JP3870234B2 - Method for breeding camptothecin-producing plant and method for producing camptothecin - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for growing a material plant for efficiently producing camptothecin, which is a raw material of an anticancer drug camptothecin derivative, and its analog, and a method for producing camptothecin and its analog using this plant.
[0002]
[Prior art]
Camptothecin is a compound useful as a raw material for pharmaceuticals such as anticancer agents and antiviral agents, and is a kind of quinoline alkaloid biosynthesized via indole alkaloids.
A common method for producing camptothecin is to harvest, extract, and purify kiju or hornbill grown in the field, but kiju and hornbill are woody plants that are difficult to rapidly reproduce and are not ready for harvest. There is a problem that a long term is required.
In addition, a method by organic synthesis has been proposed, but since a multi-step reaction is required, the yield is low and it has not been put into practical use.
[0003]
[Problems to be solved by the invention]
The present invention relates to a method for growing a camptothecin-producing plant having a high content of components such as camptothecin, vigorous growth, easy cultivation, and high productivity, and production of camptothecin and its analogs using this plant. It aims to provide a method.
[0004]
[Means for Solving the Problems]
The present invention relates to a method for cultivating a camptothecin-producing plant, characterized by artificially mating Chaphinamori (Ophiorrhiza pumila) and Ryukyuunamori (Ophiorrhiza kuroiwai).
[0005]
The present invention also relates to a camptothecin characterized in that a hybrid plant obtained by artificial mating Chaphinamori (Ophiorrhiza pumila) and Ryukyuunamori (Ophiorrhiza kuroiwai) is proliferated, grown and extracted from the grown plant. And / or a method of producing an analog thereof.
[0006]
Furthermore, the present invention relates to a camptothecin characterized by culturing a hybrid plant obtained by artificial mating of Ophiorrhiza pumila and Ophiorrhiza kuroiwai and extracting from the cultured cells. It relates to a method for producing an analog.
[0007]
That is, the present inventors paid attention to the plant belonging to the genus Ophiorrhiza in the course of earnest research in order to achieve the above object. That is, about 150 species of the genus Ophiorrhiza are distributed mainly in tropical and subtropical Asia and contain various indole alkaloids. Therefore, we collected three species of Ophiorrhiza pumila, Ophiorrhiza kuroiwai, and Ophiorrhiza japonica and their variants natively in Japan, and conducted molecular biological phylogenetic analysis and component analysis by RAPD (Random Amplified Polymorphic DNA) analysis. The genus plants formed two clusters of the O.pumila and O.kuroiwai groups and the three O.japonica group (variety) groups, consistent with the presence or absence of camptothecin. This was thought to suggest that the camptothecin biosynthetic system evolved from a common ancestor (FIG. 1).
On the other hand, as a result of the self-fabrication survey (Ishigakijima), several sterile individuals were found that were morphologically very similar to O.kuroiwai. As a result of HPLC analysis, flow site analysis, and RAPD analysis, this sterile individual is a natural hybrid of O.kuroiwai and O.pumila (tentatively called O.xkuroiwai. Although the scientific name given to the positive reference specimen stored in the study, the present inventors have revealed that this specimen plant individual is also sterile, that is, a hybrid individual. (There is no scientific name for a dwarf individual. Therefore, in this specification, a sterile individual is referred to as O.xkuroiwai.) Since this individual is larger than O.pumila and has a higher camptothecin content than O.kuroiwai, it was thought that this individual could be expected as a new camptothecin-producing resource.
As a result of further research based on the above findings, the present inventors have found that it is possible to grow two kinds of plants that are completely different in form, a hybrid of Chaboynamori and Ryukyuunamori, which is difficult to grow. However, it is possible to artificially cross Chaboinamori, which is a plant with a high camptothecin content, and Ryukyu Inamori, a plant that is easy to cultivate but has a low camptothecin content, and select individuals with high component content from the obtained hybrid individuals. And grown and cultivated to extract camptothecin and its analogs.
Separately, hybrid plants obtained by artificial mating were aseptically cultured, camptothecin and its analogs were extracted from the cultured cells, and the present invention was completed.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The method for cultivating a camptothecin-producing plant according to the present invention can be obtained by artificially pollinating O.pumila pollen with O.kuroiwai as a mother plant, or with O.pumila as a mother plant. By artificial pollination of kuroiwai pollen, hybrids between artificial species are bred.
That is, the above-mentioned two kinds of plants are cultivated in an artificial weather room, and the stamens before the flowering are removed (male) from the buds on the day before the flowering of the mother individual. The pupae after depilation are left for one day and then artificially mated using pollen collected from the pollen species. When seeded 90 days after mating and inoculated on an appropriate medium such as vermiculite medium, germination occurs at a high germination rate (about 90%). The artificial hybrid individuals obtained in this way have an external form that is difficult to distinguish from Ryukyu Winning Mori, like the sterile individuals observed in their own dough.
[0009]
The hybrid individuals obtained above are sterile and vegetative breeding is necessary because the usual seed breeding is impossible. Although “cutting” can be bred, growth by tissue culture is desirable for more stable and efficient breeding. Proliferation by tissue culture is performed, for example, as follows.
The top buds and buds of hybrid plants sterilized by a conventional method are placed on a ½ concentration MS solid medium (sucrose concentration 1%) and cultured under illumination at 25 ° C. and 4000 lux. Passage and growth are performed by cutting the elongated stem into nodes and placing it in a new medium having the same composition. At this time, an appropriate amount (0.1 μM to 10 μM) of cytokinin such as benzyladenine, isopentenyladenine, zeatin, or zeatin riboside may be added to the medium in order to perform more efficient growth.
[0010]
Such hybrids are genetically unstable, so there is a variation in the content of camptothecin, and high productivity can be achieved by selecting individuals with a high content. Moreover, this hybrid plant can be cultivated loosely and can be cultivated on a large scale.
This hybrid plant produced by crossing two kinds of plants, Chaboynamori and Ryukyuunamori, has an illuminance of 5000 Lux or higher compared to Chaboinamori, which can only be cultivated under low illuminance of 5000 Lux or less and high humidity. It withstands low humidity and exhibits a camptothecin content (1 × 10 ⁻⁴ % to 1 × 10 ⁻³ % per fresh weight, 0.01% to 0.1% dry matter weight ratio) similar to that of Chaboynamori. In addition, while Chaboinamori grows only around 10 cm in height after 6 months of sowing, hybrid plants grow to a height of 50 cm or more, and the production amount of camptothecin per individual is about 50 times.
In addition, by using this hybrid plant, it is not contained in kiju, Japanese dogwood, etc. that are currently used as camptothecin extraction materials, but is not contained in chaboside (10-glucopyranosyloxy-9-methoxycamptothecin) or kiju. It has become possible to produce camptothecin analogues such as 9-methoxycamptothecin.
[0011]
Extraction of camptothecin and the like from hybrid plants is usually carried out by pulverizing the harvested plant stems and leaves in a mortar or the like and usually 100 to 500 ml of extraction solvent (for example, methanol, ethanol, etc.) per 10 g of dry matter weight. Then, the extraction is performed. Extraction may be performed once using a large amount of solvent, but it is more preferable to divide the extraction solvent into several parts and perform the extraction a plurality of times because the extraction efficiency increases. The temperature at the time of extraction is usually room temperature, but it may be arbitrarily heated as necessary.
As a method other than the above, freeze-dried stalks and leaves of harvested plants can be used for extraction, or can be used for extraction without being dried. Further, instead of extracting from the stems and leaves, it is possible to use the underground part by washing it with water, or harvest the roots by hydroponics etc., and use it as an extraction material.
[0012]
In addition, it is also possible to produce camptothecin and its related compounds by tissue culture such as callus culture and hairy root culture using hybrid plants as materials. At this time, it is also possible to recover camptothecin and its related compounds released into the culture solution.
[0013]
【Example】
Hereinafter, the present invention will be described more specifically with reference examples and examples. However, the present invention is not limited to these reference examples and examples.
[0014]
Reference Example 1 Molecular biological system classification of plant materials (1) Plant materials
O.kuroiwai Makino is a sub-shrub that reaches a height of 1 m. According to the Ryukyu Botanical Magazine, Okinawa Main Island, Ishigaki Island, Iriomote Island, Yonaguni Island, Taiwan (southern and Hongyu Island) and It is supposed to be distributed over the Philippines.
O.pumila Champ. Is a perennial herb of 5 to 15 cm in height and is distributed in Amami Oshima, Tokunoshima, Okinawa Main Island, Ishigaki Island, Iriomote Island, Yakushima Island, Taiwan, and South China.
O. japonica v. Japonica Blume is a perennial herb with a plant height of 10 to 20 cm. In Honshu, it is widely used in the warm west of southern Chiba Prefecture, Shikoku, Kyushu, Tokunoshima, Okinoerabujima, Okinawa Main Island, Taiwan and China. Distributed.
O.japonica f.tashiroi Ohwi (Nagabanosimainamori, Nagabainamori) is longer oval, 8-15cm long and 1-3cm wide oval-shaped needle-shaped line-shaped inversion compared to the basic species It is a needle shape and is considered to be a basic stream type. The distribution area is Ishigakijima, Iriomotejima, and northern Taiwan.
O.japonica v.amamiana Hatsushima grows naturally on Amami Oshima and Okinawa main islands, and is said to have a lower plant height than the basic species. The individuals used for the experiment were distributed from the National Science Museum Tsukuba Experimental Botanical Garden and collected near the Kunigami Nagao Bridge on the main island of Okinawa.
[0015]
(2) Molecular biological phylogenetic classification of plant materials The molecular biological classification of sweet potato genus plants (Ophiorrhiza genus plants) collected from their own dough is for 10-mer random primers using whole genomic DNA extracted from fresh leaves as a template. The RAPD (Random Amplified Polymorphic DNA) method was used. Genetic similarity was determined by UPGMA from about 300 electrophoresis bands obtained with 17 kinds of random primers, and a tree diagram was created. The results are shown in FIG.
[0016]
As a result of RAPD analysis, the genus Ophiorrhiza was roughly divided into two groups: a group consisting of O.pumila and O.kuroiwai, and a group consisting of O.japonica basic species and their variants and varieties. Since two species containing camptothecin formed one cluster, the biosynthetic pathway of camptothecin is considered to be derived from the same ancestor species. O.pumila and O.kuroiwai are perennial herbs, the latter are sub-shrubs, and the morphologically different, but as described later, these two species of hybrids (tentative name: O. x kuroiwai) The presence of these two plants is considered to indicate that reproductive speciation has not progressed as much as the morphological difference.
[0017]
Example 1 An artificial interspecific hybrid was bred by artificially pollinating O.pumila from Amami Oshima with O.kuroiwai collected at Ishigakijima as an artificial cross.
The above two kinds of plants were cultivated in an artificial weather room, and the stamens before the flowering were removed from the buds on the day before flowering of O.kuroiwai together with the corolla. The pupae after depilation were artificially mated using pollen collected from O.pumila after being left for one day. After 90 days from the mating, the seeds were collected and sown in vermiculite medium. As a result, germination occurred at a high germination rate (about 90%). The artificial hybrid individuals obtained in this way had an external form that was difficult to distinguish from Ryukyu Winning Mori, as was the sterile individual observed in its own dough.
[0018]
[Component analysis of hybrid individuals]
When the leaves of the obtained hybrid individuals were extracted with methanol and the camptothecin content was measured by reverse-phase HPLC analysis, it was 1 × 10 ⁻⁴ % to 1 × 10 ⁻³ % per fresh weight, and the camptothecin content was 10%. Double individual differences were observed. Individuals with a high content were selected and cultivation was continued at 25 ° C. and 5000 lux (16 hours light period, 8 hours dark period) for one year, but no change was observed in the camptothecin content.
On the other hand, the content of camptothecin in the leaves of Chaboynamori was 5 × 10 ⁻⁴ % to 1 × 10 ⁻³ % per fresh weight, and about 2 times as many individuals were not observed.
[0019]
Example 2 Proliferation of hybrid individuals by growth tissue culture was performed as follows.
The top buds and buds of hybrid plants sterilized by a conventional method were placed on a ½ concentration MS solid medium (sucrose concentration 1%) and cultured under illumination at 25 ° C. and 4000 lux. Passage and growth were performed by cutting the elongated stems into nodes and placing them in a new medium having the same composition. In this case, in order to perform more efficient growth, an appropriate amount (0.1 μM to 10 μM) of cytokinin such as benzyladenine may be added to the medium.
[0020]
Example 3 Cultivation of hybrid individuals and camptothecin production Chaboynamori (control group) and hybrid plant seedlings grown by cuttings were cultivated for 6 months, harvested, and fresh weight and camptothecin content were measured. The cultivation area was 0.12 m ² .
Camptothecin was extracted by air-drying the harvested plant stems and leaves in a mortar and using 200 ml of methanol per 10 g of dry matter weight.
(This extraction method is an example, and freeze-drying or extraction with freshness is possible. In addition, roots can be harvested by washing the water in the underground or by hydroponics, etc., and used as an extraction material. is there.)
[0021]
[result]
Chaboynamori; fresh weight 295g, camptothecin content 1.5mg
Hybrid plant; fresh weight 520g, camptothecin content 5.2mg
[0022]
Chaboynamori was cultivated under the light of 4000 Lux and hybrid plants under the light of 5000 Lux. Chaboinamori partially browned and died at illuminance of 4000 Lux or higher, but it grew normally in hybrid plants even under illumination of 10,000 Lux.
[0023]
[Component analysis of hybrid plants]
When the components of the cultivated hybrid plant were analyzed with a high performance liquid chromatography mass spectrometer (LC-MS), in addition to camptothecin, its related compound chaboside (10-glucopyranosyloxy-9-methoxycamptothecin, contained 2 × 10 ⁻⁴ % to 2 × 10 ⁻³ %) and 9-methoxycamptothecin (content 2 × 10 ⁻⁵ % to 2 × 10 ⁻⁴ %) were found to be contained.
[0024]
【The invention's effect】
The present invention relates to a method for growing a novel camptothecin-producing plant by artificial mating of Ophiorrhiza pumila and Ophiorrhiza kuroiwai, and to cultivate or tissue-culture a hybrid plant obtained by this method. Thus, the present invention provides a method for producing camptothecin and its analogs. According to the present invention, the following effects can be obtained.
(I) High productivity is achieved by selecting a hybrid individual having a high content of components such as camptothecin and growing vigorously.
(Ii) Since production of camptothecin-related compounds that are not contained in conventionally used kiju and hornbill is observed, it can be greatly expected as a new raw material for producing camptothecin derivatives such as CPT-11 currently used as pharmaceuticals.
(Iii) The hybrid plant is a herbaceous plant that can be easily cultivated and has high productivity because it can be cultivated at a high density. Also, since a large amount of seeds can be obtained in a short period of time, the supply of seedlings is stable. Since a tissue culture technique such as hairy root culture has been established, more stable production of camptothecin can be expected regardless of natural conditions.
(Iv) Since it is possible to cultivate under low illuminance (10000 Lux or less), it is possible to produce camptothecin and its related compounds, for example, in forest floors or in shaded areas where cultivation of kiju and cedarwood is difficult.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a dendrogram showing the molecular biological affinity of a sweet potato genus plant by the RAPD method.

Claims (6)

Artificial mating of Ophiorrhiza pumila and Ophiorrhiza kuroiwai,
Select hybrid individuals with high camptothecin content and vigorous growth,
A method for cultivating a camptothecin-producing plant having a higher productivity of camptothecin than a parent plant, Chaboynamori. Artificial mating of Ophiorrhiza pumila and Ophiorrhiza kuroiwai,
Select a hybrid individual having a high component content of camptothecin, chaboside (10-glucopyranosyloxy-9-methoxycamptothecin) and / or 9-methoxycamptothecin and growing vigorously,
Propagating and cultivating the resulting hybrid individuals having higher productivity of camptothecin, chaboside (10-glucopyranosyloxy-9-methoxycamptothecin) and / or 9-methoxycamptothecin than the parent plant Chavoinamori ,
A method for producing camptothecin, chaboside (10-glucopyranosyloxy-9-methoxycamptothecin) and / or 9-methoxycamptothecin, characterized by extracting from a grown plant. Among hybrid plants obtained by artificial crossing of Chaphinamori (Ophiorrhiza pumila) and Ryukyuunamori (Ophiorrhiza kuroiwai), camptothecin, chaboside (10-glucopyranosyloxy-9-methoxycamptothecin) and / or 9-Methoxycamptothecin is high in component content and is selected for growing, growing and cultivating hybrid individuals.
It is characterized in that it is extracted from a plant having higher productivity of camptothecin, chaboside (10-glucopyranosyloxy-9-methoxycamptothecin) and / or 9-methoxycamptothecin than the parent plant Chaboynamori. A method for producing camptothecin, chaboside (10-glucopyranosyloxy-9-methoxycamptothecin) and / or 9-methoxycamptothecin. Of camptothecin, chaboside (10-glucopyranosyloxy-9-methoxycamptothecin) and / or 9-methoxycamptothecin obtained by artificial mating of Chaphinamori (Ophiorrhiza pumila) and Ryukyuunamori (Ophiorrhiza kuroiwai) Sterile culture of hybrid individuals with high component content and vigorous growth,
Extraction from the obtained cultured cells having higher productivity of camptothecin, chaboside (10-glucopyranosyloxy-9-methoxycamptothecin) and / or 9-methoxycamptothecin than the parent plant Chaboynamori A method for producing camptothecin, chaboside (10-glucopyranosyloxy-9-methoxycamptothecin) and / or 9-methoxycamptothecin, which is characterized.   The production method according to claim 4, wherein the culture is performed in a medium to which cytokinin is added.   Individuals with high component content of camptothecin, chaboside (10-glucopyranosyloxy-9-methoxycamptothecin) and / or 9-methoxycamptothecin are selected from hybrid individuals obtained by artificial mating, and aseptic culture The production method according to claim 4 or 5.

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