Alkaloids of Cell Suspensions Derived from Four Papaver spp. and the Effect of Temperature Stress

Short Communication

Alkaloids of Cell Suspensions Derived from Four Papaver spp. and the Effect of Temperature Stress G.

BRIAN LOCKWOOD

Depanment of Pharmacy, University of Manchester, Manchester, M13 9PL, UK Received October 10, 1983 . Accepted January 25, 1984

Summary Cell suspension cultures derived from Papaver bracteatum Lindl var. Arya II P. somniferum L. var. Halle and var. Berlin, P. setigerum D. c., and P. nudicaule L. were found to contain protopine, sanguinarine, isothebaine and orientalidine. Growth of P. bracteatum suspensions at 36°C followed by 5°C caused release of thebaine into the media.

Key words: Papaver; alkaloids; thebaine.

Introduction Codeine (Tam et aI., 1980) and thebaine (Kamimura et al., 1976; Zito and Stab a, 1982) have been isolated from cell suspensions of Papaver somniferum and P. brae· teatum respectively. Callus cultures derived from P. somniferum, P. braeteatum, P. setigerum, P. orientale and P. rhoeas have been shown to yield mainly benzophenanthridine, protopine and aporphine type alkaloids (Ikuta et aI., 1974; Furuya et aI., 1972) but thebaine (Kamimura and Nishikawa, 1976; Kamo et al., 1982), isothebaine and orientalidine (Lockwood, 1981) have also been isolated. The numerous attempts which have been made to induce appreciable morphinan alkaloid accumulation in cultured suspensions of Papaver spp. have resulted only in limited success (Tam et al., 1980). The purpose of this study was to identify the alkaloids present in a range of Papaver cell suspensions.

Materials and Methods Cell Cultures. Seeds of Papaver bracteatum Lindl var. Araja II, P. somniferum L. var. Halle and var. Berlin, P. setigerum D. C. and P. nudicaule L. were surface sterilised in 10m130% w/y H202 containing 1 % Triton X for 2 min, then germinated on wetted filter paper in petri dishes in darkness at 25°C. Hypocotyls of 5 day old seedlings were explanted onto Murashige and Skoog's medium (Murashige and Skoog, 1962) containing 1.0 mgl- 1 kinetin and 10 mgl- 1 2,4-dichlorophenoxyacetic acid. Funher addition of 0.1 mgl- 1 kinetin and 10 mgl- 1 asorbic acid were incorporated in subsequent media. Callus growth in darkness at 25°C was cream coloured and subculturing was undenaken every 4 weeks. Suspension cul-

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BRIAN LOCKWOOD

tures were initiated by transferring callus portions which had undergone more than 6 subcultures, to 250 ml Erlenmeyer flasks containing 50 mlliquid medium, and shaken at 150 rpm on an orbital shaker. Suspensions were maintained by shaking them at 100 rpm in a 12 h darkness/12 h light sequential regimen and subcultured weekly. Temperature treatment of P. bracteatum suspensions. Three-week-old suspensions of more than 6 subcultures were incubated either at: 5°C/l day then 36°C/l day, 5°C/3 days then 36°C/3 days, 36°C/l day then 5°C/3 days, 36°C/3 days then 5°C/3 days, under the originallight regimen (Lockwood, 1981). All 4 treatments were undertaken separately on different batches of suspensions. Extraction procedures. 50 ml suspension cultures were first separated by filtration and, after lyophilising, were extracted by grinding with 5 % acetic acid, and filtering under vacuum. The filtrate was basified by addition of 10% NI-LOH to pH 9.0 and extracted by shaking with 3 x 50 ml portions CHCh. Bulked extracts were evaporated to dryness under vacuum. Media were basified and then extracted, as for cells. Preliminary identification of alkaloids was by tic on 0.25 mm layers of silica gel, developed in a) (CH3)2CO/C6H,CH3/C2HsOH/NI-LOH (40/40112/2.5) and b) C 6H 6/(CH3)2CO/CH 30H (70120/10). Isolation was undertaken by chromatographing the extracts on 2 mm silica gel G layers in the above systems. Visualisation was effected by Dragendorff's reagent and alkaloids were isolated from silica gel bands by washing with CHCh/MeOH (Ill) and evaporated to dryness under nitrogen prior to weighing. Alkaloids were identified by comparison of tic Rr values (systems A and B) and ms data with those of standard samples. TLC identification yielded the following Rr's in two systems, isothebaine 0.70(a), 0.41 (b): orientalidine 0.82(a), 0.78(b) protopine 0.87(a) 0.10(b): sanguinarine 0.93 (a), 0.91 (b). ms. spectral measurements (70 eV, MS-902) yielded the following disintegration patterns: Isothebaine mle 311 (M+ 100%),310 (M+-l, 62%), 296 (33%), 294 (55%), 280 (42%) Orientalidine mle 397 (M+ 65%), 206 (47%), 205 (29%), 204 (41 %), 193 (29%),192 (53%), 164 (35 %), 163 (35 %), 162 (100 %) Protopine mle 353 (15%),190 (49%),163 (87%),148 (100%) Sanguinarine mle 332 (10%), 317 (100%) Thebaine mle 311 (M+ 100%),282 (11 %), 268 (17%), 255 (51 %)

Results and Discussion Various levels of alkaloids were detected in cell suspensions from the 4 Papaver spp. (Table 1). Absence of any morphinan alkaloid could be caused by lack of a functioning metabolic step as postulated by Ikuta (!kuta et al., 1974). Temperature treatment at 5 °C followed by 36°C for different periods had little effect on the alkaloid patterns of cells or media of P. bracteatum suspensions. All yielded sanguinarine, orientalidine and protopine. However, the two latter treatments, 36°C for 1 day and 3 days followed by 5 °C for 3 days caused suspension cells to yield sanguinarine alone, whilst media were found to contain 0.016 % and 0.02 % thebaine respectively along with sanguinarine. A number of plant cell cultures have been separately grown over a range up to 35°C at which virtually no growth occurred, and down to 17 °C at which growth decreased (de Capite, 1955). At a temperature of 5 °C «chilling stress» is known to cause severe inhibition of general photosynthetic reactions and disruption of organelles in both plants and plant cell cultures (Oquist, 1983). As the thebaine has been shown to be produced by P. bracteatum cultures after Z. Pjlanzenphysiol. Ed. 114. S. 361-363. 1984.

Alkaloids of cell suspensions of Papaver

363

Table 1: Alkaloid levels in suspension cultures from Papaver spp.

P. somniferum v. Halle

Cell Dry Weight (mg) 850

v. Berlin

690

P. bracteatum v. Arya II

820

P. setigerum

570

P. nudicaule

430

Cell extract (% dry wt) Protopine (0.02 %) Sanguinarine (0.05 %) Sanguinarine (0.04%) Isothebaine (0.02 %) Orientalidine (0.02 %) Sanguinarine (0.05 %) Orientalidine (0.03 %) Sanguinarine (0.04%)

Protopine (0.07%) Sanguinarine (0.07 %)

Medium extract (% cellular dry wt) Protopine (0.02 %) Isothebaine (0.02 %) Orientalidine (0.02 %) Sanguinarine (0.05 %) Orientalidine (0.03 %) Sanguinarine (0.05 %) Protopine (0.02 %) Orientalidine (0.02 %) Sanguinarine (0.02 %)

treatment at 36 °C followed by SoC, it was probably produced during the period of growth inhibition and released during «chilling». When cells were treated at SoC, initial «chilling stress» would be likely to inhibit further metabolism, with the consequence that no thebaine was produced. References DE CAPITE, L.: Am. J. Bot., 42, 765-868 (1955). FURUYA, T., A. IKUTA, and K. SYONO: Phytochem., II, 3041-3044 (1972). lKUTA, A., K. SYONO, and T. FURUYA: Phytochem., 13, 2175-2179 (1974). KAMIMuRA, S., M. AKUT SU, and M. NISHIKAWA: Agr. BioI. Chern., 40, 913-919 (1976). KAMIMURA, S. and M. NISHIKAWA: Agr. BioI. Chern., 40, 907-911 (1976). KAMo, K. K., W. KiMOTO, A. Hsu, P. G. MAHLBERG, and D. D. BILLS: Phytochem., 21, 219-222 (1982). LOCKWOOD, G. B.: Phytochem., 20, 1463-1464 (1981). MURASHIGE, T. and F. SKOOG: Physiol. Plant., 15, 473-497 (1962). OQUIST, G.: Plant, Cell Environ., 6, 281-300 (1983). TAM, W. H. J., F. CONSTABEL, and W. G. W. KURZ: Phytochem., 19, 486-487 (1980). ZITO, S. W. and E. J. STABA: Planta Medica, 45, 53-54 (1982).

Z. Pjlanzenphysiol. Ed. 114. S.361-363. 1984.