DE3007400A1 - METHOD FOR PRODUCING CODEIN FROM PLANT CELL CULTURES - Google Patents

Description

description

The invention relates to the production of the morphinan alkaloid Godein and its extraction from certain plant cell cultures. It has been found that selected cell cultures contain a new form of cytodifferentiated cells include, are able to produce codeine.

Opium contains approximately 15% anhydrous morphine. Codeine, ie morphine-3-methyl ether, is present in opium in an amount up to about 2.5% and is usually produced from morphine by methylation. The common opium poppy species is Papaver somniferum L., P. setigerum DC. however, it is also known to supply an opium product containing codeine. Codeine has antitussive and analgesic properties with little narcotic function and is normally used as a sulfate. Codeine is 7,8-didehydro-4,5-9i-epoxy-3-methoxy-17-methylmorphinan-6-of-ol (C ₁₈ H ₁ NO ₃ ) with a molecular weight of 299.36 and is a member of the morphinane group the alkaloids. Other members of this group are thebaine (C _g H ₂ NO ₃ ), the direct biosynthetic precursor of codeine, and morphine (C ₁₇ H ₁₉ NO ₃ ), which, like codeine, is a medicinally valuable active ingredient. Codeine has the structural formula

CH ₃ O

NCH ₃

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In recent years, plant cell culture methods have been used developed to the stage at which callus cultures with an ability to produce specific plant cells can be maintained in vitro. Plant cells, which are grown in vitro retain the genetic information of the original plant, i. i.e. that When induced, they develop roots and sprouts or produce metabolites that are essential for the one in question Species or variety are specific. Therefore, cell cultures based on Papaver somniferum L. and P. setigerum DC. declining as a potential source of poppy seed alkaloids, i.e. H. Morphinane, be considered. The problem, what occurs in the evaluation of this potential is to create cytophysiological conditions, which enable the exploitation of the biosynthetic potential of Papaver cell cultures. Callus tissue cultures were made produced by opium poppy, recently identifying the alkaloids present in such callus tissue have been. These callus tissues yielded benzophenanthridine, protopine, and aporphine-type alkaloids, however no morphinan-type alkaloids present in the original Plants are present, were obtained (see. Furuya et al., "Phytochemistry" 11, 3041-3044, 1972; Ikuta et al., "Phytochemistry" 13, 2175-2179, 1974). During execution of bioconversion experiments, when carried out alkaloids callus tissue cultures of Papaver somniferum L. have been added, it has been found that these cultures do not have the ability to (RS) -reticulin to thebaine, codeine and morphine, but were able to convert added (-) - codeinone to (-) - codeine to mecabolize (Furuya et al., "Phytochemistry" 17, 891-893, 1978). Therefore, using the previously known callus cultures of opium poppy, it was impossible to To synthesize codeine or morphine. A variety of another poppy species, Papaver bracteatum Lindl. (the for is known to contain thebaine), was used to obtain kailus tissues (from the seed heads), whereby

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These fabrics have been found to provide small amounts of thebaine (0.1% thebaine on a dry weight basis of the seed head tissue, see US Pat. No. 4,114,314). It was So far it has not been possible to produce codeine directly from cell cultures.

The invention is based on the knowledge that selected cell cultures which are based on the opium poppy P. somniferum L. or P. setigerum DC. can be adapted to produce codeine from staple foods. A new cell culture containing cytodifferentiated polyploid cells capable of producing codeine was produced.

The invention relates to a method for the production of codeine from plant cell cultures, which is described in the following Steps consists of: (a) Preparation of a cell inoculum from varieties of Papaver somniferum L. or Papaver setigerum DC, selected in such a way that when cells are grown in a suitable plant cell culture medium, that is growth-limited, cytodifferentiated · polyploid cells are formed which produce codeine are able, whereby these cytodifferentiated cells are larger than the undifferentiated cells, (b) Inoculation of a suitable nutrient medium for plant cell cultures with the inoculum cells and allowed to multiply subjecting these cells until at least one essential nutritional component becomes growth limiting, (c) subjecting them growth-limiting culture of a cytodifferentiation with the formation of the polyploid cells, (d) accumulation of Codeine in this cytodifferentiated cell-containing culture and (e) recovering the codeine from the obtained Culture.

The invention further comprises the new cells and the culture according to step (c).

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The invention is further illustrated by the accompanying drawings which show the growth-limited and cytodifferentiated Represents the cell culture state of cells from Papaver somniferum L. The cytodifferentiated polyploid cells are marked with A and the undifferentiated cells with B with the latter usually occurring as aggregates.

Experiments with Papaver cultures as well as with other cell strains that go back to medicinal plants have shown that cytophysiological conditions which are favorable for a metabolic production, a certain Require a degree of differentiation. In general, the higher the percentage of cells differentiated, the higher the amount of accumulated metabolites is greater. Because the differentiation and cell division among each other exclude, an extreme involvement of the cells in differentiation would prevent a culture from referring to to increase the number of cells. Differentiation of cells can be limited by a reduction in growth Factors induced, for example by nitrogen, carbohydrates or growth hormones. One culture reaches a point in time when a limiting growth factor is depleted and growth through cell division is replaced by a differentiation of cells.

In growth-limited Papaver somniferum L. cell cultures, this differentiation has been found to large cells that are polyploid and protrude from aggregates of undifferentiated smaller cells. The appearance of these cells coincides with the production of codeine in such cultures. The big cells are like it is believed to be the site of codeine fortification. A similarity between large cells and laticifers (size, polyploidy, The occurrence of multiple nucleation, pigmentation) is assumed. The initial selection of cell strains for Codeine education can therefore be a choice in terms of

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the ability of cytodifferentiation, particularly with regard to the formation of large cells as described have been.

All cell cultures that were found on transplanted pieces of tissue (inoculation materials) of Papaver somniferum L. or P. setigerum DC. decrease selected in such a way that slight cytodifferentiation in the Induction under observance of cultivation conditions, as they have been described, is possible, can are used according to the invention.

The biosynthetic potential of cells grown in vitro can be used for synthesis and enrichment made available by species-specific metabolites However, the rate of production as well as the amount of product depend at least in part on the genotype of a specific Cell line. Therefore, by selecting the cytodifferentiation method, you will be able to obtain many large cells Cell lines with superior qualities for metabolite production can be obtained. An easy way to find the genotype of varying plant material is the induction of Mutations in the cells and the selection of the mutant cell lines with regard to their ability to undergo cytodifferentiation too large cells, accompanied by an increased rate of codeine synthesis and enrichment.

The part of the opium poppy plant from which expanses or parental cells are taken can vary; H. cells can be taken from seedlings, flower parts, seed heads, leaves, stems, roots, etc. to form the starting cell culture to start. Preferably, the cells from the seedlings or flower parts including the microspores, taken.

Seedling cells (hypocotyl explants) seed head cells or other cells are particularly preferred. These cells can

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are kept on various plant cell culture media and serve as inoculum. To generate codeine these cells are inoculated on a suitable growth medium, whereupon these cells are in the presence of Light can be multiplied. To the development of the cytodifferentiated Polyploid cells, which are required according to the invention, should have at least one growth medium get exhausted, d. that is, this growth is limited. Under these limiting conditions was found that cytodifferentiation occurs which leads to the appearance of large polyploid cells. These polyploid cells are larger than the initial (undifferentiated) cells and occur most frequently on the Surface of clumps or agglomerates of the smaller cells in attached form.

The nutrient media necessary for growth, cytodifferentiation (and possibly for the codeine generation) can be the same as those used for the Starting culture or the inoculation material can be used. Any good growth medium for plant cell cultures can be used with any of the nutrients required for proportioning cell growth being depleted before the other nutrients, thus causing the medium to be growth limiting will. Typical suitable growth media are shown in Tables I and II. more details with regard to such suitable media and their production can be found in the book "Plant Tissue Culture Methods "by O. L. Gamborg and L. R. Wetter, 1975 published from National Research Council of Canada, Prairie Regional Laboratory, Saskatoon, Saskatchewan, Canada S7N OW9.

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- 10 ■■ Table I

Composition of mineral salt media for plant tissues

and cell culture MS ER B5

Macro nutrients mg / S, mM mg / i. mM mg / £ - NH ₄ NO ₃ 1650 20.6 1200 15.0 - - KNO ₃ 1900 18.8 1900 18.8 2500 25th CaCl ₂ '2H ₂ O 440 3.0 440 3.0 150 1.0 MgSO ₄ • 7H ₂ O 370 1.5 370 1.5 250 1.0 KH ₂ PO ₄ 170 1.25 340 2.5 - - (NH ₄ I ₂ SO ₄ - - - - 134 1.0 NaH ₃ PO ₄ 'H ₂ O - - - - 150 1.1

Micronutrients mg / A

mg / £

μΜ

mg / cell

AI

H ₃ BO ₃

MnSO ₄ '4H ₂ O MnSO- Ή ~ 0

ZnSO ₄ '4H ₂ O ZnSO ₄ · 7H ₂ O Zn'Na ₂ EDTA Na ₃ MoO ₄ ' 2H ₂ O CuSO ₄ '5H ₂ O CoCl ₂ ' 6H ₂ O Na ₂ 'EDTA FeSO ₄ · 7H ₂ O

Cane sugar

0. 83 5. 0 0 - 10 6th 2 100 2 .63 10 22nd 3 100 .23

8.6

0.25

0.025

0.025

37.3

27.8

30000 5.7

30 -

- 15 1.0 0.025 0.1. 0.0025 0.1 0.0025

100
100

37.3 27.8

37
0.1
0.01 0.01

100

100

40000 5.8

0.75 3.0

10
2.0

0.25

0.025

0.025

37.3

27.8

20000 5.5

4.5 50

60 7.0

1.0 0.1 0.1

100

100

Table II

Amounts and types of vitamins, hormones, and supplements to be used with the mineral salt media for agar and suspension cultures be used

MS ER B5

link mg / i, mg / A mg / £ Inositol 100 100 Nicotinic acid 0.5 0.5 1.0 Pyridoxine -HCl 0.5 0.5 1.0 Thiamine -HCl 0.1 0.5 10.0 Glycine 2.0 2.0 - IAA 1-30 - - NAA - 1.0 - Kinetin 0.04-10 0.02 0.1 2,4-D - - 0.1-2.0

The media are preferably selected for both the starting culture and the preparation of the vaccine material optimized, also for growth, cytodifferentiation and possibly for codeine production. It was found, that it is favorable, the medium especially for the growth stage with a source material for organic Nitrogen fortify, like casein hydrolyzate or coconut milk, with a cytokinin, these materials being each in a concentration range of about 0.1 to 5 ppm are suitable. Usually contains the medium a carbon source, a nitrogen source, sources for Ca, Mg, P, S and K as well as trace elements, vitamins and phytohormones. 2,4-dichlorophenoxyacetic acid is common added phytohormone, but it can also be caused by 1-naphthalene acetic acid or 3-indole acetic acid, usually be replaced in amounts of about 0.1 to 2 mg / l.

The cells can be easily removed by transferring them to fresh Hold medium or by refreshing the medium at approximately 1 to 4 week intervals. This growing

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Cells provide the necessary inoculum or, if left in a medium which becomes growth-limiting and undergoes cytodifferentiation after growth has ceased, accumulate codeine which can then be recovered. The conventional temperatures for the plant cell cultures be conveniently, for example, approximately from 20 to 40 ⁰ C for Impfmaterialzubereitung, the growth and Codeinanreicherung. The time from inoculation to the attainment of the growth-limiting state is usually about 1 to 3 weeks, depending on the size of the inoculum, the medium and the growth conditions.

The growth-limiting condition in this medium leads to the development of cytodifferentiated polyploids Cells marked with A in the drawing. These polyploid cells are usually around 5 to 10 times or more times larger than the undifferentiated cells and have average At least about 100 microns in diameter. Elongated large cells were observed, their size in the longitudinal direction were up to about 1000 microns or more. The undifferentiated cells are in contrast. diploid to these polyploid cytodifferentiated cells. Repeated microscopic examinations have shown that the "large polyploid cells often accumulate around clumps or aggregates of the undifferentiated cells, as can be seen, for example, from the drawing using the reference characters A and B. Since these polyploid Cells were always observed when codeine production was achieved, it is believed that they were related to codeine synthesis coincide and are necessary for this. At least about 10% of the total cells should be eligible for codeine fortification correspond to this polyploid type.

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The accumulation of codeine is allowed to proceed under the action of light in the growth-limited culture. Usually This codeine fortification takes about 15 to about 30 days before production rates drop, but this depends on many factors. The codeine will then obtained, for example by extraction with a suitable organic solvent, with subsequently optionally an isolation from the extract is carried out. Suitable organic solvents are ethyl acetate, Chloroform, methanol, ethanol and isopropanol. In a known manner, chromatography methods are very suitable for the isolation of the codeine from the extracted material.

example Production of a cell culture

Papaver somniferum L. cv. Marianne seeds are superficially sterilized and placed on a moistened filter paper sprouted in bowls. Hypocotyls from 5 to 6 day old seedlings are grown on nutrient medium B5 (cf. Tables I. and II) explanted, which contains 1 mg / 1 2,4-dichlorophenoxyacetic acid and has been solidified with 0.6% agar. The medium has a pH of 5.5 before sterilization by autoclaving. Generate within 6 weeks the explanted hypocotyl callus (neoplastic growth) with a diameter of 3 to 10 mm. The callus is transferred to fresh B5 medium supplemented with 1 g / l casein hydrolyzate and 2 mg / l kinetin (1-B5C medium) has been. The callus material is every 4 weeks using small pieces of callus as inoculation material in a Subculture bred. 6 months after growing in the subculture, the callus material is transferred to liquid 1BSC medium and from there in the form of 50 ml batches in 250 ml Delong flasks on freeze-drying shakers (150 opm) grown in minor crops. The cell suspension obtained and the cell aggregates are in weekly Transfer intervals to fresh 1B5C medium.

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All of these cultures are allowed to grow continuously under light with an intensity of 800 to 1000 lux at 25 to 28 ° C. For the production of biomass and for the cytodifferentiation of the culture, which leads to the formation of codeine, inoculum is collected from these suspension cultures (on sterile filters). Approximately 2 g of the cell mass is incubated with approximately 300 ml of the 1B5C medium in 1 liter kettles.

The 50 ml batch cultures, which are transferred at weekly intervals, consist mainly of small aggregates of cells, up to approximately 1 mm in diameter. These aggregates occasionally contain less than 5% cells of a large size (average diameter > 100 microns) with one or more polyploid nuclei (these cells are not growth limited and are unable to produce codeine). These aggregates vary in their pigmentation from dark brown to white. If these cultures (or the 300 ml production cultures) are completely growth-limited, then cytodifferentiation occurs, which leads to the appearance of large cells in increasing numbers. About 10 to about 50% or more cells can be of this large type.

A typical cell arrangement from one of the growth-limited differentiated 300 ml cultures is shown in the drawing, with A representing the large cells and B representing the small cells. This drawing is based on various photomicrographs.

After the incubation and the enrichment of codeine in the 1-BSC medium over a period of 3 weeks, the medium (300 ral) is extracted with ethyl acetate (2 × 50 ml), while the cells are treated with boiling methanol (150 ml) will. Both the methanol and the ethyl acetate extract are combined and evaporated. A yellowish to slightly brown material is obtained, which again

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in ethyl acetate / 1N HCl {1: 1) (2 50 ml) is dissolved. The two layers are collected separately. The ethyl acetate solution is washed with 1N HCl (10 ml). The wash solutions are collected and combined with the acidic layer obtained previously. The acidic solution is treated with sodium bicarbonate to adjust the pH to 7.5 to 8.0 and filtered. After filtration, the filtrate is extracted with ethyl acetate (2 × 25 ml). These extracts are combined with the ethyl acetate extract obtained previously. Evaporation gives a yellowish brown material (98 mg) which can be determined by TLC on silica gel plates (0.25 mm) using EtoAc: MeOH: NH ₄ OH (85: 10: 5) for development and CHCl ₃ : MeOH (8: 2) is purified as an eluent. This gives codeine of good purity (1.2 mg) with the same Rp value (0.22) as authentic codeine which has been simultaneously chromatographed by thin layer chromatography (TLC) in the manner described above (F 146 ° to 148 ° C, not corrected, literature: 154 - 155 ° C). The yield is approximately 0.15% based on the weight of the dry cells (722.2 mg). The mass spectrometric values are also used to identify the codeine product. The mass spectrum shows a molecular ion at m / e 299 corresponding to C ₁ OH ₂ -NO ₃ , the molecular formula for codeine, and is identical to that of an authentic codeine sample.

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Claims (13)

MÜLLKR-HOIiE - DiJUFBL · SCHÖN · HKItTKL DR. WOLFGANG MU LLER-BORE (PATENT ADVOCATE FROM 1S27-I975) DR. PAUL DEUFEL. DIPL.-CHEM. DR. ALFRED SCHÖN, D1PL.-CHEM. WERNER HERTEL, DIPL.-PHYS. APPROVED REPRESENTATIVES TO THE EUROPEAN PATENT OFFICE REPRESENTATIVES BEFORE THE EUROPEAN PATENT OFFICE MXNDATAIRES AGR ££ s PRES L1OFFICE EUROPEEN DES BREVETS C 3060 2 Feb. 7, 1980 Canadian Patents and Development Limited, 275 Slater Street, Ottawa, Ontario, Canada Codeine production process Plant cell cultures claims 1. Process for the production of codeine from plant cell cultures, characterized in that (a) a cell inoculum from varieties of Papaver somniferum L. or Papaver setigerum DC. manufactured , the varieties being selected such that they are used in cell growth in one suitable plant cell culture medium, which is limited in terms of its growth, cytodifferentiated polyploid cells are able to produce codeine, whereby the cytodifferentiated cells are larger than the undifferentiated cells, 030047/0602 S MUNICH SO-SlKnEIiTSTR. * -POST BOX K607B0 · CABLE: MÜEBOPAT · TEL ·. (OSfI) 474005 • TELKX 3-S4 (b) a suitable nutrient medium for plant cell cultures is inoculated with the inoculum cells, whereupon these cells can be multiplied until at least an essential nutrient component is limited in growth, (c) this growth-limited culture is left to cytodifferentiate to form the polyploid cells, (d) Codeine contained in the cytodifferentiated cells Culture enriches and (e) recovering codeine from the culture obtained. 2. The method according to claim 1, characterized in that the nutrient medium used in Tables I and II corresponds to the types MS, ER or B5 described. 3. The method according to claim 2, characterized in that the medium used is an added nutrient source of organic nitrogen for the propagation stage. 4. The method according to claim 1, characterized in that the nitrogen nutrient is growth-limiting. 5. The method according to claim 1, characterized in that the variety is a selected Papaver somniferuiu L. 6. The method according to claim 1, characterized in that the large cytodifferentiated cells at least approximately Make up 10% of the total cell population " = 7 The method of claim 6, characterized in that the _B cells have a cytodifferensierten düren-average diameter of at least about 100 microns. 030047/0602 8. The method according to claim 1, characterized in that the inoculation material used is applied to a seedling tissue going back. 9. The method according to claim 1, characterized in that the inoculum used on flower parts or pollen going back. 10. Plant cell culture from Papaver somniferum L. or Papaver setigerum DC., Characterized in that the culture limits growth by exhausting the growth nutrient material and at least 10% of the cells cytodifferentiate to form large polyploid cells are. 11. The culture of claim 10, characterized in that about 10 to about 50% or more of the total Cells are large polyploid cells. 12. Cultured cytodifferentiated plant cells that are based on Papaver somniferum L. or Papaver setigerum DC. declining and growth-limited polyploid cells are capable of producing codeine. 13. Cells according to claim 12, characterized in that they are about 5 to 10 times or several times larger than the undifferentiated parent cells. 030047/0602