JPS6296088A - Production of antitumor substance - Google Patents

Abstract

PURPOSE:To obtain the titled substance in high concentration and yield, independent of natural environmental condition, etc., by culturing a tissue or cell group of a plant belonging to a specific genus and accumulating an antitumor substance in the adventitious bud and/or adventitious root of the cultured plant. CONSTITUTION:Tissue or cell group of a plant belonging to Putterlickia, Camptotheca, Nothapodytis or Podophyllum genus is washed and sterilized. The sterilized cell group, etc., (e.g. Pterliquia genus) is inoculated in a medium composed of a base culture medium (e.g. White medium), sugars, vitamins, 10<-6>-10<-7>M of auxin and 10<-5>-10<-6>M of cytokinin, etc., and cultured by alternating the irradiation with light of 5,000-10,000 lux for 16-24hr and the maintenance in dark place for 8-0hr to effect the differentiation induction of an adventitious bud or root. The produced bud, etc., is extracted to obtain an antitumor substance such as maytansine from a plant of Putterlickia genus, camptothecin from a plant of Camptotheca genus or Nothapodytis genus or podopyllotoxin, etc., from a plant of Podophyllum genus.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing an antitumor substance, and more specifically,
Anti-II produced and accumulated in certain types of higher plants [! ! Concerning a method for efficiently obtaining injectable substances using tissue culture at °C. [Prior technology] In addition to antitumor substances derived from lower plants such as fungi, alkaloids newly found in higher plants have recently been introduced. It has been found that there are some compounds that exhibit strong antitumor activity [e.g., Dourus et al., Recent Results.
In Cancer Research (Recent Re5u)
lts in Cancer Research),
70, pp. 21-44 (1980)], its application to therapeutic drugs is being investigated.

However, in general, there are the following limitations or problems in obtaining and utilizing active ingredients in higher plants, including the above-mentioned anti-cancer substances.

That is, in the case of higher plants, the active ingredients are extracted by solvent extraction from the whole plant, fruit, or seed of the plant, which is usually cultivated from seeds sown in the soil or by vegetative propagation of plant parts such as cuttings. However, in the case of methods that involve cultivating plants, in addition to the basic limitation that the vegetation distribution is limited by temperature, climate, soil quality, etc. Since the growth of plants is greatly influenced by weather conditions, the yield and the content of active substances in the plants vary from year to year, making it extremely difficult to obtain the substances stably and efficiently.

As a method to solve the above-mentioned problems encountered when separating and obtaining active substances in higher plants, there is a method using so-called tissue culture, in which plant tissues are cultured and active substances produced and accumulated in the culture are collected. Camptotheca is known for its antitumor properties.
It has been proposed to collect camptothecin, an antitumor substance contained in plants belonging to the genus, from amorphous cultures (so-called callus) by tissue culture methods (Sakato et al. Ral and Biological Chemistry (Agr.
Cultural & Biological Che
mistry), 88(1), 217-218(19
74)), L/Cal, but anti from callus! 1lI
In the case of the method of collecting the tumorous substance, the content of the antitumor substance in the callus, including the above-mentioned example of camptothecin, is generally 1. The problem is that the amount is only a few tenths or less of that in the mother plant, so it has not yet been put to practical use.

On the other hand, it is possible to differentiate adventitious buds by tissue culture and collect antitumor substances from the adventitious buds of plants of the genus Cephalota (active substance: harringtonine).
(Japanese Society of Plant Physiologists 1982)
Proceedings of the annual conference, p. 156), but in this case as well, the content of effective substances in the culture was only a fraction of that in the mother body, as in the callus mentioned above. .

[Problem that the invention seeks to solve]

In view of the current situation, the present inventors have continued their intensive research into a more advantageous method of obtaining antitumor substances in higher plants. In the adventitious shoots and roots differentiated from tissue culture, anti-tumor substances are produced and accumulated in the same amount as in the mother plant under natural cultivation, which is combined with the high proliferation rate that is an advantage of tissue culture. Therefore, the inventors have discovered that it is possible to produce the substance more efficiently in this case than by using cultivated plants, and have completed the present invention.

The purpose of the present invention is to develop antitumor substances derived from higher plants in a highly reproducible and more efficient manner by using a tissue culture method in a completely controlled environment that is not affected by the natural environment. The purpose is to provide a manufacturing method.

[Means for solving problems]

That is, the present invention relates to buterlichia (putterlick).
-1a) Ijill, Camptotheca
eca) genus, Nothapodytes
es ) or Podophyllu
m) Cultivating tissues or cell groups of plants belonging to the genus, inducing differentiation of adventitious buds and/or adventitious roots from the culture, and collecting antitumor substances produced and accumulated in the adventitious buds and/or roots. This is a method for producing an antitumor substance characterized by the following.

In the present invention, adventitious buds and adventitious roots refer to stems that are induced to differentiate from the cultured tissue or cell group itself or from dedifferentiated cells (callus) derived from the cultured tissue or cell group; It refers to tissues with shapes such as leaves, roots, whiskers, root hairs, etc.

In addition, the antitumor substances contained in plants according to the present invention include maytansine in plants of the genus Ptyrrhychia, camptothecin in plants of the genus Camptotheca and Notapodites, and plants of Podophyllum genus. Among them, podophyllotoxin is cited as a representative example.

In the plant according to the present invention, the antitumor substance is accumulated in the adventitious buds (roots) grown by tissue culture to the same extent as the content of the mother plant; This has only become clear after looking at the above, and is completely unexpected considering the prior art described above.

Examples of plants of the genus Pterlichia to which the method of the present invention is applied include Pterlichia vulcosa (Pu-tterl).
ickia verrucosa), and Camptotheca genus such as Camptotheca acuminata (Ca.
-mptotheca acuminata) etc.
Examples of the genus Nothapodytes include Nothapodytes foetida.
) (The genus Notapodites is Mappia
The genus Notapodites foetida is also referred to as the genus Mappia foetida.
tida), but in this specification,
The former is used as a uniform name], and Podophyllum peltatum is used as a uniform name.
Podophyllum pθ-1tatum),
Podophyllum blainku (Podophyll-11um)
pleintha), Bodophyllum emodi (P
o-dh711 dish emodi), etc. Among these plants, plants belonging to the genus Camptothecar and Podophyllum have particularly good propagation properties of adventitious shoots (roots), and the desired anti-PMM substance can be produced with particularly high efficiency.

In the present invention, tissue culture of these plants is performed, for example, as follows.

That is, after thoroughly washing the plant under water, it is cut into leaves, stems, roots, etc., which are sterilized using ethanol, benzalkonium chloride, sodium hypochlorite, etc., and then washed with sterile water. Place M on a medium in a test tube or Erlenmeyer flask.Mediums include basic media such as Linsmeyer-Skoog, Murashige-Skoog, B5, Niche & Niche, White, etc., supplemented with sugars, vitamins, and growth regulators. A medium containing substances, etc. is used.As the basic medium, any medium commonly used in braided tissue culture can be used, and is not limited to the above.

Examples of growth regulators include indole-8-acetic acid (
IAA), indole-8-butyric acid (IBum), α-naphthalene vinegar 1! J(NAA), 2,4-dichlorophenoxy i! +1:1l (2,4-D), etc., kinetin (Kn), 6-benzyladenine (6-BA)4, and other cytokinins, which can be used alone or in isolation. Used in combination as appropriate.

When culture is continued at 22 to 80°C in a medium like this, approximately 1
Differentiation of adventitious shoots or roots occurs from the culture in about 1 to 4 weeks. At this time, the generation of adventitious shoots (roots) mainly depends on the amount of growth regulators auxin and cytokinin, the plant tissue subjected to tissue culture, and the light irradiation conditions, but these km conditions are as follows: The temperature varies depending on the type of plant.

Showing these conditions for the plants according to the present invention, first of all, in plants of the genus Pterlichia, the temperature ranges from lo-4 to l0-
5.00 for 16-24 h in medium containing 7 M auxin and 10-'-10-' M cytokinin.
Adventitious buds are easily obtained by starting the culture from C leaves, which are alternately irradiated with light at 0 to 0 to 0 lux and kept in clear sunlight for 8 to θ hours, while adventitious roots are produced at lo-s to 10 lux.
0 to 4 o'clock, 1,000 to 5 m in a medium containing M auxin and 8 M cytokinin.
,000 lux and holding for 24 to 20 hours alternately to start culturing from leaf tissue.

In addition, the odor of plants of the genus Cambutoteca ranges from lO'' to 1
between 8 and 16113 and 2.000 to 5 in medium containing 0 M auxin and 10 = lOM cytokinin.
For the differentiation of adventitious shoots, it is appropriate to start from seedlings (sprouting) that are exposed to light at 1,000 lux and kept in the sun for 16 to 8 hours.
0-6 to 10-'M auxin and lo-7 to 10-'
1 for 0 to 4 h in medium containing y of cytokinin.
It is best to start from seedlings by alternating light irradiation of ,000 to 5,000 lux and keeping the sun clear for 24 to 20 hours.

For plants of the genus Notapodites, the same culture conditions as for plants of the genus Camptotheca can be used.

Furthermore, in the Podophyllum cormorant plant, auxin and cytokinin were mixed in a ratio of 1:10 to 1:100, and 10-7 to 10-8M and 1O-6, respectively.
Adventitious buds are easily obtained when cultured in a medium containing a concentration range of 1 O-'M, while for differentiation of adventitious roots, auxin and cytokinin are cultured in a mixing ratio of 100:1 to 1:1, and each IO Cultivation in a medium containing a concentration range of -' to 10-'M and 10 to 10M is suitable.

In any of the above cases, it is preferable to induce differentiation of adventitious roots rather than adventitious buds, as this generally results in better proliferation.

Also, during the differentiation of adventitious shoots or roots.

Depending on the type of plant), callus formation may precede C, but in this case, if you remove some of the adventitious shoots (roots) that have regenerated from the callus and transplant them into a medium, new germination and rooting will occur. Therefore, the culture can be continued under the same conditions as above.

The adventitious buds and roots obtained in this way contain the antitumor substance of the mother plant at approximately the same content as the mother plant, and are treated with a bath medium such as C alcohol according to a conventional method. The substance can be easily separated and collected by extraction treatment.

According to the above-described method of the present invention, it is possible to obtain higher plant tissue containing a high content of antitumor substances by a tissue culture method that is not affected by the natural environment and has excellent proliferative properties. Thus, a method for producing anti-tumor substances is provided which is much more reproducible and has improved efficiency compared to conventional cultivation methods.

The present invention will be specifically described below with reference to Examples.

In the Examples, the degree of proliferation of adventitious buds (roots) was measured, and the antitumor substance was purified and quantified as follows.

(1) Multiplication rate of adventitious buds (roots) The fresh fiA (f) of adventitious buds (roots) at the time of subculture in the 8th subculture and the adventitious buds (roots) when cultured for 3 weeks after subculture. ) fresh] tB(f), the degree of proliferation (c times) was determined from the following formula.

(2) Purification and quantification of antitumor substances Adventitious buds (roots) were freeze-dried and extracted with 95% ethanol, and the resulting extract was subjected to liquid chromatography (ODS silica column, eluent niacetonitrile/water = 50150 )
[1] After isolation and purification of the target antitumor substance by chromatography (ODS silica) and comparison with the standard, the purified product was analyzed for ultraviolet absorption spectra, fluorescence Identification and quantification were performed by spectra and mass spectrometry spectra.

Example 1 70 seedlings of Cambutotheca acuminata were thoroughly washed with water.
% ethyl alcohol for 2-3 minutes, followed by 0.2% benzalkonium chloride aqueous solution for 2 minutes, and 1% sodium hypochlorite aqueous solution for 5-IO minutes. Seedlings from which the drug was completely removed with water were used as explants under sterile conditions. This is IEA 10”
M, 6-BAIO'M was added and placed on a Linsmeyer-Skoog medium containing 3% sucrose and 1% agar adjusted to pH 5.7, and incubated at 25°C for 16 hours at 5,000 lux. When culturing under white light irradiation and culturing in the dark for 8 hours were performed alternately, adventitious buds differentiated after one week.

This was subcultured several times to increase the number of adventitious buds, and camptothecin was extracted, purified, and quantified using this material as a material (Example 1 of the present invention).

For comparison, camptothecin was extracted, purified and quantified from calli obtained by subculture in the same manner as above, except that the amounts of IBA and 6-BA added were 10-'M each (Comparative Example 1).

Table 1 shows those results and the test results for the leaves of the mother plant Camptothecum acuminata (Control Example 1).

Table 1 [: From the results in Table 1, adventitious buds obtained by tissue culture of explants of Camptotheca acuminata (Example 1 of the present invention) have the same level of antitumor activity as the mother plant. (camptothecin) and its proliferation rate was also found to be good. In addition, the callus of Comparative Example 1 contained 1. Only the following anti-tumor substance (camptothecin) is contained in the callus, but according to the test results of 5akato et al., the content rate of the anti-tumor substance (camptothecin) in callus is 0.0002.
5%, which is 1/10 of that in the mother plant, clearly demonstrating the superiority of the method of the present invention.

Example 2゜Rinsmeyer Skoog medium was replaced with B as the basic medium.
5 medium and IBAIO-' as a growth regulator.
NAAIO-4M and K instead of M and 6-BAIO-'M
Adventitious roots were induced to differentiate by culturing under the same conditions as in Example 1 except that n l O-' M was added, and these were subcultured.

At this time, the proliferation rate of adventitious roots was extremely high, 12.2 times, and the content of anti-mm substance (camptothecin) in adventitious roots was 0.007%, which was that of the roots of the mother plant (Camptotheca acuminata). 0,008%) showed the same value.

Example 3 Notabodytes foetida was used instead of Camptotheca acuminata as the test plant, and White medium was used instead of C Linsmeyer Skoog medium as the basic medium, and IBAIO- as the growth regulator.
2.4-D l instead of 'M and 6-BAIO-'M
Culture was carried out under the same conditions as in Example 1 except that O-@M and 6-BAIQ-''M were added to the above basic medium to induce differentiation of adventitious roots, which were then subcultured.

Regarding the adventitious roots obtained in this way, the proliferation rate and antitumor properties Ij
When the content of camptothecin (camptothecin) was determined, the multiplication rate was 9.6 times higher, and the content of anti-intestinal substances was as high as 0.007%, the same as that of the roots of the mother plant (Cnotabodytes foetida). The value was shown.

Example 4 Using roots of a variety of the genus Bodophyllum, Podophyllum bell, Tatum, the same sterilization process as in Example 1 was carried out, and then the sections were placed in white basic medium with 8% sucrose,
2.4-D l O-”M, 6-BA l O-”
When placed on a 1% agar medium supplemented with M, callus (dedifferentiated cells) was formed, and this callus further induced redifferentiated adventitious roots. This tissue continues to proliferate in the form of callus → adventitious roots, and when subcultured on the same medium, callus/adventitious roots form.
A large amount of adventitious roots can be obtained. The proliferation rate of adventitious roots at this time is 8
．． It was twice that amount. In addition, 13 η of an antitumor substance (podophyllotoxin) was obtained from 375 g of this adventitious root by one extraction and purification operation, and the yield of the substance was 0.0085%.
Met.

On the other hand, the yield of podophyllotoxin from the roots of the mother plant (Bodophyllum peltatum) used as a control was o,
It was found that the culture (adventitious roots) contained the same or more podophyllotoxin than the plant roots.

Example 5 A product of the genus Pterlichia U Using leaves of Pterlichia vulcosa, the sterilization process was carried out in the same manner as in Example 1, and then the sections were placed in Niche & Niche basic medium with 8% sucrose and IAA.
When the cells were placed on a 1% agar medium supplemented with t O-'M and Knt O-'M, callus was formed, but regenerated adventitious buds were mixed in from this callus, and callus → adventitious buds were formed. Repeated proliferation. When subcultured on the same medium, a large number of adventitious buds were obtained.

At this time, the degree of increase in adventitious buds was 6.4 times.

In addition, by extraction and purification from this adventitious bud 400f,
Approximately 5 capes of an anti-intestinal substance (maytansine) were obtained with a yield of 0.00125%.

On the other hand, as a control, the yield of maytansine obtained from the stems and leaves of the mother plant (Pterlichia vulcosa) was 0.00.
120%, and the culture (adventitious buds) is the plant body (stems and leaves).
It was found that it contains maytansine equivalent to

In addition, when the callus produced when culturing was carried out in the same manner as above except that IAA was changed to 10-'M and Kn was changed to IO'M, maytansine was undetectable.

Claims (1)

[Claims] (1) Putterlickia genus,
Tissues or cell groups of plants belonging to the Camptotheca genus, No-thapodytes genus, or Podophyllum genus are cultured, and adventitious buds and/or cell groups are isolated from the culture.
Alternatively, a method for producing an antitumor substance, which comprises inducing differentiation of adventitious roots and collecting the antitumor substance produced and accumulated in the adventitious buds and/or roots.