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Alkaloid Production by Ochrosia elliptica Cell Suspension Cultures
Alkaloid Production by Ochrosia elliptica Cell Suspension Cultures KouASsi KouAmo 1}, JoEl. CREcHE1}, jEAN-CLAUDE CHfNmux 1}, MARc RmEAu 1) and CLAUDE VIEL2) 1) 2)
Laboratoire de Biotechnologie Vegetale, Faculte de Pharmacie, 2 his bld Tonnelle F-37032 Tours cedex, France Laboratoire de Chimie Tberapeutique II, ERA-CNRS 317, Faculte de Pharmacie, 3, rue J.B. Clement F-92290 Chitenay-Malabry, France
Received October 5, 1984 ·Accepted October 29, 1984
Summary Ochrosia elliptica cell suspension cultures were obtained from callus cultures using an original «cell washing technique». These suspensions accumulated ellipticine, 9-methoxyellipticine, el-
liptinine, isoreserpiline, and reserpiline (a new alkaloid for this species) during the stationary growth phase. Alkaloid production can be enhanced by cloning small cell aggregates.
Key words: Ochrosia elliptica, bioproductWn, cloning, plant cell cultures, pyridocarbazole alkaloids.
Introduction
Some pyridocarbazole alkaloids such as ellipticine or 9-methoxyellipticine are DNA-intercalating drugs with antitumor activities (Dalton et al., 1967; Paoletti et al., 1978}. In an earlier paper {Kouadio et al., 1984}, we showed that Ochrosia elliptica callus tissues produce these two alkaloids in similar quantities as in in vivo propagated plants. We have now investigated both metabolic regulation and selection of cell lines for production of large amounts of pyridocarbazole alkaloids in cultured 0. elliptica cells. In this note we report establishment of cell suspension cultures by a «cell washing method», isolation, and identification of alkaloids and the selection of high-producing lines after cloning of small cell aggregates. Materials and Methods Callus cultures Three Ochrosia elliptica strains, each initiated from a young leaf taken from 4-6-week-old
different seedlings, were cultured on Gamborg et al. (1968) medium with addition of vitamins of this medium, 20g I- 1 saccharose and 100mgl- 1 casein hydrolysate (cB5-C medium»). The growth substances were 2,4-D (10mgi- 1) and K (1 mgi- 1). Media were gelified by agar (8 g I- 1) and the tissues were subcultured every three weeks in continuous light (2500 lux) at 24 °C for at least one year before establishing the suspensions.
Abbreviations: 2,4 D
=
2,4-dichlorophenoxyacetic acid; K = kinetin.
J Plant. PhysioL VoL 118. pp. 277-283 (1985)
278
KouASSI KouADIO et al.
Suspension cell cultures Two-week-old callus (6g fresh weight) from each of the three precedent strains were dissociated in 250 ml shaken Erlenmeyer flasks containing 50 ml Bs-C liquid medium. A variety of adsorbents or antioxydants were added to prevent intense darkening of the cells and the media: active charcoal (300 mgi- 1), polyvinyl-pyrrolidone (1 gi- 1), or cysteine (30 mgl- 1). In other experiments suspension cultures were established stepwise: 6 g dissociated callus were shaken for 48 h in 50 ml Bs-C medium. Cells were removed from the medium by aseptic filtration through Miracloth (40!£m) and were washed with 250ml B5 medium (without the growth substances). The cells were shaken in 50 mlliquid medium (2,4-D concentration was diminished to 5 mgl- 1) for another 48 hours and then filtered. The same operation was repeated 3 times every 48 hours. The suspensions were returned to the shaker for 15 days incubation and were filtered and washed a last time. The cells were then cultured in B5-C medium with one of the following combinations of growth substances: 5mgi- 1 2,4-D+1mgi- 1 K: Bs-Ct medium; 5mgl- 1 2,4D + 0.1 mg I- 1 K: Bs-C2 medium; 1 mg I- 1 2,4-D + 0.1 mg I- 1 K: Bs-C3 medium. During the ftrst two months the cells were subcultured every two weeks and later at intervals of 7 days (10 ml of suspension was added to 40ml fresh medium) in 250ml Erlenmeyer flasks (100rpm, 24°C, 2500 lux). Cell viability was estimated with trypan blue (Weber and Lark, 1979).
Alkaloid isolation and identification 21-day-old cells (60g fresh weight) of the strain OEtb (see Table!) were freeze-dried and powdered and the alkaloids were extracted as previously described for callus tissues (Kouadio et al., 1984). We isolated four alkaloids after chromatography on preparative Silicagel 60 plates with 95 % ethanol as developing solvent. They were compared to standard compounds and identified as ellipticine, 9-methoxyellipticine, isoreserpiline, and reserpiline by UV, MS, IR, and fluorometry spectra. Another alkaloid was also present in these extracts but in very small amounts and therefore cannot be isolated. It migrated at the same Rf as elliptinine in two solvent systems on silicagel plates: ethanol (95 %) or CH2Ch: MeOH (9: 1, v/v). All these alkaloids could be shown in extracts from other strains on analytical Sigel chromatographs with the two precedent solvent systems. Table!: Pyridocarbazole alkaloid content (~·g- 1 dr. wt.) in five suspension cultures (Cells were in stationary growth phase). Suspension cultures OEtb OMIA2 OMtAJ OMI~
OM3A2
Medium
Ellipticine
9-Methoxyellipticine
BsC2
33 36 35 54 28
40 46 47 98 39
BsCt
BsC2 BsC3
BsCt
Time-course of cell suspension growth The growth curves of two liquid strains subcultured for 1~ years, were studied over a period of 4 weeks. The cells were cultured in 250ml shaken-flasks (containing 50ml of OEtb suspension) or in a 2liter Biolafitte bioreactor (with 1.21 OM1A2 suspension). The procedure of cell counting and fresh or dry biomass determination was performed as described previously for Catharanthus roseus cell cultures (Merillon et al., 1983). ]. Plant. Physiol. Vol. 118. pp. 277-283 {1985)
Alkaloids of Ochrosia elliptica cell suspensions
279
Alkaloid content Pyridocarbazole alkaloids were extracted by 5 ml methanol from 100 mg freeze-dried cells and quantified by a fluorometric technique (Kouadio et al., 1984). Organic extracts were concentrated and chromatographed over Silicagel 60 analytical plates (Merck n° 5553) with CH2Ch-MeOH (92-8, v/v) as developing system. Ellipticine and 9-methoxyellipticine were eluted from the plates using methanol. They were quantified by fluorometry (Aexc. 398 nm, l\em. 444nm for ellipticine; l\exc. 412nm, l\em. 470nm for 9-methoxyellipticine) and calibration curves were established with standard alkaloids. The detection limit was below 0.01 p.g and the curves were linear up to 0.40 JLg for the two alkaloids. Reserpiline and isoreserpiline were semi-quantitatively estimated after spraying Dragendorff's reagent over the chromatoplates. Cloning with small cell aggregates 7-day-old OM1A2 cell suspension culture was filtered through a 250/Lffi steel mesh. The filtrate contained small, fine cell aggregates of 1-40 cells. 0.25ml Ge 105 aggregates) were plated over a thin layer of agar nutrient medium (of same composition as the liquid nutrient medium of the strain) in 9 em Petri dishes. The plated aggregates were cultured at 24 °C, under continuous light (2500 lux) for 20 days. Then 48 callii, now 1-2 mm in size were isolated and transferred to fresh B5C1 agar medium. The tissues were subcultured three times on the same medium before alkaloid quantifications were made.
Results and Discussion
Suspension cultures of Ochrosia elliptica 0. elliptica suspension cultures were difficult to establish: in fact, calli could easily be dissociated but the liquid medium darkened very intensely within 24 hours. 40% of the cells were still alive on the 9th day but the living cells did not grow and the whole culture became necrotic within three weeks. A variety of adsorbents or antioxydant agents used by different authors to reduce browning in tissue cultures (Siegel and Enns, 1979; Hunter, 1979; Weatherhead et al., 1978) have been added to the medium but these substances were inefficient for 0. elliptica cells. We have developed a «cell washing method» for the establishment of suspensions: the cultures were filtered every two days and the cells were washed with fresh medium. The cells did not grow for 1-2 weeks but the filtrates of the successive washings became less and less coloured. Progressively, the growth of the washed cells started again. With this method, five different suspension cultures were obtained from three callus strains (Table 1). They are now subcultured every 7 days. After a period of 1~ years we studied the growth characteristics of two of these suspensions. The OE1b cells were cultured in Erlenmeyer flasks and the OM1A2 cells in a 2 liter bioreactor. The growth curves (Figs. 1 and 2) and growth parameters (Table2) are similar for the two suspensions, with a stationary growth phase starting after the 7th day. Cells are yellowish but they become brownish after the 21th day if they are not subcultured. ]. Plant. PhysioL Vol. 118. pp. 277-283 (1985}
280
KouASSI KouADIO et al. Fig I A
S' S'
Fig 2
c:i...:
5
1:' ..Id 0 Ill
ai ~
30
50
V)
Q
§
15
"'(
:...: _,
"'(
16
24
DAYS
3 5 7 9 12
17
25
Figs. 1 and 2: Growth curves of Ochrosia elliptica cell suspension cultures (A) and time-course of pyridocarbazole alkaloid accumulation (B). Cultures of OE,b cells in Erlenmeyer flasks (Fig.1) and OM 1A2 cells in bioreactor (Fig. 2). Fresh weight (0-0), dry weight (•-•), ellipticine (0--D), 9-methoxyellipticine
<•--->·
Table2: Maximal biomass for the OE,b and OM1A2 cell suspension cultures. Suspension cultures
Conditions of culture
fr. wt. g
dr. wt. g
cells 106 • mi- 1
Yg
Erlenmeyer flask Bioreactor
180 123
10.6 9.5
2.3 2.2
0.53 0.47
Isolation ofalkaloids and production time-course The alkaloids of cell suspensions were analysed. As for callus cultures, the cell cultures accumulate the two pyridocarbazole alkaloids, ellipticine and 9-methoxyellipticine, which were identified by UV, IR, and mass spectra by comparison with the spectra given by standard compounds (Kouadio et al., 1984). We have also isolated reserpiline and isoreserpiline from cell cultures. They were identified by comparison with literature data for UV, IR, and mass spectra (Gabetta and Mustich, 1975) and this was confirmed by comparison with authentic samples. The IR spectrum of isoreserpiline shows bands of Bohlmann (between 2760 and 2840 em -I) which are characteristic of trans-quinolizidinic structure (Bohlmann, 1957, 1958; Nakanishi, 1962). These bands were not detected for reserpiline. It is worth mentioning that reserpiline ]. Plant. PhysioL Vol. 118. pp. 277-283 (1985)
Alkaloids of Ochrosia elliptica cell suspensions
281
Table3: Pyridocarbazole alkaloids amounts (p.g·g- 1 dr. wt.) in OMt.A.t cells during subcultures. Alkaloids were always dosed on the 20th day of cell growth. Alkaloids ellipticine 9-methoxyellipticine
Subcultures 6th 10th 56 54 98 90
14th 47 87
23th 50
92
has not been isolated in differentiated plants of 0. elliptica but has been detected as a minor alkaloid in a related species 0. balansae (Bruneton and Cave, 1972 a, b). Elliptinine has been tentatively identified by comparison of the Rf values with a standard compound. Time-course of alkaloid accumulations was determined with OEtb and OM1A2 strains. A preliminary qualitative analysis by thin layer chromatography over Silicagel 60 showed that the four alkaloids gave spots of maximal intensities after revelation by Dragendorff's reagent during the stationary growth phase. This was confrrmed for the two pyridocarbazole alkaloids by spectrofluorometric quantifications (Figs. 1 and 2). We compared the amounts of ellipticine and 9-methoxyellipticine in cells of the five suspension strains and the results of dosages made during the stationary growth phase are given in Table 1. In terms of pyridocarbazole alkaloid production, these suspensions are at present stable. For example Table3 gives the alkaloid contents of OMt~ cells at 4 different subcultures.
Cloning ofcell aggregates Assuming that the cell suspension cultures exhibited differences between the cells because the cell culture itself generates genetic and/or epigenetic variations, we used the technique developed by Sato and Yamada (1984) to obtain high-berberine producing cultures of Coptis japonica. We cloned OM1A2 small cell aggregates and we selected 48 colonies of homogeneous appearance. These cell lines were subcultured three times and the pyridocarbazole alkaloid contents were then determined. Results are shown in Fig. 3: the lines had different alkaloid amounts on a dry weight basis. Ellipticine varied in a ratio 1-20 and 9-methoxyellipticine in a ratio 1-16. There was poor correlation between the two alkaloids: for example, high-ellipticine producing strains could be found with low 9-methoxyellipticine content and vice versa, but the two highest producing strains accumulated more ellipticine and 9-methoxyellipticine than did the non-selected strain cultured under the same conditions, i.e. on agar medium. We have now repeated the cloning of these strains, assuming that same cell lines with stable producitivity can be obtained. In conclusion, to our knowledge, this is the first isolation of pyridocarbazole alkaloids from suspension cell cultures of 0. elliptica. It is evident from the results that in vitro selected cells can produce these alkaloids in greater amounts than in vivo cells, ]. Plant. Physiol Vol. 118. pp. 277-283 {1985)
282
KouAssr KouADro et al. 9 Methoxyel lipticine
Ellip ticine
40
40
i:3 7,85
i.: 89,40 s. =54,03
•• .:42,00
I
I
I
Cmax= 266,70
I
20
20
I
I
Cmax-::. 193,70
I
\ I
I
' 30
JiO
Alkaloids . pg .g~ dr wt
Fig. 3: Distribution of ellipticine and 9-methoxyellipticine in 0. elliptica cell lines derived from OMrA2 suspension. x = average value; Sx = standard deviation; Cmax = maximum alkaloid amounts;(~): values of the unselected strain cultivated on an agar medium (ellipticine = 65, 9-methoxyellipticine = 32J&g·g- 1 dr. wt.). and reserpiline, not yet detected in the trees. Studies of the influenced of changes or physiological conditions and nutrients in the culture medium are also in progress for production optimization. Acknowledgements The authors are thankful to Prof. Dr. J. Poisson, for a gift of reserpiline and isoreserpiline, and to Dr. N. Boord for correcting the English text. References BoHLMANN, F.: Zur Konfigurationsbestimmung von Chinolizin-Derivaten. Angew. Chern., 69, 641-642 (1957). - Zur Konfigurationsbestimmung von Chinolizidin-Derivaten. Chern. Ber., 91, 2157-2167 (1958). B&UNETON, J. etA. CAvf: Plantes de Nouvelle-Caledonie. Alcaloides des feuilles d'Ochrosia ,baJ. ansae (Apocynacees). Ann. Pharm. Fr., 30, 629-636 (1972a). - - Alcaloides d'Ochrosia balansae. Phytochem., 11, 846-847 (1972 b). DALTON, L. K., S. DEMERAc, B. C. ELMES, J. W. LooER, J. M. SwAM, and T. TETEI: Synthesis of the tumor inhibitory alkaloids, ellipticine, 9-methoxyellipticine, and related pyrido [4,3-b] carbazole. Aust. J. Chern., 20, 2715-2725 (1967). GABETTA, B. and G. MusncH: Spectral data of indole alkaloids. Inverni della Beffa, Milan, 1975. GAMBORG, 0. L., R. A. Mu.LER, and K. 0JIMA: Nutrient requirements of suspension cultures of Soybean root cells. Exp. Cell. Res., 50, 151-158 (1968). ]. Plant. Physiol. Vol. 118. pp. 277-283 {1985)
Alkaloids of Ochrosia elliptica cell suspensions
283
HUNTER, C. S.: IN VITRO culture of Cinchona ledgeriana L. J. Hort. Sci., 54, 111-114 (1979}. KouADIO, K., J. C. CHENIEUX, M. RIDEAu, and C. VIEL: Antitumor alkaloids in callus cultures of Ochrosia elliptica. J. Nat. Products, 47, 872-874 (1984}. MERILLON, J. M., J. c. CHENIEUX, et M. RIDEAu: Cinetique de croissance, evolution du metabolisme glucido-azote et accumulation alcaloidique dans une suspension cellulaire de Catharanthus roseus. Planta med., 47, 169-176 (1983}. NAKANISHI, K.: Infrared absorption spectroscopy practical. 159-160. Holden-Day, San Francisco, 1962. PAOLETII, C., P. LECOINTE, P. LESCA, S. CRos, D. MANsUY, and N. DAT-XuONG: Metabolism of ellipticine and derivatives and its involvement in the antitumor action of drugs. Biochemie, 60, 1003-1009(1978}. SATO, F. andY. YAMADA: High berberine-producing cultures of Coptis japonica cells. Phytochem., 23, 281-285 (1984}. SIEGEL, M. R. and R. K. ENNs: Soluble polyvinyl pyrrolidine and bovine serum albumin adsorb polyphenols from soybean suspension cultures. Plant physiol., 63, 206-208 (1979}. WEATHERHEAD, M. A., J. BURDON, and G. G. HENSHAW: Some effect of activated charcoal as an additive to plant tissue media. Z. Pflanzenph., 89, 141-147 (1978}. WEBER, G. and K. G. LARK: An efficient plating system for rapid isolation of mutants from plant cell suspensions. Theor. appl. genet., 55, 81-86 (1979}.
]. Plant. Physiol. Vol. 118. pp. 277-283 (1985)
Laboratoire de Biotechnologie Vegetale, Faculte de Pharmacie, 2 his bld Tonnelle F-37032 Tours cedex, France Laboratoire de Chimie Tberapeutique II, ERA-CNRS 317, Faculte de Pharmacie, 3, rue J.B. Clement F-92290 Chitenay-Malabry, France
Received October 5, 1984 ·Accepted October 29, 1984
Summary Ochrosia elliptica cell suspension cultures were obtained from callus cultures using an original «cell washing technique». These suspensions accumulated ellipticine, 9-methoxyellipticine, el-
liptinine, isoreserpiline, and reserpiline (a new alkaloid for this species) during the stationary growth phase. Alkaloid production can be enhanced by cloning small cell aggregates.
Key words: Ochrosia elliptica, bioproductWn, cloning, plant cell cultures, pyridocarbazole alkaloids.
Introduction
Some pyridocarbazole alkaloids such as ellipticine or 9-methoxyellipticine are DNA-intercalating drugs with antitumor activities (Dalton et al., 1967; Paoletti et al., 1978}. In an earlier paper {Kouadio et al., 1984}, we showed that Ochrosia elliptica callus tissues produce these two alkaloids in similar quantities as in in vivo propagated plants. We have now investigated both metabolic regulation and selection of cell lines for production of large amounts of pyridocarbazole alkaloids in cultured 0. elliptica cells. In this note we report establishment of cell suspension cultures by a «cell washing method», isolation, and identification of alkaloids and the selection of high-producing lines after cloning of small cell aggregates. Materials and Methods Callus cultures Three Ochrosia elliptica strains, each initiated from a young leaf taken from 4-6-week-old
different seedlings, were cultured on Gamborg et al. (1968) medium with addition of vitamins of this medium, 20g I- 1 saccharose and 100mgl- 1 casein hydrolysate (cB5-C medium»). The growth substances were 2,4-D (10mgi- 1) and K (1 mgi- 1). Media were gelified by agar (8 g I- 1) and the tissues were subcultured every three weeks in continuous light (2500 lux) at 24 °C for at least one year before establishing the suspensions.
Abbreviations: 2,4 D
=
2,4-dichlorophenoxyacetic acid; K = kinetin.
J Plant. PhysioL VoL 118. pp. 277-283 (1985)
278
KouASSI KouADIO et al.
Suspension cell cultures Two-week-old callus (6g fresh weight) from each of the three precedent strains were dissociated in 250 ml shaken Erlenmeyer flasks containing 50 ml Bs-C liquid medium. A variety of adsorbents or antioxydants were added to prevent intense darkening of the cells and the media: active charcoal (300 mgi- 1), polyvinyl-pyrrolidone (1 gi- 1), or cysteine (30 mgl- 1). In other experiments suspension cultures were established stepwise: 6 g dissociated callus were shaken for 48 h in 50 ml Bs-C medium. Cells were removed from the medium by aseptic filtration through Miracloth (40!£m) and were washed with 250ml B5 medium (without the growth substances). The cells were shaken in 50 mlliquid medium (2,4-D concentration was diminished to 5 mgl- 1) for another 48 hours and then filtered. The same operation was repeated 3 times every 48 hours. The suspensions were returned to the shaker for 15 days incubation and were filtered and washed a last time. The cells were then cultured in B5-C medium with one of the following combinations of growth substances: 5mgi- 1 2,4-D+1mgi- 1 K: Bs-Ct medium; 5mgl- 1 2,4D + 0.1 mg I- 1 K: Bs-C2 medium; 1 mg I- 1 2,4-D + 0.1 mg I- 1 K: Bs-C3 medium. During the ftrst two months the cells were subcultured every two weeks and later at intervals of 7 days (10 ml of suspension was added to 40ml fresh medium) in 250ml Erlenmeyer flasks (100rpm, 24°C, 2500 lux). Cell viability was estimated with trypan blue (Weber and Lark, 1979).
Alkaloid isolation and identification 21-day-old cells (60g fresh weight) of the strain OEtb (see Table!) were freeze-dried and powdered and the alkaloids were extracted as previously described for callus tissues (Kouadio et al., 1984). We isolated four alkaloids after chromatography on preparative Silicagel 60 plates with 95 % ethanol as developing solvent. They were compared to standard compounds and identified as ellipticine, 9-methoxyellipticine, isoreserpiline, and reserpiline by UV, MS, IR, and fluorometry spectra. Another alkaloid was also present in these extracts but in very small amounts and therefore cannot be isolated. It migrated at the same Rf as elliptinine in two solvent systems on silicagel plates: ethanol (95 %) or CH2Ch: MeOH (9: 1, v/v). All these alkaloids could be shown in extracts from other strains on analytical Sigel chromatographs with the two precedent solvent systems. Table!: Pyridocarbazole alkaloid content (~·g- 1 dr. wt.) in five suspension cultures (Cells were in stationary growth phase). Suspension cultures OEtb OMIA2 OMtAJ OMI~
OM3A2
Medium
Ellipticine
9-Methoxyellipticine
BsC2
33 36 35 54 28
40 46 47 98 39
BsCt
BsC2 BsC3
BsCt
Time-course of cell suspension growth The growth curves of two liquid strains subcultured for 1~ years, were studied over a period of 4 weeks. The cells were cultured in 250ml shaken-flasks (containing 50ml of OEtb suspension) or in a 2liter Biolafitte bioreactor (with 1.21 OM1A2 suspension). The procedure of cell counting and fresh or dry biomass determination was performed as described previously for Catharanthus roseus cell cultures (Merillon et al., 1983). ]. Plant. Physiol. Vol. 118. pp. 277-283 {1985)
Alkaloids of Ochrosia elliptica cell suspensions
279
Alkaloid content Pyridocarbazole alkaloids were extracted by 5 ml methanol from 100 mg freeze-dried cells and quantified by a fluorometric technique (Kouadio et al., 1984). Organic extracts were concentrated and chromatographed over Silicagel 60 analytical plates (Merck n° 5553) with CH2Ch-MeOH (92-8, v/v) as developing system. Ellipticine and 9-methoxyellipticine were eluted from the plates using methanol. They were quantified by fluorometry (Aexc. 398 nm, l\em. 444nm for ellipticine; l\exc. 412nm, l\em. 470nm for 9-methoxyellipticine) and calibration curves were established with standard alkaloids. The detection limit was below 0.01 p.g and the curves were linear up to 0.40 JLg for the two alkaloids. Reserpiline and isoreserpiline were semi-quantitatively estimated after spraying Dragendorff's reagent over the chromatoplates. Cloning with small cell aggregates 7-day-old OM1A2 cell suspension culture was filtered through a 250/Lffi steel mesh. The filtrate contained small, fine cell aggregates of 1-40 cells. 0.25ml Ge 105 aggregates) were plated over a thin layer of agar nutrient medium (of same composition as the liquid nutrient medium of the strain) in 9 em Petri dishes. The plated aggregates were cultured at 24 °C, under continuous light (2500 lux) for 20 days. Then 48 callii, now 1-2 mm in size were isolated and transferred to fresh B5C1 agar medium. The tissues were subcultured three times on the same medium before alkaloid quantifications were made.
Results and Discussion
Suspension cultures of Ochrosia elliptica 0. elliptica suspension cultures were difficult to establish: in fact, calli could easily be dissociated but the liquid medium darkened very intensely within 24 hours. 40% of the cells were still alive on the 9th day but the living cells did not grow and the whole culture became necrotic within three weeks. A variety of adsorbents or antioxydant agents used by different authors to reduce browning in tissue cultures (Siegel and Enns, 1979; Hunter, 1979; Weatherhead et al., 1978) have been added to the medium but these substances were inefficient for 0. elliptica cells. We have developed a «cell washing method» for the establishment of suspensions: the cultures were filtered every two days and the cells were washed with fresh medium. The cells did not grow for 1-2 weeks but the filtrates of the successive washings became less and less coloured. Progressively, the growth of the washed cells started again. With this method, five different suspension cultures were obtained from three callus strains (Table 1). They are now subcultured every 7 days. After a period of 1~ years we studied the growth characteristics of two of these suspensions. The OE1b cells were cultured in Erlenmeyer flasks and the OM1A2 cells in a 2 liter bioreactor. The growth curves (Figs. 1 and 2) and growth parameters (Table2) are similar for the two suspensions, with a stationary growth phase starting after the 7th day. Cells are yellowish but they become brownish after the 21th day if they are not subcultured. ]. Plant. PhysioL Vol. 118. pp. 277-283 (1985}
280
KouASSI KouADIO et al. Fig I A
S' S'
Fig 2
c:i...:
5
1:' ..Id 0 Ill
ai ~
30
50
V)
Q
§
15
"'(
:...: _,
"'(
16
24
DAYS
3 5 7 9 12
17
25
Figs. 1 and 2: Growth curves of Ochrosia elliptica cell suspension cultures (A) and time-course of pyridocarbazole alkaloid accumulation (B). Cultures of OE,b cells in Erlenmeyer flasks (Fig.1) and OM 1A2 cells in bioreactor (Fig. 2). Fresh weight (0-0), dry weight (•-•), ellipticine (0--D), 9-methoxyellipticine
<•--->·
Table2: Maximal biomass for the OE,b and OM1A2 cell suspension cultures. Suspension cultures
Conditions of culture
fr. wt. g
dr. wt. g
cells 106 • mi- 1
Yg
Erlenmeyer flask Bioreactor
180 123
10.6 9.5
2.3 2.2
0.53 0.47
Isolation ofalkaloids and production time-course The alkaloids of cell suspensions were analysed. As for callus cultures, the cell cultures accumulate the two pyridocarbazole alkaloids, ellipticine and 9-methoxyellipticine, which were identified by UV, IR, and mass spectra by comparison with the spectra given by standard compounds (Kouadio et al., 1984). We have also isolated reserpiline and isoreserpiline from cell cultures. They were identified by comparison with literature data for UV, IR, and mass spectra (Gabetta and Mustich, 1975) and this was confirmed by comparison with authentic samples. The IR spectrum of isoreserpiline shows bands of Bohlmann (between 2760 and 2840 em -I) which are characteristic of trans-quinolizidinic structure (Bohlmann, 1957, 1958; Nakanishi, 1962). These bands were not detected for reserpiline. It is worth mentioning that reserpiline ]. Plant. PhysioL Vol. 118. pp. 277-283 (1985)
Alkaloids of Ochrosia elliptica cell suspensions
281
Table3: Pyridocarbazole alkaloids amounts (p.g·g- 1 dr. wt.) in OMt.A.t cells during subcultures. Alkaloids were always dosed on the 20th day of cell growth. Alkaloids ellipticine 9-methoxyellipticine
Subcultures 6th 10th 56 54 98 90
14th 47 87
23th 50
92
has not been isolated in differentiated plants of 0. elliptica but has been detected as a minor alkaloid in a related species 0. balansae (Bruneton and Cave, 1972 a, b). Elliptinine has been tentatively identified by comparison of the Rf values with a standard compound. Time-course of alkaloid accumulations was determined with OEtb and OM1A2 strains. A preliminary qualitative analysis by thin layer chromatography over Silicagel 60 showed that the four alkaloids gave spots of maximal intensities after revelation by Dragendorff's reagent during the stationary growth phase. This was confrrmed for the two pyridocarbazole alkaloids by spectrofluorometric quantifications (Figs. 1 and 2). We compared the amounts of ellipticine and 9-methoxyellipticine in cells of the five suspension strains and the results of dosages made during the stationary growth phase are given in Table 1. In terms of pyridocarbazole alkaloid production, these suspensions are at present stable. For example Table3 gives the alkaloid contents of OMt~ cells at 4 different subcultures.
Cloning ofcell aggregates Assuming that the cell suspension cultures exhibited differences between the cells because the cell culture itself generates genetic and/or epigenetic variations, we used the technique developed by Sato and Yamada (1984) to obtain high-berberine producing cultures of Coptis japonica. We cloned OM1A2 small cell aggregates and we selected 48 colonies of homogeneous appearance. These cell lines were subcultured three times and the pyridocarbazole alkaloid contents were then determined. Results are shown in Fig. 3: the lines had different alkaloid amounts on a dry weight basis. Ellipticine varied in a ratio 1-20 and 9-methoxyellipticine in a ratio 1-16. There was poor correlation between the two alkaloids: for example, high-ellipticine producing strains could be found with low 9-methoxyellipticine content and vice versa, but the two highest producing strains accumulated more ellipticine and 9-methoxyellipticine than did the non-selected strain cultured under the same conditions, i.e. on agar medium. We have now repeated the cloning of these strains, assuming that same cell lines with stable producitivity can be obtained. In conclusion, to our knowledge, this is the first isolation of pyridocarbazole alkaloids from suspension cell cultures of 0. elliptica. It is evident from the results that in vitro selected cells can produce these alkaloids in greater amounts than in vivo cells, ]. Plant. Physiol Vol. 118. pp. 277-283 {1985)
282
KouAssr KouADro et al. 9 Methoxyel lipticine
Ellip ticine
40
40
i:3 7,85
i.: 89,40 s. =54,03
•• .:42,00
I
I
I
Cmax= 266,70
I
20
20
I
I
Cmax-::. 193,70
I
\ I
I
' 30
JiO
Alkaloids . pg .g~ dr wt
Fig. 3: Distribution of ellipticine and 9-methoxyellipticine in 0. elliptica cell lines derived from OMrA2 suspension. x = average value; Sx = standard deviation; Cmax = maximum alkaloid amounts;(~): values of the unselected strain cultivated on an agar medium (ellipticine = 65, 9-methoxyellipticine = 32J&g·g- 1 dr. wt.). and reserpiline, not yet detected in the trees. Studies of the influenced of changes or physiological conditions and nutrients in the culture medium are also in progress for production optimization. Acknowledgements The authors are thankful to Prof. Dr. J. Poisson, for a gift of reserpiline and isoreserpiline, and to Dr. N. Boord for correcting the English text. References BoHLMANN, F.: Zur Konfigurationsbestimmung von Chinolizin-Derivaten. Angew. Chern., 69, 641-642 (1957). - Zur Konfigurationsbestimmung von Chinolizidin-Derivaten. Chern. Ber., 91, 2157-2167 (1958). B&UNETON, J. etA. CAvf: Plantes de Nouvelle-Caledonie. Alcaloides des feuilles d'Ochrosia ,baJ. ansae (Apocynacees). Ann. Pharm. Fr., 30, 629-636 (1972a). - - Alcaloides d'Ochrosia balansae. Phytochem., 11, 846-847 (1972 b). DALTON, L. K., S. DEMERAc, B. C. ELMES, J. W. LooER, J. M. SwAM, and T. TETEI: Synthesis of the tumor inhibitory alkaloids, ellipticine, 9-methoxyellipticine, and related pyrido [4,3-b] carbazole. Aust. J. Chern., 20, 2715-2725 (1967). GABETTA, B. and G. MusncH: Spectral data of indole alkaloids. Inverni della Beffa, Milan, 1975. GAMBORG, 0. L., R. A. Mu.LER, and K. 0JIMA: Nutrient requirements of suspension cultures of Soybean root cells. Exp. Cell. Res., 50, 151-158 (1968). ]. Plant. Physiol. Vol. 118. pp. 277-283 {1985)
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HUNTER, C. S.: IN VITRO culture of Cinchona ledgeriana L. J. Hort. Sci., 54, 111-114 (1979}. KouADIO, K., J. C. CHENIEUX, M. RIDEAu, and C. VIEL: Antitumor alkaloids in callus cultures of Ochrosia elliptica. J. Nat. Products, 47, 872-874 (1984}. MERILLON, J. M., J. c. CHENIEUX, et M. RIDEAu: Cinetique de croissance, evolution du metabolisme glucido-azote et accumulation alcaloidique dans une suspension cellulaire de Catharanthus roseus. Planta med., 47, 169-176 (1983}. NAKANISHI, K.: Infrared absorption spectroscopy practical. 159-160. Holden-Day, San Francisco, 1962. PAOLETII, C., P. LECOINTE, P. LESCA, S. CRos, D. MANsUY, and N. DAT-XuONG: Metabolism of ellipticine and derivatives and its involvement in the antitumor action of drugs. Biochemie, 60, 1003-1009(1978}. SATO, F. andY. YAMADA: High berberine-producing cultures of Coptis japonica cells. Phytochem., 23, 281-285 (1984}. SIEGEL, M. R. and R. K. ENNs: Soluble polyvinyl pyrrolidine and bovine serum albumin adsorb polyphenols from soybean suspension cultures. Plant physiol., 63, 206-208 (1979}. WEATHERHEAD, M. A., J. BURDON, and G. G. HENSHAW: Some effect of activated charcoal as an additive to plant tissue media. Z. Pflanzenph., 89, 141-147 (1978}. WEBER, G. and K. G. LARK: An efficient plating system for rapid isolation of mutants from plant cell suspensions. Theor. appl. genet., 55, 81-86 (1979}.
]. Plant. Physiol. Vol. 118. pp. 277-283 (1985)