1979, Vol. 35, pp. 162-166, O Hippokrates Verlag GmbH
N?taJournalor Medicinal
med~caPlant Research
Metabolism of Digitoxin in Individual Plants of Digitalis lanata Elmar W. Weiler
Key Word Index: Digitalis lanata; Cardenolides; Digitoxin Metabolism; 12-Hydroxylation; Screening Techniques.
Abstract
Leaf discs of one and two year old plants of Digitalis lanata EHRH.metabolize 3H-digitoxin to desacetyl lanatoside A, lanatoside A, and the corresponding C-12 hydroxylated compounds, desacetyl lanatoside C and lanatoside C. Individual plants from different sources show wide variations in their metabolic profile. A simple test for the screening of plant individuals for high C-12 hydroxylation ability is described. Introduction
Whereas the general pathway of cardenolide biosynthesis is well understood [I-31, comparatively little is known about the course of subsequent transformations of the basic cardenolide skeleton from which a number of heart glycosides are synthesized. More than 60 cardenolides may occur in one plant species [4] and the reactions involved
include specific hydroxylations a t various positions of the aglycone (e. g. C-12, C-16) and the formation of glycosides with rare sugars some of which are found exclusively in this class of compounds 151. In order to investigate transformation reactions occuring at the stage of the cardenolide tridigitoxoside, tritiumlabeled digitoxin was fed to leaf discs of Digitalis lanata EHRH.An examination of a number of individual plants revealed that considerable variability existed with regard to the metabolic pattern. Since C-12 hydroxylated cardenolides (e. g. digoxin) are of much greater pharmaceutical interest than the corresponding deoxy compounds (e. g. digitoxin) a simple test for the C-12 hydroxylation ability of individual plants which permits a convenient selection of plants (e. g. for cell culture and breeding work) is described. The procedure can be used with cell cultures and intact plants.
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Lehrstuhl fur Pflanzenphysiologie, Ruhr-Universitst Bochum, Federal Republic of Germany.
Metabolism of Digitoxin Results
In ethanolic extracts of leaf discs of one and two year old plants of Digitalis lanata, w h i h had been incubated with SH-digitoxin for 1 to 4 days, four radioactive metabolites could be detected in addition to digitoxin. These were identified by TLC, co-crystallization of their acetylated derivatives with authentical compounds and analysis of their acid hydrolysis products, as desacetyl lanatoside A (I), lanatoside A (11), desacetyl lanatoside C (111) and lanatoside C (IV). Kinetic experiments showed that after one day of incubation, (I) and (11) were predominate in the extracts but then declined with a concomitant accumulation of (111) and (IV) by day four. Thus, exogeneously applied digitoxin appears to undergo glucosylation at C'-16, acetylation at C'-15 and hydroxylation at C-12, but: not hydroxylation at C-16. Since no radioactive digoxin could be detected in the extracts, glucosylation either preceeds hydroxylation
or occurs a t a much faster rate. Acetylation may occur either before or after C-12 hydroxylation. Compared to the controls, kept in dim light of 700 lux, dark incubations showed a 20010 decrease in the hydroxylation reaction, but did not differ significantly in the amount of glucosylation and acetylation after 2 days of incubation. The tissue was extremely sensitive to ethanol in the incubation mixture. At 1010 ethanol, the uptake and metabolism of SH-digitoxin were significantly reduced and at 5010 ethanol both were completely inhibited. This effect limits the amount of soluble digitoxin which could be added to the system for saturation experiments. At an optimal substrate concentration of 34 pg digitoxin/ml(4 O/o ethanol) the rate of digitoxin uptake was found t o be 50 pgl day/g fresh leaf material for an average leaf disc sample. However, there was a substantial and significant variation in digitoxin metabolism when individual plants were analyzed for their metabolic pattern.
Table I Mean values and ranges for metabolites of digitoxin in Digitalis lanata leaf discs formed after 48 hrs of incubation. Distribution of radioactivityb Uptake of Desacetyl- LanatoDesacetyl- Lanatolanatw'de eside C digitoxin" lanatoside Aside A
Digitoxin
plants 238 plants 250
58.9 60.6
13.7 10.4
11.0 12.6
11.3 8.5
36.2 37.0
28.4 31.0
plants 238 plants 250
24.9-81.8 18.8-100
0-74.8 0-50.6
0-40.8 0-35.2
0-34.4 0-55.0
0-72.1 0-69.3
0-82.4 0-100
Number of plants
Mean values: l-yr old 2-yr old Ranges: 1-yr old 2-yr old
" = in percent of digitoxin added
= in percent of digitoxin taken up
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163
A technique was developed which permitted the recording of a complete metabolite profile of a plant after a single TLC run, thus providing an efficient screening system for these experiments. In Table I, mean values and ranges found for the various metabolites are listed for 250 two year old plants from nine different sources and for 238 one year old plants from three other sources. As can be seen, one and two year old plants show on an average almost identical metabolic behavior but as the ranges indicate, individual variation was very great. From the 456 plants analyzed, a number of individuals have been isolated which showed extremely different metabolic patterns. Whereas some plants were apparently unable to
metabolize the incorporated 3H-digitoxin, others metabolized it completely within the standard incubation time of 2 days. Also, plants were found which lacked either the desacetyl lanatosides (I, 111) or the lanatosides (11, IV). The amount of C-12 hydroxylated metabolites produced from incorporated digitoxin after 2 days by individual plants varied from 0 % to 82O/o. Fig. 1 shows the frequency distribution for the C-12 hydroxylated metabolites (I11 IV). It is apparent that only a small fraction of the plants analyzed posessed efficient digitoxin hydroxylation systems (0.2 'J/o of the total showed more than 80% C-12 hydroxylated compounds (I11 IV) in the screening test). Experiments repeated over a period of a least 3-4 weeks proved the stability of these extreme types.
+
+
Discussion
-
~
12-Hydroxylation
["/o]
Fig. I. Frequency distribution of formation of C-12 hydroxylated digitoxin metabolites (III+ IV) C-12 hydroxylated digitoxin in leaf discs of Digitalis lanata.
The results presented here demonstrate that exogeneously applied digitoxin undergoes glucosylation, acetylation and hydroxylation at G I 2 but not at C-16 in Digitalis lanata leaf tissue. These observations support the findings of TSCHESCHE et al. [I] and FRANZand MEIER[6] and they are in accord with results obtained from cell cultures of D. lanata [7]. However, the inter-individual variation of the metabolic pattern which had not yet been investigated was found to be considerable. This variation appears to be a stable character of the individual plant, especially as far as the C-12 hydroxylation is concerned. Unfortunately, it is not known at present to what
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Weiler
Metabolism of Digitoxin
Experimental Plant material Digitalis lanata seeds were obtained from various botanical gardens and individuals. Plants were grown in the field in 1974. Leaves of 2 year old plants were harvested in June and July, leaves of one year old plants in August and September. Chemicals 3H-digitoxi'n was purchased from Amenham (spec. act. 5.3 mCi/mmol). Lanatosides were purchased from Merck and secondary glycosides from Roth. Desacetyl lanatosides were
prepared from lanatosides by ammonolysis followed by chromatographic purification. Incubation and extraction procedure 500 mg leaf discs (10 mm diameter) punched out from freshly harvested, fully expanded leaves were incubated in 7.25 ml 0.05 M phosphate buffer, p H 5.5 containing 5 pCi (1 nmol) 3H-digitoxin-(G). Except when otherwise stated, the samples were shaken under dim light (700 lux) for 48 hrs at room temperature (23' C). The leafs discs were then removed from the incubation mixture, washed with water and extracted two times with 12.5 ml 80°/o ethanol. The volume of the combined extracts was brought to 25 -ml and after determination of total activity, was reduced to near dryness. The material soluble imn1 ml 80~10ethanol was used for TLC. Thin layer chromatography System I: Desacetyl lanatoides and lanatosides were separated in the solvent system tetrahydrofurane : CHCls : formamide = 50 : 50 : 6.5. The adsorbent was ,Kieselgur Gu from Merck. 0.25 mm layers were impregnated in acetone : formamide = 90:lO and used immediately after evaporation of the acetone. Developed plates were heated for 90 min at 90° C to remove the formamide. System 11: Formamide impregnated silica gel plates were used in combi,nation with the solvent system xylol : ethyl methyl ketone : formamide = 40:60:5 to separate secondary glycosides and aglycones. Identification o f metabolites Extracts from various one and two year old plants were combined. Four metabolites of SH-digitoxin could be detected inTLC-system I when incubation times varied from 1 to 4 days: A (Rf = 0.83), B (Rf = 0.52), C (Rf = O.39), D (Rf = 0.17), digitoxin (Rf = 0.95). A cochromatographed with lanatoside A, B with lanatoside B and desacetyl lanatoside A, C cochromatographed with lanatoside C and D with desacetyl lanatoside C. The metabolites were eluted and hydrolyzed in a mixture of methanol: 0.1 N H2S0, (1:l) for 20 min. After neutralization, chloroform extracts were made and chromatographed in System 11. The hydrolysis product of A and B showed the
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degree an individual one year old plant retains its metabolite profile in the second year of growth and data on the inheritance of the extreme metabolic patterns are not available. Preliminary studies indicate that there is no close correlation between the actual content of C-12 hydroxylated cardenolides present in a specific plant and the hydroxylation rate of the same plant as determined by the method used in this study. On the contrary, however plants exhibiting a high hydroxylation capacity also contained high amounts of C-12 hydroxylated cardenolides in the leaves. The broad variability in the ultimate metabolic fate of digitoxin in the plants assayed stresses the need to conduct careful biosynthesis studies with many plants of diverse origin. This will be necessary to obtain a picture representative for the plant species on the whole and to elucidate individual variabilities. The method reported here should be useful in selecting plants as starting material for breeding work or, more important, for cell cultures to be used for biotransformation reactions (especially C-12 hydroxylation).
Weiler
Screening test: For the screening of individual plants, TLC aliquots of the consystem I was used. 20 centrated leaf disc extracts were applied as a 2 cm band on the plates. The plates were developed for 15 cm and dried. Complete separation of the four metabolites could thus be aduieved. The distribution of radioactivity on the plates was then recorded with a TLC scanner (Berthold) and the percentage of metabolites formed calculated from the peak areas.
Acknowledgment I wi.sh to thank Dr. CORDUAN and Prof. REINHARD, Tiibingen, who provided seeds of
Digitalis lanata, and Prof. R. MANSELL, Tampa, for checking the English version of the manuscript. This work was supported by a grant of the .BundesrnG~ster fiir Forschung und Technologieu, Bonn to Prof. ZENK,Bochum.
References 1. Tschesche, R., Hombach, R., Scholten, H. and Peters, M.: Phytochemistry 9, 1505 (1972). 2. Tschesche, R.: Proc. R. Soc. (London) 180, 187 (1972). 3. Aberhard, D. J., Lloyd-Jones, J. G. and Caspi, E.: Phytochemistry 12, 1065 (1973). 4. Kaiser, F.: Arch. Pharmaz. 299, 263 (1966). 5. Reichstein, T.: Naturwissenschaften 54, 53 (1967). 6. Franz, G. and Meier, H.: Planta Medica 17, 396 (1969). 7. Rainhard, E, Boy, H. M. and Kaiser, F.: Planta Medica, Suppl. 163 (1975). Address: Dr. E . Weiler, Ruhr-Universitat Bochum, Institut fiir Pflanzenphysiologie, Postfach 2148, 0-4630 Bochum
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same Rf as digitoxi,genin (Rf = 0.56) and the C and D product co-chromatographed with digoxigenin (Rf = 0.22). These results were confirmed in 10 other solvent systems. Metabolites A and B were acetylated in the presence of 50 mg lanatoside A in py/Ac,O and metabolites C and D in the presence of 50 mg lanatoside C. The acetylated compounds were recrystallized from ethylacetatelpetroleumether, acetonelwater and acetic acidJwater. Specific radioactivities were constant after the second crystallization.
N?taJournalor Medicinal
med~caPlant Research
Metabolism of Digitoxin in Individual Plants of Digitalis lanata Elmar W. Weiler
Key Word Index: Digitalis lanata; Cardenolides; Digitoxin Metabolism; 12-Hydroxylation; Screening Techniques.
Abstract
Leaf discs of one and two year old plants of Digitalis lanata EHRH.metabolize 3H-digitoxin to desacetyl lanatoside A, lanatoside A, and the corresponding C-12 hydroxylated compounds, desacetyl lanatoside C and lanatoside C. Individual plants from different sources show wide variations in their metabolic profile. A simple test for the screening of plant individuals for high C-12 hydroxylation ability is described. Introduction
Whereas the general pathway of cardenolide biosynthesis is well understood [I-31, comparatively little is known about the course of subsequent transformations of the basic cardenolide skeleton from which a number of heart glycosides are synthesized. More than 60 cardenolides may occur in one plant species [4] and the reactions involved
include specific hydroxylations a t various positions of the aglycone (e. g. C-12, C-16) and the formation of glycosides with rare sugars some of which are found exclusively in this class of compounds 151. In order to investigate transformation reactions occuring at the stage of the cardenolide tridigitoxoside, tritiumlabeled digitoxin was fed to leaf discs of Digitalis lanata EHRH.An examination of a number of individual plants revealed that considerable variability existed with regard to the metabolic pattern. Since C-12 hydroxylated cardenolides (e. g. digoxin) are of much greater pharmaceutical interest than the corresponding deoxy compounds (e. g. digitoxin) a simple test for the C-12 hydroxylation ability of individual plants which permits a convenient selection of plants (e. g. for cell culture and breeding work) is described. The procedure can be used with cell cultures and intact plants.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Lehrstuhl fur Pflanzenphysiologie, Ruhr-Universitst Bochum, Federal Republic of Germany.
Metabolism of Digitoxin Results
In ethanolic extracts of leaf discs of one and two year old plants of Digitalis lanata, w h i h had been incubated with SH-digitoxin for 1 to 4 days, four radioactive metabolites could be detected in addition to digitoxin. These were identified by TLC, co-crystallization of their acetylated derivatives with authentical compounds and analysis of their acid hydrolysis products, as desacetyl lanatoside A (I), lanatoside A (11), desacetyl lanatoside C (111) and lanatoside C (IV). Kinetic experiments showed that after one day of incubation, (I) and (11) were predominate in the extracts but then declined with a concomitant accumulation of (111) and (IV) by day four. Thus, exogeneously applied digitoxin appears to undergo glucosylation at C'-16, acetylation at C'-15 and hydroxylation at C-12, but: not hydroxylation at C-16. Since no radioactive digoxin could be detected in the extracts, glucosylation either preceeds hydroxylation
or occurs a t a much faster rate. Acetylation may occur either before or after C-12 hydroxylation. Compared to the controls, kept in dim light of 700 lux, dark incubations showed a 20010 decrease in the hydroxylation reaction, but did not differ significantly in the amount of glucosylation and acetylation after 2 days of incubation. The tissue was extremely sensitive to ethanol in the incubation mixture. At 1010 ethanol, the uptake and metabolism of SH-digitoxin were significantly reduced and at 5010 ethanol both were completely inhibited. This effect limits the amount of soluble digitoxin which could be added to the system for saturation experiments. At an optimal substrate concentration of 34 pg digitoxin/ml(4 O/o ethanol) the rate of digitoxin uptake was found t o be 50 pgl day/g fresh leaf material for an average leaf disc sample. However, there was a substantial and significant variation in digitoxin metabolism when individual plants were analyzed for their metabolic pattern.
Table I Mean values and ranges for metabolites of digitoxin in Digitalis lanata leaf discs formed after 48 hrs of incubation. Distribution of radioactivityb Uptake of Desacetyl- LanatoDesacetyl- Lanatolanatw'de eside C digitoxin" lanatoside Aside A
Digitoxin
plants 238 plants 250
58.9 60.6
13.7 10.4
11.0 12.6
11.3 8.5
36.2 37.0
28.4 31.0
plants 238 plants 250
24.9-81.8 18.8-100
0-74.8 0-50.6
0-40.8 0-35.2
0-34.4 0-55.0
0-72.1 0-69.3
0-82.4 0-100
Number of plants
Mean values: l-yr old 2-yr old Ranges: 1-yr old 2-yr old
" = in percent of digitoxin added
= in percent of digitoxin taken up
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163
A technique was developed which permitted the recording of a complete metabolite profile of a plant after a single TLC run, thus providing an efficient screening system for these experiments. In Table I, mean values and ranges found for the various metabolites are listed for 250 two year old plants from nine different sources and for 238 one year old plants from three other sources. As can be seen, one and two year old plants show on an average almost identical metabolic behavior but as the ranges indicate, individual variation was very great. From the 456 plants analyzed, a number of individuals have been isolated which showed extremely different metabolic patterns. Whereas some plants were apparently unable to
metabolize the incorporated 3H-digitoxin, others metabolized it completely within the standard incubation time of 2 days. Also, plants were found which lacked either the desacetyl lanatosides (I, 111) or the lanatosides (11, IV). The amount of C-12 hydroxylated metabolites produced from incorporated digitoxin after 2 days by individual plants varied from 0 % to 82O/o. Fig. 1 shows the frequency distribution for the C-12 hydroxylated metabolites (I11 IV). It is apparent that only a small fraction of the plants analyzed posessed efficient digitoxin hydroxylation systems (0.2 'J/o of the total showed more than 80% C-12 hydroxylated compounds (I11 IV) in the screening test). Experiments repeated over a period of a least 3-4 weeks proved the stability of these extreme types.
+
+
Discussion
-
~
12-Hydroxylation
["/o]
Fig. I. Frequency distribution of formation of C-12 hydroxylated digitoxin metabolites (III+ IV) C-12 hydroxylated digitoxin in leaf discs of Digitalis lanata.
The results presented here demonstrate that exogeneously applied digitoxin undergoes glucosylation, acetylation and hydroxylation at G I 2 but not at C-16 in Digitalis lanata leaf tissue. These observations support the findings of TSCHESCHE et al. [I] and FRANZand MEIER[6] and they are in accord with results obtained from cell cultures of D. lanata [7]. However, the inter-individual variation of the metabolic pattern which had not yet been investigated was found to be considerable. This variation appears to be a stable character of the individual plant, especially as far as the C-12 hydroxylation is concerned. Unfortunately, it is not known at present to what
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Weiler
Metabolism of Digitoxin
Experimental Plant material Digitalis lanata seeds were obtained from various botanical gardens and individuals. Plants were grown in the field in 1974. Leaves of 2 year old plants were harvested in June and July, leaves of one year old plants in August and September. Chemicals 3H-digitoxi'n was purchased from Amenham (spec. act. 5.3 mCi/mmol). Lanatosides were purchased from Merck and secondary glycosides from Roth. Desacetyl lanatosides were
prepared from lanatosides by ammonolysis followed by chromatographic purification. Incubation and extraction procedure 500 mg leaf discs (10 mm diameter) punched out from freshly harvested, fully expanded leaves were incubated in 7.25 ml 0.05 M phosphate buffer, p H 5.5 containing 5 pCi (1 nmol) 3H-digitoxin-(G). Except when otherwise stated, the samples were shaken under dim light (700 lux) for 48 hrs at room temperature (23' C). The leafs discs were then removed from the incubation mixture, washed with water and extracted two times with 12.5 ml 80°/o ethanol. The volume of the combined extracts was brought to 25 -ml and after determination of total activity, was reduced to near dryness. The material soluble imn1 ml 80~10ethanol was used for TLC. Thin layer chromatography System I: Desacetyl lanatoides and lanatosides were separated in the solvent system tetrahydrofurane : CHCls : formamide = 50 : 50 : 6.5. The adsorbent was ,Kieselgur Gu from Merck. 0.25 mm layers were impregnated in acetone : formamide = 90:lO and used immediately after evaporation of the acetone. Developed plates were heated for 90 min at 90° C to remove the formamide. System 11: Formamide impregnated silica gel plates were used in combi,nation with the solvent system xylol : ethyl methyl ketone : formamide = 40:60:5 to separate secondary glycosides and aglycones. Identification o f metabolites Extracts from various one and two year old plants were combined. Four metabolites of SH-digitoxin could be detected inTLC-system I when incubation times varied from 1 to 4 days: A (Rf = 0.83), B (Rf = 0.52), C (Rf = O.39), D (Rf = 0.17), digitoxin (Rf = 0.95). A cochromatographed with lanatoside A, B with lanatoside B and desacetyl lanatoside A, C cochromatographed with lanatoside C and D with desacetyl lanatoside C. The metabolites were eluted and hydrolyzed in a mixture of methanol: 0.1 N H2S0, (1:l) for 20 min. After neutralization, chloroform extracts were made and chromatographed in System 11. The hydrolysis product of A and B showed the
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degree an individual one year old plant retains its metabolite profile in the second year of growth and data on the inheritance of the extreme metabolic patterns are not available. Preliminary studies indicate that there is no close correlation between the actual content of C-12 hydroxylated cardenolides present in a specific plant and the hydroxylation rate of the same plant as determined by the method used in this study. On the contrary, however plants exhibiting a high hydroxylation capacity also contained high amounts of C-12 hydroxylated cardenolides in the leaves. The broad variability in the ultimate metabolic fate of digitoxin in the plants assayed stresses the need to conduct careful biosynthesis studies with many plants of diverse origin. This will be necessary to obtain a picture representative for the plant species on the whole and to elucidate individual variabilities. The method reported here should be useful in selecting plants as starting material for breeding work or, more important, for cell cultures to be used for biotransformation reactions (especially C-12 hydroxylation).
Weiler
Screening test: For the screening of individual plants, TLC aliquots of the consystem I was used. 20 centrated leaf disc extracts were applied as a 2 cm band on the plates. The plates were developed for 15 cm and dried. Complete separation of the four metabolites could thus be aduieved. The distribution of radioactivity on the plates was then recorded with a TLC scanner (Berthold) and the percentage of metabolites formed calculated from the peak areas.
Acknowledgment I wi.sh to thank Dr. CORDUAN and Prof. REINHARD, Tiibingen, who provided seeds of
Digitalis lanata, and Prof. R. MANSELL, Tampa, for checking the English version of the manuscript. This work was supported by a grant of the .BundesrnG~ster fiir Forschung und Technologieu, Bonn to Prof. ZENK,Bochum.
References 1. Tschesche, R., Hombach, R., Scholten, H. and Peters, M.: Phytochemistry 9, 1505 (1972). 2. Tschesche, R.: Proc. R. Soc. (London) 180, 187 (1972). 3. Aberhard, D. J., Lloyd-Jones, J. G. and Caspi, E.: Phytochemistry 12, 1065 (1973). 4. Kaiser, F.: Arch. Pharmaz. 299, 263 (1966). 5. Reichstein, T.: Naturwissenschaften 54, 53 (1967). 6. Franz, G. and Meier, H.: Planta Medica 17, 396 (1969). 7. Rainhard, E, Boy, H. M. and Kaiser, F.: Planta Medica, Suppl. 163 (1975). Address: Dr. E . Weiler, Ruhr-Universitat Bochum, Institut fiir Pflanzenphysiologie, Postfach 2148, 0-4630 Bochum
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same Rf as digitoxi,genin (Rf = 0.56) and the C and D product co-chromatographed with digoxigenin (Rf = 0.22). These results were confirmed in 10 other solvent systems. Metabolites A and B were acetylated in the presence of 50 mg lanatoside A in py/Ac,O and metabolites C and D in the presence of 50 mg lanatoside C. The acetylated compounds were recrystallized from ethylacetatelpetroleumether, acetonelwater and acetic acidJwater. Specific radioactivities were constant after the second crystallization.
