JPH02138984A - Production of bisbenzylisoquinoline alkaloid and culture medium used therefor - Google Patents

Abstract

PURPOSE:To produce the subject alkaloid with excellent productivity by carrying out organ culture of a root or induced root of Stephania cepharantha Hayata and separating bisbenzylisoquinoline alkaloid from the cultured induced root. CONSTITUTION:A culture medium for organ culture containing 0.3-0.7mM (NH4)2SO4, 10-23mM KNO3 and 0.6-1.5mM MgSO4 is prepared. A root of Stephania cepharantha Hayata, a root directly induced from a tuberous root slice thereof or a root obtained from a callus prepared from a plant body slice thereof is subjected to organ culture in the culture medium prepared as described above. Bisbenzylisoquinoline alkaloid is then separated and collected from the above-mentioned cultured induced root. If gibberellin is added to the organ culture medium, vigorous propagation is preferably initiated stably for a long period.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to a method for producing bisbenzylisoquinoline alkaloids, and more specifically, the present invention relates to a method for producing bisbenzylisoquinoline alkaloids, and more specifically, the roots of T. spp. The present invention relates to a method for producing bisbenzylisoquinoline alkaloids by separating and collecting bisbenzylisoquinoline alkaloids, and a medium used therein.

[Conventional technology and its problems]

It is known that the T. chinensis, which grows naturally in the mountains and forest areas of Okinawa, Taiwan, and southern China, produces bishencylisoquinoline alkaloids containing physiologically active substances such as cephalanthine, isotetrandrine, and berbamine.

However, wild and natural plants, including this one, are difficult to obtain and expensive due to various natural restrictions such as geographical distribution and growth period. However, this has been a bottleneck in the use of T. chinensis as a raw material for physiologically active substances.

Therefore, attempts have been made to produce bisbenzylisoquinoline alkaloids by synthetic methods, but since all of these alkaloids have complex chemical structures, the synthesis requires multiple steps, the operations are complicated, and the final yield is The disadvantage was that the rate was also extremely low.

In recent years, plant tissue culture methods that effectively utilize the biological functions of plant cell systems have been used to produce useful substances that are scarce in resources and difficult to chemically synthesize. And, as an example of applying this method to the white-spotted fish, there is a report by Akasu et al. (P.
Hytochemistry 15, 471 (1976
)). According to this report, as a result of culturing the callus derived from the tuberous roots of T. spp. on an agar medium,
It has been confirmed that two ffl alkaloids, allomoline and berbamine, were produced among bishensylisoquinoline alkaloids. However, this method does not produce pharmacologically important cephalanthine, isotetrandrine, etc., and it is presumed that some kind of enzyme system deficiency or inhibition of enzyme expression occurs. In addition, the yields of allomoline and berbamine produced were too low to be of practical use.

[Means to solve the problem]

The present inventors have conducted extensive research into a method for producing bisbenzyl-isoquinoline alkaloids, which are extremely useful as physiologically active substances, by plant tissue culture, and have recently discovered that cephalanthine , that various bisbenzylisoquinoline alkaloids including isotetrandrine are produced, and that the productivity of bisbenzylisoquinoline alkaloids can be significantly improved by adjusting the components contained in the medium used in organ culture. heading,
The invention has been completed.

That is, the present invention provides a method for producing bisbenzylisoquinoline alkaloids, which comprises organ-culturing the roots or induced roots of P. japonica, and separating and collecting bisbenzylisoquinoline alkaloids from the cultured induced roots. .

Furthermore, the present invention provides a medium containing a predetermined amount of specific inorganic salts and capable of producing bisbenzylisoquinoline alkaloids at a high rate.

In the present invention, the roots used as the starting material for culture include the roots of T. japonicus (refers to fine roots developed from tuberous roots), the roots directly grown from the tuberous root sections of T. c. Examples include roots derived from callus obtained from plant sections.

Among the above roots, to obtain derived roots from tuberous root sections, sterilize the tuberous root sections of T. japonica according to the conventional method, wash with sterile water, cut into approximately 1 cm squares, and add carbon sources, auxin, cytokinin, etc. They may be placed and cultured in a basic medium for plant tissue culture supplemented with plant hormones, agar, and if necessary a nitrogen source such as amino acids.

The tuberous roots preferably have a growth period of March to June, and are cut into rings with a width of 0.1 to 5 cm, preferably 0.5 to 2 cm to obtain sections. Particularly preferred are slices cut into rings with a width of about 1 cm. Sterilization of this section is performed, for example, with 70% Et.
This is done by stirring for 10 min in O)1 followed by 10 min in a 1% Na0CJ2 solution. Basic media for plant tissue culture include LS, MS, B5, White.
Among these, LS medium is preferred. As the carbon source to be added to the basic medium for plant tissue culture, monosaccharides such as sucrose, glucose, fructose, maltose, mannose, rhamnose, trisaccharides, soluble starch, etc. are preferable, and sucrose and glucose are particularly preferable. Furthermore, as the auxin added to the basic medium,
naphthaleneacetic acid, indoleacetic acid, indolebutyric acid, 2
．． Examples include 4-dichlorophenoxyacetic acid, among which naphthaleneacetic acid is preferred. Its concentration is 0.1 xlo-
5-10 x 10-5 M, preferably 0.5 X1o-5
~5X10-5M. Further, examples of the cytokinin include hensyl adenine, kinetin, ceatin, etc. Among them, hensyl adenine is preferred. Its concentration is 0. IX 10-6 to 10X 10-6M, preferably 0.5XIO-6 to 5X 10-6M. In addition, the culture temperature in this culture is 20 to 30 °C,
Preferably, the temperature is 25 to 28°C, and it is preferable that no light be irradiated during culturing.

In addition, in order to obtain callus from a plant section of T. japonicum, the plant section is sterilized according to a conventional method, and a carbon source, plant hormones such as auxin and cytokinin, agar, and nitrogen such as amino acids are added as necessary. It is best to place and culture on a basic medium for plant tissue culture supplemented with a source. Formation of callus begins one week after the start of this culture, and adventitious roots are induced during subculturing of the callus, so this is used as the starting material for culture.

In the above-mentioned callus culture and adventitious root induction, the basic medium for plant tissue culture, the carbon source added, the culture conditions, etc. are the same as in the case of culturing the tuberous root slices of T. japonicus, and the concentration of plant hormones is also the same. However, for auxin, it is 10-3 to 10-'M, especially 10-5 to 10-JJ.
or preferably 10-4 to 10-8M for cytokinin
, preferably 10-6 to 10-'M, is the same as in the case of the above culture.

For organ culture of the roots or induced roots of T. japonicum , the roots or induced roots obtained as described above are cultured at 05 to 1 c.
This is carried out by cutting the plants into m-long pieces, planting them in a basic medium for plant tissue culture to which a carbon source, plant hormones such as auxin, and a nitrogen source are added if necessary, and culturing them.

The types of carbon sources and auxin added to the culture medium are as described above.
The procedure is the same as in the case of culturing the tuberous root slices of T. japonicus, but the concentration of auxin is 10-4 to 10-'M, especially 1
0-5 to 10-6M is preferable, and among the auxins, indolebutyric acid is particularly preferable.

Although growth and enlargement of induced roots can be observed even with the addition of auxin, browning occurs during long-term culture and productivity decreases. However, by further adding gibberellin to the medium, the above problem is solved and stable and vigorous growth occurs over a long period of time, which is preferable. Gibberellin is a substance with a shebang ring, G8, 7 to G, 30 to G. Several ten species have been identified from natural sources, such as 32 to G. There are no particular restrictions on the gibberellin used, or G8. and mixtures of GA3 and others are preferred. The concentration of gibberellin added is 10-' ~ 10
-''M, particularly preferably 10-5 to 10-'M. The culture conditions are 20 to 30°C, preferably 25 to 28°C.
It is preferable to perform shaking culture at a culture temperature of 80 to 100 rpm.

In the present invention, the production amount of alkaloids can be significantly improved by adjusting the concentration of specific inorganic salts in the medium used for organ culture.

That is, 0.3-1.5mM, preferably 0.3-1.5mM
0.7mM (N114)2SO4, 10-45
KNO3 in mM, preferably 10-23mM and 0.6
~3.0mM, preferably 0.6-1.5mM Mg
Adjust to contain SO4 or 10-45mM
It is suitable for the present invention to use a medium that is adjusted to contain KNO3 and 0.6 to 3,0 mM MgSO4 and not contain (NH4)2S04.

When culturing is carried out according to the method described above, differentiation and elongation of lateral roots are observed after about one week, and these roots can be harvested after about four weeks.

The bisbenzylisoquinoline alkaloids from the culture-induced roots thus obtained are separated and collected, for example, as follows. That is, first, the culture-induced roots are dried,
Grind and extract with organic solvent. Preferred organic solvents include lower alcohols such as methanol and ethanol; fatty acid esters such as methyl acetate and ethyl acetate; polar organic solvents such as alkyl halides such as chloroform and methylene chloride; among these, lower alcohols, particularly methanol, are preferred. be. Further, the extraction method may be a conventional extraction operation, but a method of immersion and applying ultrasonic waves is particularly preferred because it is simple. Next, after distilling off the extraction solvent, extraction is performed with an acidic aqueous solution having a pH of 3 or less, an alkali is added to the aqueous extract to adjust the pH to 9 to 10, and the mixture is extracted again with an organic solvent.

As the acidic aqueous solution, aqueous solutions such as hydrochloric acid, sulfuric acid, and citric acid, particularly citric acid, are suitable. Further, as the alkali, NaOH, OH, NH3, etc., and particularly ammonia are suitable. Further, as the organic solvent, chloroform is preferred, and lower alcohols are unsuitable because they are miscible with water. By distilling off the solvent from the extract thus obtained, a mixture of bisbenzylisoquinoline alkaloids is obtained.

C Effects of the Invention] According to the method of the present invention, various bisbenzylisoquinoline alkaloids such as berbamine, allomoline, isotetrandrine, and cephalanthine can be easily produced, as shown in Examples below. Furthermore, alkaloid productivity can be significantly improved by adjusting the content of inorganic salts in the medium used.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 (1) After thoroughly washing the tuberous roots of T. spp. collected in early April with water, cut them into approximately 1 cm wide pieces, and cut them into 70%
It was immersed in ethanol for 10 minutes and 1% antiformin solution for 10 minutes, and then washed with sterile water. Approximately 1 c sterilized tuberous roots
[[It was cut into one corner, placed on LS basic medium supplemented with 10-5M naphthaleneacetic acid, 10-6M benzyladenine, 3% sucrose, and 1% agar, and cultured at 27°C in the dark. After 30 days of culture, numerous roots and callus formation were confirmed from the explants. Adventitious roots were cut from the explants and placed in Gamborg's 8-5 basal medium (1mM (NH4)2SO4.30) supplemented with 10-5M indolebutyric acid and 2% sucrose.
(containing mM KNO3 and 1 mM MgSO4), and subcultured with shaking at 27° C. and 85 rpm. The cultured root root gradually shows browning, but it is between 10-6 and 10-5.
This can be prevented by adding M gibberellin (manufactured by Wako Pure Chemical Industries, Ltd.). After subculture, the culture-induced roots were freeze-dried to obtain a raw material for extraction.

(3ml for 50mg of raw material for extraction obtained in (1) above)
L methanol was added, and extraction was performed at 40° C. for 5 minutes while applying ultrasound. After repeating this extraction twice, methanol was distilled off to obtain a crude extract. 2 mA to this crude extract,
Add 3% citric acid solution, separate the insoluble matter in a furnace, and add 1 m of furnace liquid.
After adjusting Il to about 9 with concentrated aqueous ammonia, it was extracted with chloroform. Chloroform was distilled off to obtain a bisbenzylisoquinoline alkaloid mixture.

(++i) The bisbenzylisoquinoline alkaloid mixture obtained in (11) above was analyzed by high performance liquid chromatography (IPLC), and the results shown in Figure 1 were obtained, confirming the production of seven types of alkaloids.

Note that the conditions for performing HPLC are as follows.

Column Develosil 005-3 (manufactured by Nomura Chemical) Eluent Water/methanol/28% ammonia water (
100/400/1) Flow rate: 0.3 wl/min Temperature: 25°C Among these, allomoline, berbamine, homoallomoline, isotetrandrine, and cycleanine were also confirmed by MS as shown in Figures 2 to 6. Ta.

Example 2 Roots or plant organs obtained by differentiating from tuberous root sections or callus of T. japonicus were cut out and weighed 0.1 g.
Fresh weight 100mf1. In Erlenmeyer flask, SB5 (described below)
When cultured in a medium at 25 m DEG C. in the dark with shaking at 80-90 rpm, the cells multiplied about 20 times in 30 days, and significant amounts of useful alkaloids were produced, including about 2.5 mg of allomoline and about 1 mg of berbamine. Figure 7 shows changes in cultured root weight and allomoline and berbamine content. The growth curve was obtained by harvesting and weighing the cultured roots after culturing for a predetermined period of time. Further, the alkaloid content was obtained by freeze-drying the cultured roots and then treating them in a conventional manner and analyzing the basic fraction by HPLC. The alkaloid is
Identification was achieved by cochromatography with standard specimens and mass spectroscopy.

Example 3 Indolebutyric acid 1μM, gibberellin 1μM and sucrose 2
100 in Gamborg B 5 basal medium supplemented with %
0.1 g of fresh weight of cultured roots of T. japonicum were transplanted into a ++l flask and cultured for 30 days. Table 1 shows a comparison of the proliferation rate and the alkaloid content with those without the addition of gibberellin.

Note that each numerical value is an average value of two tests.

Table 1 Example 4 Indolebutyric acid 10 in Gamborg B 5 basal medium
A medium supplemented with 1 μM gibberellin and 3% sucrose, and an improved SB5 medium (0.5 mM
NH4)2SO4, 12.5mM KNO, and 0
．． containing 8mM MgSO4) and NB5 medium (25mM
Contains M KNO3 and 1mM Mg5Oa, (NH
4) using 2SO4-free), 50n each
In a +fL flask, 0.1 g of fresh weight of the cultured root of T. japonicum was transplanted to each 10 mJZ medium and cultured for 14 days. Table 2 shows a comparison of the growth rate and alkaloid content when using each medium.

In addition, each numerical value shows an average value.

Table 2 (B) is a drawing showing IPLC. In addition, the standard specimen (A
) is a mixture of equal amounts (by weight) of each alkaloid.

2 to 6 are drawings showing MS for allomoline, berbamine, homoallomoline, isotetrandrine, and cycreanin, respectively.

FIG. 7 is a drawing showing changes over time in the weight and alkaloid content of the roots of T. spp. in Example 2.

that's all

Claims (1)

[Scope of Claims] 1. A method for producing bisbenzylisoquinoline alkaloids, which comprises organ-culturing the roots or induced roots of P. japonica, and separating and collecting bisbenzylisoquinoline alkaloids from the cultured induced roots. 2. The manufacturing method according to claim 1, wherein organ culture is performed in a medium containing gibberellin. 3, 0.3-1.5mM (NH_4)_2SO_4,
The manufacturing method according to claim 1 or 2, wherein organ culture is carried out in a medium containing 10-45 mM KNO_3 and 0.6-3.0 mM MgSO_4. 4, 0.3-0.1mM (NH_4)_2SO4,1
0-23mM KNO_3 and 0.6-1.5mM M
The manufacturing method according to claim 1 or 2, wherein organ culture is performed in a medium containing gSO_4. 5, 10-45mM KNO_3 and 0.6-3.0m
The method according to claim 1 or 2, wherein organ culture is carried out in a medium containing MgSO_4 and not containing (NH_4)_2SO_4. 6. Carbon source, 0.3-0.7mM (NH_4)_2S
O_4, 10-23mM KNO_3 and 0.6-1.
1. A medium used for organ culture of roots or induced roots of a plant belonging to the family Tiliaceae, characterized by containing 5mM MgSO_4. 7. Carbon source, 10-45mM KNO_3 and 0.6-
Contains 3.0mM MgSO_4, (NH_4)_2S
A medium used for organ culture of roots or induced roots of a plant of the family Tuscanidae, characterized in that it does not contain O_4. 8. A sixth or seventh compound further containing an effective amount of gibberellin.
The medium according to the claims.