Characterization of a Suspension Culture of Chelidonium majus L. on Growth and Accumulation of Sanguinarine

Biochem. Physiol. Pflanzen 188, 116-120 (1992) Gustav Fischer Verlag Jena

Characterization of a Suspension Culture of Chelidonium majus L. on Growth and Accumulation of Sanguinarine FRANCA TOMEI) and MARIA LAURA COLOMB02) 1) Dipartimento di Biologia, 2) 1st. Sc. Farmacologiche, Milano, Italy

Key Term Index: alkaloid accumulation kinetics, cell cultures growth, sanguinarine production; Chelidonium majus L.

Summary Cell suspension cultures of C. majus L. - obtained from callus - were characterized on growth and on accumulation of isoquinoline alkaloids. The liquid batch cultures consisted of coarse cell aggregates; their fresh volume becames triple each 14 days after subcultures. Cell cultures were found to produce relatively large amounts of isoquinoline alkaloids; sanguinarine is accumulated as main alkaloid and also released into the medium.

Introduction Chelidonium majus L. (fam. Papaveraceae) drew the attention of several authors for its use in folk medicine. Cytotoxic, antiviral, antifungal (LENFELD et al. 1981; HBJMANKOVA et al. 1984) and anti-inflammatory (AMOROS et al. 1977) activities have been found in crude extract of this plant. C. majus is well known as a source of benzophenanthridine (sanguinarine, chelidonine, chelerytrine), protoberberine (berberine) and protopine (coptisine) alkaloids (W AGNER 1982). Cell cultures of C. majus are reported only in a Patent (BOEHM et al. 1980) in a Schenk and Hildebrandt (SH) medium plus auxin. We have reported previously that calli of C. majus were able to produce alkaloids (COLOMBO and TOME 1991). In order to continue the characterization of alkaloid accumulation in C. majus cell cultures, further experiments were done in liquid medium. In this work we analyze the time course of cell growth and sanguinarine production in cell suspension cultures of C. majus L.

Materials and Methods Cell suspension cultures of C. majus were initiated by transferring callus obtained from seeds subcultured on solid modified Gamborg B5 medium (COLOMBO and TOME 1991) - to the same medium without agar in lOO rnl Erlenmeyer flasks . Cells were cultured on rotatory platform shaker at 140 rpm, on darkness at 25°C. Suspension culture was subcultured every three weeks by adding 25 ml of culture to 50 ml fresh medium. Suspension-grown cells were harvested during the growth cycle. To determine the fresh volume of cells, an aliquot of 10 ml was transferred to a calibrated conical tube followed by centrifugation for 5 min at 1,500 x g. 116

BPP 188 (1992) 2

The medium pH and the sucrose values were routinely measured in the resulting supernatant. Sucrose was analyzed by the phenol sulphuric method (DoBors et al. 1956). Cell dry weight (d. w.) was calculated from the weight difference of a glass filters before and after suction filtration on an aliquot of cell suspension culture and subsequent drying at 60°C for 24h. The extraction of isoquinoline alkaloids, from cells and from medium separately, was performed as previously reported (BUGATTI et al. 1991). Alkaloids were separated by HPLC, using a standard system consisting of a L-6200 Intelligent Pump Merck-Hitachi, a L-4200 UV -Vis Detector Merck-Hitachi and a gradient program. Eluent A=H 20: ACN (75:25) + 0.001 % H3P04 ; eluent B = H20: ACN (5: 95) + 0.001 % H3P04 , on a C 18 LiChrospher 5!tm (250 X 4 mm i. d.). The elution profile was: 0-5 min, 100 % A (isocratic); 5-20 min, from 0 to 25% B in A; 20-35 min, 25% B in A (isocratic); 35-45 min, from 25% to 100% B in A; 45-55 min, 100% B (isocratic). Flow rate was 1.5 ml X min-I. In order to determine the peak purity, alkaloids containing extracts were analyzed by means of diode array detection (L-3000-Merck-Hitachi) from 200 to 520 nm, usually monitored at 280 nm. Alkaloids were checked also by a fluorescence detector Jasco 820-FP from 200 to 700 nm. For the quantification of sanguinarine, an external standard method has been elaborated.

Results and Discussion

Time Course Growth From established callus, a suspension culture as almost brownish was easily obtained in undifferentiated form. The cell cultures presented cell filament, small cell cluster and isolated cell club (37.5-500/lm). A growth curve is outlined in Fig. 1. Cell culture growth starts immediately and reaches - if measured as dry weight - the maximum after 14 days; the dry weight slows down from the 14th to the 23rd day. Cell fresh volume increase stops later, 20th day; this is probably due to a higher cell hydration. Sucrose concentration decreases at constant rate untill the 20th - 23rd day, when it is almost exhausted; during the culture period the pH values of the suspension increases gradually to 6.3, after an initial decrease from pH 6.5 to pH 5.0-5.3 (Fig. 1).

Alkaloid Production The chloroform extract of both cells and medium was analyzed by means of HPLC; it was necessary to adopt a gradient program since the recovered metabolites have very similar chromatographic properties. One of the major components of the cell alkaloid chloroform extract was sanguinarine; its identity was confirmed by spectroscopic methods (HPLC Diode Array, HPLC fluorescence, IR). Besides sanguinarine nine other alkaloids, all present in minor amounts and more polar than sanguinarine, could be detected in HPLC chromatograms; UV spectra of these compounds are typical of the isoquinoline alkaloids; with diode - array - detection further information was obtained on the chromophore of the alkaloids and on peak purity. The fluorescence spectra of unknown compounds had generally an emission wavelength of 440 nm, with an excitation of 350 nm. The identity of these compounds is under study. BPP 188 (1992) 2

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Time Course of Alkaloid Accumulation

The time course of sanguinarine production shows an increase almost in parallel with cell growth; the highest increase has been detected from the 3rd to the 10th-14th day of growth, with a maximum content of 8.85 mg per 100 mg dry cell weight (Fig. 2). The sanguinarine accumulation decreases after the 14th day. Minor metabolites have different time courses of production during the culture period (Fig. 3). Cultured cells release also a considerable amount of alkaloids into the medium. The release of sanguinarine is maximum (37.3 mg X I-I culture) at the early phase of cell growth (Fig. 2). Besides sanguinarine five alkaloids are released into the liquid medium. The compound with r. t. 17.70 min as relative percentage was the product preferentially excreted by cells at the end of the growth phase, reaching a maximum at 20th day (Fig. 4). From the above results, it can be concluded that undifferentiated cell suspensions derived from C. majus are able to produce sanguinarine in larger amount than callus culture (COLOMBO and TOME 1991) and that the release of this alkaloid in the medium is noticeable, since it reaches - when measured per liter of culture - an amount higher than 50 % of the cell content. Acknowledgements This work was supported by research grant from the Ministero dell' Universita e della Ricerca Scientifica e Tecnologica of Italy.

References AMOROS, M., FAUCONNIER, B., and GIRRE, L.: Proprietes antivirales de quelque extraits de plantes indigenes. Ann. pharm. fran". 35, 371-376 (1977). BOEHM, H., FRANKE, J., and LAMMEL, L.: Patent Ger. (East) 143270 (1980). BUGATTI, C., COLOMBO, M. L., and TOME, F.: A new method for alkaloid extraction from Chelidonium majus L. Phytochemical Analysis 2, 65-67 (1991). COLOMBO, M. L., TOME, F.: Production of sanguinarine by Chelidonium majus callus cultures. Planta Med., 57, 428-429 (1991). DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., and SMITH, F.: Colorimetric method for determination of sugars and related substances. Anal. Chern. 28, 350-356 (1956). HBJAMANKOVA, N., WALTEROWA, D., PREININGER, V., and SIMANEK, V.: Antifungal activity of quaternary benzo[c]phenanthridine alkaloids from Chelidonium majus. Fitoterapia IV, 291-294 (1984). LENFELD, J., KROUTIL, M., MARSALEK, E., SLAVIK, J., PREININGER, V. and SIMANEK, V.: Antiinflammatory activity of quaternary benzophenanthridine alkaloids from Chelidonium majus. Planta Med. 43, 161-165 (1981). WAGNER, H.: Pharmazeutische Biologie. G. Fischer Verlag. Stuttgart 1982. Received July 30, 1991; accepted October 29, 1991.

Authors' addresses: Dr. FRANCA TOME, Dipartimento di Biologia, Via Celoria 26, 20133 Milano; Dr. MARIA LAURA COLOMBO, 1st. Sc. Farmacologiche, Via Balzaretti 9, 20133 Milano, Italy. 120

BPP 188 (1992) 2