Agr. Biol. Chem., 40 (5), 907"911, 1976

Growth and Alkaloid Production of the Cultured Cells

of Papaver bracteatum•õ

Shoji KAMIMURAand Minoru NISHIKAWA

Fermentation Research Laboratories, Sankyo Co., Ltd., Hiromachi, Shinagawa-ku, Tokyo Received November 4, 1975

Callus cells were derived from seedlings of Papaver bracteatum Lindl. and maintained on agar media for more than 2 years. Liquid cultures have been established and maintained for more than 2 years and successful growth of considerable amounts of callus cells in suspension culture has been accomplished. The callus cells thus obtained were harvested, lyophilized and alkaloids were extracted. Alkaloids were also extracted from culture filtrates. Separation and identification of alkaloids was accomplished by TLC and GLC. The main alkaloids produced in the callus cells were identified by gas chromatographic-mass spectrometry as L-stylopine and . The production of by the cells was almost negligible in the cultural condition used here.

A Papaver bracteatum Lindl. (strain) has Japan, and showed that the alkaloids con been reported to contain a alkaloid, tained in these callus cells were mainly benzo thebaine (A), (Fig. 1) as the major alkaloid, phenanthridine, protopine and aporphine. by Sharghi and Lalezari,1) Neubauer and They thought that the callus derived from Mothes2) and Fairbairn and Hakim.3) The this strain of P. bracteatum seemed to form baine has been used as a material source of simpler alkaloidal components than those of . Recent research on the cultivation the original plants, and to lack the biosynthetic and alkaloidal analysis of P. bracteatum has versatility of the original plant. been conducted by Ogiso et al. (unpublished The study reported here was undertaken to data). They showed that the strain of P. produce large amounts of callus cells of P. bracteatum obtained from West Germany bracteatum in liquid cultures and to obtain contained thebaine abundantly as the major fundamental information on the capacity of alkaloid, however, the other 7 common the callus cells to produce secondary consti strains of the plant cultured in Japan con tuents. tained isothebaine as the major alkaloid, but MATERIALS AND METHODS not thebaine. Ikuta et al." have reported the detection of benzophenanthridines alkaloids Plant tissue culture and media. Callus cells derived (Norsanguinarine, oxysanguinarine, dihydro from sterile seedlings of P. bracteatum Lindl. (obtained sangunarine, sanguinarine and chelirubine), from Firma Ernst Benary, 351, Hann Miinden, West Germany) were induced in the agar medium as pre protopine and aporphine from P. bracteatum viously described.5 They have been continuously cells. The strain of this plant used in their subcultured on an agar medium at 40-day intervals studies was obtained from Kasukabe Experi for more than 2 years and in a liquid medium at 10-day mental Station of Medicinal Plants, National intervals for more than 2 years. Suspension cultures Hygienic Laboratories. They investigated the were established by inoculating a liquid medium with callus cells which had been grown on an agar medium. presence of alkaloids in callus cells derived The agar medium used was Murashige and Skoog•Ls from 11 species of Papaveraceae growing in and contained 3 % sucrose, 1 mg/liter NAA, 0.1 mg/

liter kinetin and 10% coconut milk. The liquid •õ Tissue Culture of Papaver bracteatum. Part medium for suspension culture had the same com- II. See References 5, 6). 908 S. KAMIMURA and M . NISHIKAWA position as the agar medium except that it contained chloroform, by shaking. The combined chloroform no agar or coconut milk, and the plant growth extracts were concentrated under vacuum to almost regulators used were I mg/liter 2,4-D and 0.1 mg/liter dryness, dissolved in 10 ml chloroform, and washed kinetin. three times, each with 10 ml of distilled water. The washed chloroform extract was concentrated under Suspension culture. Cells from well established vacuum to dryness, and subjected to GLC and TLC suspension cultures, which had been reported in a analyses using thebaine as a standard sample. previous paper,') were aseptically transferred to 100-m1 b) Alkaloid (B) and (C) (Fig. 1). After con Erlenmeyer flasks, 500-m1 Sakaguchi flasks or 5-liter siderable preliminary work on TLC and GLC analyses, Fernbach flasks containing 25, 80 or 1000 ml of liquid it was found that two new alkaloids (B) and (C), medium, respectively. The cell suspension was filtered which had not been detected in the parent plant, could through a platinum sieve of 80 mesh (250ƒÊm) (Ikemoto be found in the callus cells. Both alkaloids were Chemical Industrial Co., Ltd., Tokyo, Japan). A extractable by the method of thebaine extraction. glass scoop was used to transfer 21.2 mg of fresh cells However, when a small amount of material was (1.6 mg dry wt.)/10 ml medium to each new flask. The used, neither alkaloid could be extracted well; there- flasks were incubated in the dark at 26°C on a reci fore, a slight modification of the procedure was made. procal shaker at 120 rpm. The fine powder of callus cells (5 g) was extracted The remaining callus cells were weighed and frozen three times, each with 30 ml of 5% acetic acid solu at -5°C for subsequent chemical analysis. The tion for 1 hr by shaking. The combined extracts were culture filtrate was concentrated under vacuum to dried and resuspended in 30 ml fresh 5 % acetic acid about one-tenth its original volume and stored under solution overnight at 40°C. Further treatment of the refrigeration for further chemical assay. combined acetic acid extracts followed the procedures described in the section on thebaine extraction. Extraction and isolation of alkaloids. The frozen c) Extraction of culture filtrates. The culture tissue from agar and liquid cultures was lyophilized in filtrates were adjusted to pH 10 with 28% ammonia a Virtis freeze drier (N.Y., U. S. A.). Lyophilized cells solution and extracted three times with chloroform were ground to a fine powder. (three times their volume), and these chloroform ex a) Thebaine (A). The fine powder (100 g) of the tracts were concentrated. Further treatment of the callus cells was moistened with a few ml of 5 chloroform extracts were carried out as described Na2CO3 aqueous solution and extracted with 300 ml above. of methylene chloride in a Soxhlet extractor for 10 hr. After filtration, the methylene chloride extract was Separation and identification of alkaloids. The concentrated under reduced pressure to 10ml. This existence of thebaine in various extracts was con extract was shaken three times, each with 20ml of 5 firmed on a preparative silica gel F TLC (Merck, acetic acid, and the combined acetic acid extracts were 0.25 mm) in solvent systems (I), (II) and (III), and made basic to pH 10 with 28% ammonia solution. by GLC. The solvent systems were (I); benzene: This was extracted three times, each with 20ml of methanol (4:1, v/v), (II); n-butanol: acetic acid: water Tissue Culture of Papaver bracteatum 909

(7:1:2, v/v), (111); benzene: ethyl acetate: ether: 10% P. bracteatum plants was treated by the same procedure ammonia solution (4:6:1:0.3, v(v) and (IV); n- as the callus cells to isolate thebaine. hexane: ethyl acetate: methanol (15: 3: 1, v/v). Visua b) Alkaloid (B), L-stylopine. It is well known lization of developed TLC plate was accomplished that Coptis japonica Makino contains , with UV light (bluish violet fluorescence) and with palmatine and coptisine. The methanol extracts of Dragendorff's spray reagent. the fine powdered root of the plant were applied to Gas liquid chromatography was carried out on a a silica gel column and eluted with solvent system 2 mm, (LD.) by 60 cm-long glass column coated with (II), and with methanol. The yellow colored fractions 2% OV-17 on 80- to 100-mesh Chromosorb AW. were collected. The combined fractions were evaporat The column was operated isothermally at 250°C (M & ed to almost dryness, spotted on preparative silica F 104, Chicago, USA) with a hydrogen flame detector. gel F TLC (Merck, 2 mm, 20 x 40 cm), and developed Optical rotation was recorded on a polarimeter in solvent system (II). Two yellow bands were

(Perkin-Elmer 141, Uberlingen, West Germany). Mass observed on TLC and they were eluted with methanol. spectra were obtained with a JMS-D 100 (JEOL, The methanol extracts were reduced with sodium Tokyo, Japan) combined gas liquid chromtographic- borohydride, and evaporated to dryness under vacuum.

mass spectrometry instrument equipped with a 2mm, The dried extract was partitioned in chloroform-water

(I.D.), by 1 m-long glass column coated with 3% OV-1 solution. The chloroform solution, after separation on 80- to 100-mesh Chromosorb W, operated isother by centrifugation, were concentrated, and chromato mally at 250°C (GC-LC, Shimazu Co., Kyoto, Japan) graphed on preparative silica gel F TLC (Merck,

with a hydrogen flame detector. The electron energy 0.25 mm) in solvent system (IV). The three reduced

was 75 eV, trap current was 300ƒÊA and source alkaloids were observed as pale yellow fluorescent temperature was 200°C. spots under UV light. They were eluted with chloro The isolation of alkaloids (B) and (C) was accom form. After repeated purification on TLC, each spot

plished by silica gel column chromatography, TLC was analyzed by GLC and their chemical structures and GLC. The chloroform extracts were applied to were determined by gas chromatographic-mass spectro a silica gel column. Elution was accomplished step- metry as L-stylopine (L-tetrahydroeoptisine), tetrahydro

wise with benzene-methanol solution at a flow rate berberine and tetrahydropalmatine. L-Stylopine, thus of I ml/min. Fractions of approximately 50 nil were obtained was used as a reference for identification

collected. On the basis of TLC analysis, appropriate of the alkaloid isolated from the callus cells.

fractions were combined and concentrated under

vacuum to a volume of 1 ml. The concentrated eluent RESULTS was spotted on preparative silica gel F TLC (Merck, 2 mm, 20 x 40 cm) and developed for 38 cm in a glass Growth of the cells in solid and liquid media jar using solvent system (1). The location of alkaloids The callus cells derived from seedlings of was detected under UV light and by Dragendorff•Ls reagent. Spots were eluted from the silica gel with P. bracteatum were initially white in color but

methanol. Alkaloids (B) and (C) were isolated on turned brown in a few weeks on the solid

preparative silica gel F TLC (Merck, 0.25 nom) in medium or after subcultures on the medium solvent system (IV). They were then eluted from which contained 1 mg/liter of 2,4-D, 0.1 mg/ the developed TLC plate with chloroform, and crystal liter of kinetin and 10% of coconut milk. lized to yield pale yellow needles. However, subculturing on the medium con taining 1 mg/liter of NAA, 0.1 mg/liter of Extraction of referential samples a) Thebaine (A). The fine powdered root of kinetin and 10% coconut milk brought about

TABLE L GROWTH OF P. bracteatum CALLUS CELLS IN FLASK SUSPENSION CULTURE Each flask was incubated for 3 weeks at 26°C in the dark on a reciprocal shaker which operated at 120 rpm. The average fresh and dry cell weight per flask was made from each treatment with 4 flasks/per treatment and each treatment was carried out in triplicate. 910 S . KAMIMURA and M. NISHIKAWA

a white callus, and the maximum growth was identified as . L-stylopine (L-tetrahydrocopti usually reached in about 50 days ." sine). The results on the cell growth in liquid medium are summarized in. Table I. Experi Alkaloid (C). The chloroform extract was ments were carried out in triplicate , with 4 prepared by the same procedures as for the flasks/replicate. With few exceptions, cell isolation of (B). (C) was a pale yellow needle- growth was abundant and the average fresh like crystal which gave a positive color reac and dry weight of the cells harvested from tion to Dragendorff=s spray reagent. Rf each flask was fairly constant. values on silica gel TLC were 0.200, 0.300 and 0.214 in solvent systems (I), (II) and (III), Alkaloid production respectively, and the Rt value on GLC was The existence of thebaine in the callus cells 11 min at 250°C. In gas chromatographic- was confirmed by TLC and GLC analyses. mass spectrometry, the parent peak was found However, it was limited to the callus cells at to be M+ at m/e=353 (5 %), the base peak at early stages of subculturing. About 30m pas- /e=148 and the fragment peak at m/e=205 sages of subculturing resulted in reduced re (I %). By comparing the typical fragments of covery of this alkaloid, although trace amounts (C) and their relative intensities with protopine, of it existed in the callus cells even at the later as described by Doljes et al." and Porter and stages of serial passages of subculturing.6) In Baldas,9) alkaloid (C) was identified to be the course of this study, it was found that the protopine. callus cells formed new alkaloids (B and C), The production of both alkaloids , L-stylo after long periods of subculturing which had pine and protopine, was approximately 150-

not been isolated from the parent plants . 200 u,g and 100•`150ƒÊg , respectively, per g Alkaloids (B) and (C) were formed fairly con dry weight cells of P. bracteatum. In the cul stantly in. the cells over the period of 2 years . tural filtrates, L-stylopine was produced about 1 ƒÊg/ml, and protopine was slightly less than

Identification of alkaloids (B) and (C) L-stylopine. Alkaloid (B). The callus cells derived from seedlings of P. bracteatum were extracted with

acetic acid. The chloroform soluble fraction DISCUSSION obtained from the acetic acid extracts gave (B) Studies were conducted on the develop by chromatography. (B) was a pale yellow ment of techniques for the cultivation of P . needle-like crystal which gave a positive color bracteatum cells on a relatively large scale . reaction to Dragendorff•Ls spray reagent . Rf The growth of cells in various types of flasks values on silica gel TLC were 0 .696, 0.500 and was studied repeatedly , and the results are 0.905 in solvent systems (1) , (II) and (III), listed in Table I. In Fernbach flasks , a growth respectively, and the Rt value on GLC was rate on the order of about 4 g fresh weight/ 9.4 min at 250°C. Measurement of optical liter/day was obtained in Murashige and rotation showed that (B) was an L-form . In Skoog•Ls medium containing 3 % sucrose , 1 mg/ gas chromatographic-mass spectrometry, the liter 2,4-D and 0.1 mg/liter kinetin . Growth parent peak was found to be M+ at in/e=323, rate was 0.285 g dry weight of cells/liter/ day . the base peak at m/e=148 and the fragment Productivity in terms of the ratio of dry cell peak at in/e=1.74 (Fig. 1). The fragmentations weight/fresh cell weight was higher in the in mass spectrometry, as well as TLC and cultures in Erlenmeyer flasks (7 .5 %), followed GLC analyses, were in good agreement with by Sakaguchi flasks (7.26 %) and lower those of the authentic sample , in F , which was ernbach flasks (7.01 %). derived from coptisine and described by Tani" The growth rate obtained was similar to the (private communication). Thus, (B) was results of others. 10-12) Tissue Culture of Papaver bracteatum 911

An important observation on the contents of alkaloids in cultured cells was made previ Acknowledgement. The authors thank Dr. C. Tani, Professor of Kobe Pharmaceutical University, Moto ously by Konoshima et al."' in Datura stramo yama Kitamachi, Higashinada-ku, Kobe City, and nium var. tatula stem callus. They showed Dr. J. Slavik, Professor of University J. EV. Purkyne, that the alkaloid content in the cells was low Komenskeho nam. 2, Czechoslovakia, for their kind when cell growth was large. In our results, as presentation of L-stylopine. They also wish to express well, the production rate of alkaloids per their thank to Dr. T. Hayashi, College of General weight of dry cells was higher in Erlenmeyer Education, Univesity of Tokyo, Komaba, Meguro-ku, Tokyo, Dr. R. N. Goodman and Co-workers, Depart- and Sakaguchi flask cultures, and lower in ment of Plant Pathology, Univesity of Missouri, Colum Fernbach flask cultures. bia, Missouri, USA, and Dr. A. Ogiso, Central A certain strain of P. bracteatum contains Research Laboratories, Sankyo Co., Ltd., Hiromachi, thebaine as the major alkaloid.'-" Shinagawa-ku, Tokyo, for their helpful discussion and The plant used here was the strain of P. critically reviewing the manuscript. bracteatum obtained from West Germany. By comparison of the alkaloidal constituents in REFERENCES various strains of this species, Ogiso et al. 1) N. Sharghi and I. Lalezari, Nature, 213, 1244 showed that the strain from West Germany (1967). contained only thebaine as the major alkaloid, 2) D. Neubauer and K. Mothes, Planta Med., 11, whereas the other seven strains contained 387 (1963). isothebaine as the major alkaloid, but not 3) J. W. Fairbairn and F. Hakim, J. Pharm. thebaine. Pharmc., 25, 353 (1973). 4) A. Ikuta, K. Syono and T. Furuya, Phytochemi Based on their findings, a series of experi stry, 13, 2175 (1974). ments was carried out in an attempt to dis 5) S. Kamimura and M. Akutsu, Agr. Biol. Chem., cover the potential of cultured cells of P. 40, 899 (1976). bracteatum to produce thebaine. It was found, 6) S. Karnimura, M. Akutsu and M. Nishikawa, however, that P. bracteatum cells formed L- ibid., 40, 913 (1976). 7) C. Tani and N. Takao, J. Pham. Soc. Japan, 82, stylopine and protopine in their cells and 598 (1962). cultural filtrates as the major alkaloids under 8) L. Dolejs, V. Hanus and J. Slavik, Collection the cultural conditions used here, and that the Czech. Chem. Commun., 9, 2479 (1964). production of thebaine in the cells and filtrates 9) Q. N. Porter and J. Baldas, "Mass spectrometry was almost negligible. of heterocyclic compounds," Wiley-Interscience, a Division of John Wiley & Sons, Inc., New York, The formation of thebaine in the cells, as N.Y., 1966, pp. 434. described in the following paper," was ob- 10) B. D. Patterson and D. P. Carew, Lloydia, 32, served at the early stage of the callus growth 131 (1969). soon after induction, but was reduced to trace 11) C. Wang and E. J. Staba, J. Pharm. Sci., 52, amounts in the cells after 20-30 passages of 1058 (1963). 12) P. K. Lee, D. P. Carew and J. Rossaga, Lloydia, serial subculturing, each at 10-day intervals. 35, 150 (1972). The results we obtained in cell culture 13) M. Konoshima, M. Tabata, H. Yamamoto and using the different strain of P. bracteatum N. Hiraoka, Yakugaku Zasshi, (J. Pharm. Soc. were similar to that of Ikuta et al.4) Japan), 90, 370 (1970).