and 480 hours or less, more specifically 24 hours or more and 168 hours or less.

In addition to the light source (first light source) that mainly emits light in the wavelength range of 370 nm or more and 400 nm or less, the lighting device of the present invention includes at least red light (more specifically, a wavelength of 600 nm or more and 700 nm or less) A light source (second light source) that emits light containing light in the wavelength range of . The second light source can be, for example, an incandescent lamp, a white or red fluorescent lamp, a white or red lamp, a high pressure sodium lamp, a metal halide lamp, a xenon lamp, a red LED, sunlight, or the like. In this case, the lighting device of the present invention may further include a second control unit that controls the timing of dimming and/or lighting and extinguishing of the second light source. light source may be controlled.
The light from the second light source has a PPFD of, for example, 50 μmol/m ² /s or more and 500 μmol/m ² /s or less, more specifically 100 μmol/m ² /s or more and 400 μmol/m ² /s or less. obtain. The lighting device of the present invention may have only one second light source, or may have a plurality of second light sources.
In one embodiment, the lighting device of the present invention comprises an LED or LD array or an LED cluster composed of a plurality of LEDs or LDs as first light sources and a plurality of LEDs or LDs as second light sources. be prepared.

In the above method of the present invention, the illumination device of the present invention irradiates the catharanthus with light in a wavelength range of 370 nm or more and 400 nm or less, and irradiates the Catharanthus roseus with light in a wavelength range of 200 nm or more and less than 370 nm during the irradiation. to less than 20% of the light irradiation amount in the wavelength range of 370 nm or more and 400 nm or less.
The lighting device of the present invention can be used in any manner as long as it can irradiate a plant body (especially leaves) of Catharanthus roseus with light in a wavelength range of 370 nm or more and 400 nm or less (e.g., above, below, or to the side of Catharanthus roseus). , obliquely upward and/or obliquely downward).
The lighting device of the present invention can be used for Madagascar periwinkle in a cultivated state as well as for Madagascar periwinkle in a non-cultivated state or after harvesting, and thus may be used, for example, in plant cultivation facilities, plant factories, and the like. , in post-harvest storage facilities for Madagascar periwinkle and in dedicated facilities for increasing the amount of vinblastine in Madagascar periwinkle.

The present invention also provides
a container for containing the whole plant and/or parts thereof of Madagascar periwinkle;
A first light source for irradiating the periwinkle in the container with light in a wavelength range of 370 nm or more and 400 nm or less, which emits light in a wavelength range of 370 nm or more and 400 nm or less, and emits light in a wavelength range of 200 nm or more and less than 370 nm. a first light source emitting less than 20% of the light emission in the wavelength range of 370 nm or more and 400 nm or less;
The illuminance of light in the wavelength range of 370 nm or more and 400 nm or less to the Catharanthus roseus in the container is 0.5 W/m ² or more and 500 W/m ² or less, or the light irradiation amount is 150 kJ/m ² or more and 150000 kJ/m ² or less. a first control unit that controls the first light source so that
a second light source for irradiating the periwinkle in the container with light comprising red light;
and a second control unit that controls the second light source so that the photosynthetically active radiant flux density (PPFD) for Catharanthus roseus in the container is 50 μmol/m ² /s or more and 500 μmol/m ² /s or less. A device for increasing the amount of vinblastine in Madagascar periwinkle is provided.

The container is not particularly limited as long as it has an internal space capable of accommodating the whole and/or part of the Catharanthus roseus plant (preferably the part including the leaves) (hereinafter simply referred to as "Catharanthus roseus"), for example , tank-shaped, drum-shaped with at least one opening, which may or may not be closable, eg by a lid or the like. The opening may be provided anywhere on the container (eg, at the top or side of the container).
The container may be placed in an upright position or may be placed in an inclined position. Alternatively, the container may be supported by a support mechanism so that it can be both upright and tilted (0° to 90° with respect to the vertical).
The container may have a structure capable of storing water therein and/or may include a water supply section for supplying water to Madagascar periwinkle in the container. The water supply can be of any type, for example of the pouring or spraying type. The water supply may also be a water circulation system having a filtration system along the way. A water circulation mechanism may include a pump.
The container itself may be rotated (rotating axis: 0° (vertical axis) to 90° (horizontal axis) relative to the vertical) so as to stir the Madagascar periwinkle contained therein, or the stirring mechanism may be attached. The stirring mechanism can be, for example, a rotating blade or a bubbling mechanism (bubbling gas: dry air or CO ₂ gas, for example).

Regarding the first light source, all of the matters described above in connection with the method of the present invention for the "light source that mainly emits light in the wavelength range of 370 nm or more and 400 nm or less" apply. The first light source is preferably an LED or LD, more preferably an LED or LD that emits light having a wavelength spectrum with a peak wavelength of 385±10 nm and a half width of 5 nm or more and 20 nm or less.
For the second light source, all that has been said above for "light comprising at least red light" in relation to the method of the invention applies. The second light source is, for example, an incandescent lamp, a white or red fluorescent lamp, a white or red lamp, a high pressure sodium lamp, a metal halide lamp, a xenon lamp, a red LED, sunlight.
The first and second light sources can be installed in any manner as long as the manner is such that predetermined light can be applied to the Catharanthus roseus in the container. For example, the first and second light sources may be provided such that predetermined light is emitted from the opening of the container to the Madagascar periwinkle.
For the first and second controls, all that has been said above with respect to the lighting device according to the invention applies.

The vinblastine bulking device of the present invention may include, for example, a temperature controller, a pH controller, an _O2 and/or _CO2 supply, and may further include sensors to monitor the environment inside the container. The sensors can be, for example, water temperature sensors, pH sensors, conductivity sensors, absorbance sensors, gas sensors (eg _O2 and/or _CO2 sensors). The vinblastine bulking device of the present invention may be configured to automatically maintain the environment inside the container at predetermined conditions based on information from these sensors.

<Experiment 1>
Catharanthus roseus (cultivated variety “Titan” Pure White) was hydroponically cultivated under the following conditions.
<Hydroponic cultivation conditions>
White fluorescent lamp: 100 μmol/m ² /s
Light/dark cycle: 16 hours/8 hours Room temperature: 23°C
Hydroponic solution: OAT house A prescription (pH: 6.0; EC: 1.0ds/m)

Leaves were harvested from Madagascar periwinkle 40-60 days after sowing. Leaf discs were prepared by cutting the collected leaves into 35 mm squares.
While immersed in ultrapure water (pH: 6.3; EC: 0.005 ds/m), the leaf disc was irradiated with the following light for 5 days (light/dark cycle: 16 hours/8 hours; room temperature: 23°C). The white fluorescent lamp and the LED light were irradiated at the same time.
White fluorescent lamp (150 μmol/m ² /s) only White fluorescent lamp (150 μmol/m ² /s) + LED light (peak wavelength: 280 nm; 5 W/m ² )
White fluorescent lamp (150 μmol/m ² /s) + LED light (peak wavelength: 310 nm; 5 W/m ² )
White fluorescent lamp (150 μmol/m ² /s) + LED light (peak wavelength: 365 nm; 5 W/m ² )
White fluorescent lamp (150 μmol/m ² /s) + LED light (peak wavelength: 385 nm; 5 W/m ² )
White fluorescent lamp (150 μmol/m ² /s) + LED light (peak wavelength: 405 nm; 5 W/m ² )

The types of LEDs used for irradiation with LED light of each wavelength are as follows.
<LED type>
・280nm: NCSU234BU280 FWHM 10nm Deep UV-LED
・310nm: NCSU234BU310 FWHM 10nm Deep UV-LED
・365nm: NCSU276AU365 FWHM 9nm UV-LED
・385nm: NVSU119CU385 FWHM 11nm UV-LED
・405nm: NVSU119CU405 FWHM 12nm UV-LED
Figure 1 shows the emission spectrum of each LED.

After irradiation, each leaf disc was freeze-ground and solvent-extracted with methanol. The obtained extract was subjected to LC-MS/MS analysis under the following analysis conditions to quantify vinblastine in the extract. For comparison, a leaf disc not irradiated with light was also analyzed in the same manner.
<LC-MS/MS analysis conditions>
・LC system ACQUITY UPLC system (Waters)
・Column ODS column (ACQUITY UPLC BEH C18 (2.1×150mm, 1.7μm), Waters)
・Column temperature 40℃
・Flow rate 0.3ml/min ・Injection volume 5μl
・Mobile phase Solvent A: 0.1% aqueous formic acid; Solvent B: Acetonitrile Time (min) 0 7 9 14 14.5 19.8
Solvent B (%) 20% 30% 95% 95% 20% 20%
・Detector SYNAPT HDMS (Waters, Q-TOF-MS)

result:
Figure 2 shows the amount of vinblastine obtained by LC-MS/MS analysis.
The amount of vinblastine in Catharanthus roseus that was irradiated with light having a wavelength of around 385 nm increased approximately 66-fold compared to that of Catharanthus roseus that was not irradiated with light ("0 day" in the figure).
In Madagascar periwinkle irradiated with white fluorescent light or light with a wavelength of around 280, 310, 365 or 405 nm, the amount of vinblastine was equal to or less than that in periwinkle that was not irradiated with light.

From these results, it was concluded that irradiation with light around 385 nm is effective for increasing the amount of vinblastine in Madagascar periwinkle, whereas irradiation with white light and light around 280, 310, 365, and 405 nm does not contribute to increasing the amount of vinblastine. It can be understood.

<Experiment 2>
Leaf discs prepared in the same manner as in Experiment 1 were immersed in ultrapure water (pH: 6.3; EC: 0.005 ds/m) and exposed to white fluorescent light (150 μmol/m ² /s) only (control) or white fluorescent light. Lamp (150 μmol/m ² /s) + LED light (peak wavelength: 385 nm) was irradiated for 5 days at various illuminances (5, 25, 50 and 150 W/m ² ) (light/dark cycle: 16 hours/8 hours; Room temperature: 23°C). The white fluorescent lamp and the LED light were irradiated at the same time. The type of LED used is NVSU119CU385 FWHM 11nm UV-LED.
After light irradiation, vinblastine extracted from each leaf disc was quantified by LC-MS/MS analysis in the same manner as in Experiment 1.

result:
The amounts of vinblastine obtained are shown in FIG. 3 (μg/g dry weight) and Table 1 (% by weight).

As is clear from FIG. 3 and Table 1, in Catharanthus roseus that was irradiated with light having a wavelength of about 385 nm for 5 days, vinblastine was obtained at an illuminance of 5 W/m ² to 150 W/m ² (irradiation dose of 1440 to 43200 mol/m ² ). A significant increase in the amount was confirmed. In particular, when the illuminance is 25-50 W/m ² (irradiance 7200-14400 mol/m ² ), the amount of vinblastine increases by about 220- to about 370-fold compared to Madagascar periwinkle (“Cont.”) irradiated only with white light. (Vinblastine content per dry weight: about 0.32% to about 0.54%).

<Experiment 3>
Leaf discs prepared in the same manner as in Experiment 1 were immersed in ultrapure water (pH: 6.3; EC: 0.005 ds/m) and exposed to white fluorescent light (150 μmol/m ² /s) only (control) or white fluorescent light. Lamp (150 μmol/m ² /s) + LED light (peak wavelength: 385 nm; 50 W/m ² ) was irradiated (light/dark Cycle: 16h/8h; room temperature: 23°C). The white fluorescent lamp and the LED light were irradiated at the same time. The type of LED used is NVSU119CU385 FWHM 11nm UV-LED.
After irradiation, each leaf disc was freeze-ground and solvent-extracted with methanol. The resulting extract was subjected to LC-MS/MS analysis and HCLP analysis to quantify vinblastine and vindoline and catharanthine, respectively, in the extract. The LC-MS/MS analysis conditions are the same as those used in Experiment 1. HCLP analysis conditions are as follows.

・検出：190～800nm <HCLP analysis conditions>
・Column: ODS column (Triart C18 (150×4.6mm, S-5μm), YMC)
・Column temperature: 40℃
・Flow rate: 1 ml/min ・Injection volume: 5 μl
・Mobile phase: Eluent A 0.1% formic acid aqueous solution
Eluent B 0.1% formic acid acetonitrile solution

・Detection: 190 to 800 nm

result:
The amounts of vinblastine obtained are shown in Figure 4 (μg/g dry weight) and Table 2 (% by weight).

As is clear from FIG. 4 and Table 2, in Catharanthus periwinkle irradiated with light having a wavelength of about 385 nm at an illuminance of 50 W/m ² , the irradiation time of 1 hour to 15 days (360 hours) (approximately 180 to 43200 kJ /m ² ), a significant increase in the amount of vinblastine was confirmed. In particular, during irradiation periods of 3-7 days (72-168 hours) (irradiance of about 8640-20160 kJ/ ^m2 ), vinblastine doses were significantly higher than those of Madagascar periwinkle (“Cont.”) irradiated only with white light. about 240- to about 290-fold increase (vinblastine content by dry weight: about 0.32% to about 0.39%).

The vinblastine synthesis rate calculated from the amount of vinblastine obtained by LC-MS/MS analysis and the amounts of vindoline and catharanthine obtained by HCLP analysis (= amount of vinblastine/(amount of vinblastine + amount of vindoline + amount of catarathine)) is shown in FIG. show.
As is clear from FIG. 5, in Catharanthus periwinkle irradiated with light having a wavelength of about 385 nm at an illuminance of 50 W/m ² , the synthesis rate of vinblastine was reduced up to 5 days of irradiation (irradiation dose of about 14400 kJ/m ² ). It was confirmed that it increased and then remained almost constant.
In addition, discoloration was observed in leaf discs irradiated for 10 days or 15 days. This discoloration is considered to be due to long-term irradiation with short-wavelength light.

<Experiment 4>
Leaf discs prepared in the same manner as Experiment 1 were immersed in ultrapure water (pH: 6.3; EC: 0.005 ds/m) and irradiated with the following light for 5 days (light/dark cycle: 16 hours/8 time; room temperature: 23°C). The white fluorescent lamp and the LED light were irradiated at the same time.
White fluorescent lamp (150 μmol/m ² /s) only (control)
White fluorescent lamp (150 μmol/m ² /s) + LED light (peak wavelength: 375 nm; 50 W/m ² )
White fluorescent lamp (150 μmol/m ² /s) + LED light (peak wavelength: 396 nm; 50 W/m ² )
The type of LED used for light irradiation of each wavelength is NVSU119CU375 half width 9 nm UV-LED for peak wavelength 375 nm, and NVSU119CU395 half width 12 nm UV-LED for peak wavelength 396 nm. FIG. 6 shows the emission spectrum of each LED.
After light irradiation, vinblastine extracted from each leaf disc was quantified by LC-MS/MS analysis in the same manner as in Experiment 1.

result:
The amount of vinblastine obtained is shown in FIG.

As is clear from FIG. 7 and Table 3, a significant increase in the amount of vinblastine was also confirmed in Catharanthus periwinkle irradiated with light having a wavelength of around 375 nm and around 396 nm. In particular, when irradiated with light having a wavelength of around 375 nm, the amount of vinblastine increased approximately 800-fold compared to that of Madagascar periwinkle (“Cont.”) irradiated only with white light (vinblastine content per dry weight: approximately 1.09 %).
From these results, it was confirmed that irradiation with light within a wavelength range of 370 nm or more and 400 nm or less (more specifically, wavelengths of 375 nm or more and 396 nm or less) is effective for increasing the amount of vinblastine in Madagascar periwinkle.

<Experiment 5>
Leaf discs prepared in the same manner as in Experiment 1 were immersed in ultrapure water (pH: 6.3; EC: 0.005 ds/m) and irradiated with the following light for 5 days (light/dark cycle: none [continuous irradiation ]; room temperature: 25° C.).
White fluorescent lamp (150 μmol/m ² /s) only (control)
LED light (peak wavelength: 375nm; 50W/m ² )
LD light (peak wavelength: 376nm; 50W/m ² )
The type of the light-emitting diode used for the irradiation of the LED light is NVSU119CU375 9 nm half width UV-LED, and the type of the laser diode slot module used for the irradiation of the LD light is NUU102E. FIG. 6 shows the emission spectra of the used LED and LD slot module.
After light irradiation, vinblastine extracted from each leaf disc was quantified by LC-MS/MS analysis in the same manner as in Experiment 1.

result:
The amount of vinblastine obtained is shown in FIG. 8 and Table 4.

As is clear from FIG. 8 and Table 4, the LD light-irradiated Madagascar periwinkle also showed a significant increase in the amount of vinblastine, as in the LED light-irradiated Madagascar periwinkle. LD light irradiation increased the amount of vinblastine in Madagascar periwinkle by about 280-fold (vinblastine content per dry weight: about 0.38%) compared to irradiation with white light only (“Cont.”).
From these results, it was confirmed that the amount of vinblastine in Madagascar periwinkle remarkably increases by irradiating with light within the predetermined wavelength range (370 nm or more and 400 nm or less) of the present invention, regardless of the type of light source.

<Experiment 6>
In experiment 3, vincristine was quantified by LC-MS/MS analysis using leaf discs from control and 1 day and 3 day irradiation times of Madagascar periwinkle. The LC-MS/MS analysis conditions are the same as those used in Experiment 1.

result:
Figure 9 shows the amounts of vinblastine and vincristine obtained by LC-MS/MS analysis.
As is clear from FIG. 9, the amount of vincristine increases as the amount of vinblastine increases in Madagascar periwinkle irradiated with light of a specific wavelength.
From this result, it was confirmed that irradiation with light within the predetermined wavelength range of the present invention (370 nm or more and 400 nm or less) is also effective in increasing the amount of vincristine in Madagascar periwinkle.

Claims (14)

Catharanthus roseus in its entirety or at least a portion including leaves is irradiated with light in a wavelength range of 370 nm or more and 400 nm or less in an amount effective to increase the amount of vinblastine in the catharanthus roseus, during the irradiation A method for treating Madagascar periwinkle, wherein the irradiation amount of light in a wavelength range of 200 nm or more and less than 370 nm with which said Madagascar periwinkle is irradiated is less than 20% of the irradiation amount of light in a wavelength range of 370 nm or more and 400 nm or less. 2. The method according to claim 1, wherein leaves of Madagascar periwinkle are irradiated with the light in the wavelength range of 370 nm or more and 400 nm or less. 3. The method according to claim 1, wherein the irradiation amount of light in the wavelength range of 200 nm or more and less than 370 nm is less than 10% of the irradiation amount of light in the wavelength range of 370 nm or more and 400 nm or less. 4. The method according to any one of claims 1 to 3, wherein the illuminance of the light in the wavelength region of 370 nm or more and 400 nm or less is 0.5 W/m ² or more and 500 W/m ² or less. 5. The method according to any one of claims 1 to 4, wherein the illuminance of the light in the wavelength range of 370 nm or more and 400 nm or less is 5 W/m ² or more and 150 W/m ² or less. The method according to any one of claims 1 to 5, wherein the irradiation time of the light in the wavelength region of 370 nm or more and 400 nm or less is 1 hour or more and 480 hours or less. The method according to any one of claims 1 to 6, wherein the irradiation time of the light in the wavelength region of 370 nm or more and 400 nm or less is 24 hours or more and 168 hours or less. The method according to any one of claims 1 to 7, wherein the amount effective for increasing the amount of vinblastine in Madagascar periwinkle is an irradiation dose of 150 kJ/m ² or more and 150000 kJ/m ² or less. The method according to any one of claims 1 to 8, wherein the amount effective for increasing the amount of vinblastine in Madagascar periwinkle is an irradiation dose of 2000 kJ/m ² or more and 30000 kJ/m ² or less. 10. The method according to any one of claims 1 to 9, wherein the light in the wavelength region of 370 nm or more and 400 nm or less has a wavelength spectrum with a peak wavelength of 385±10 nm and a half width of 0.5 nm or more and 20 nm or less. 11. The method according to any one of claims 1 to 10, wherein the light in the wavelength region of 370 nm or more and 400 nm or less is light emitted from a light emitting diode or a laser diode. A method for obtaining vinblastine, which comprises extracting and/or purifying vinblastine from Madagascar periwinkle treated by the method according to any one of claims 1 to 11. A light source that can emit light mainly in a wavelength range of 370 nm or more and 400 nm or less, and that emits less than 20% of the light in the wavelength range of 200 nm or more and less than 370 nm. When,
The illuminance of light in the wavelength range of 370 nm or more and 400 nm or less to Catharanthus roseus is 0.5 W/m ² or more and 500 W/m ² or less, or the irradiation amount of the light is 150 kJ/m ² or more and 150000 kJ/m ² or less. and a control unit for controlling a light source, and is used for increasing the amount of vinblastine in Madagascar periwinkle. A container for housing the whole plant body of Madagascar periwinkle or at least a portion containing the leaves;
A first light source for irradiating the periwinkle in the container with light in a wavelength range of 370 nm or more and 400 nm or less, which emits light in a wavelength range of 370 nm or more and 400 nm or less, and emits light in a wavelength range of 200 nm or more and less than 370 nm. a first light source emitting less than 20% of the light emission in the wavelength range of 370 nm or more and 400 nm or less;
The illuminance of light in the wavelength range of 370 nm or more and 400 nm or less to the Catharanthus roseus in the container is 0.5 W/m ² or more and 500 W/m ² or less, or the light irradiation amount is 150 kJ/m ² or more and 150000 kJ/m ² or less. a first control unit that controls the first light source so that
a second light source for irradiating the periwinkle in the container with light comprising red light;
and a second control unit that controls the second light source so that the photosynthetically active radiant flux density (PPFD) for Madagascar periwinkle in the container is 100 μmol/m ² /s or more and 400 μmol/m ² /s or less. A device for increasing the amount of vinblastine in Madagascar periwinkle, characterized by:

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