Plant Cell Reports

Plant Cell Reports (1990) 9:101 104

9 Springer-Verlag1990

Alkaloids of hairy root cultures of a Datura candida hybrid Philippe Christen 1, Margaret E Roberts z, J. David Phillipson 2, and William C. Evans 3 i Department of Pharmacognosy, University of Geneva Sciences II, 30 Quai E.-Ansermet, CH-1211 Geneva 4, Switzerland 2 Department of Pharmacognosy, The School of Pharmacy, 29 39 Brunswick Square, London WC1N lAX, UK 3 Department of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, UK Received March 6, 1990/Revised version received April 24, 1990 - Communicated by M. H. Zenk

ABSTRACT From the in vitro hairy root cultures of a Datura candida hybrid, 19 tropane alkaloids have been identified using capillary gas-liquid chromatography and mass spectrometry. As in the parent plants, scopolamine is the major alkaloid. Two hitherto undescribed alkaloids have been detected and their structure tentatively eharacterised on the basis of their mass spectral fragmentations.

investigation. In addition to the above alkaloids, the hairy roots contain 17 other alkaloids and a more detailed investigation of these minor alkaloids is presented here. MATERIALS AND METHODS The plant material a n d the establishment of the hairy root cultures from a hybrid of D. candida x D. candida cultivar have been described previously (Christen et al. 1989).

INTRODUCTION In Wettstein's classification of the Solanaceae (Wettstein 1895), the tribe Daturae comprises the two genera Datura and Solandra, Within the genus Datura, some species are herbaceous, some aquatic and others arborescent (Safford 1921). They all produce a wide range of tropane alkaloids, the tree daturas exhibiting no marked differences from the other sections in this respect, Hybridization followed by selection offers a means of development of new chemical races w i t h biosynthetic capacity possibly superior to that of either of the parents. EIDabbas and Evans (1982) reported on the alkaloid content of the F I hybrid involving reciprocal crossew of the tree daturas D. candida and D. aurea. The scopolamine content (up to 0.72% dry wt.) of this hybrid was superior to that of the parents D. aurea (0.66% dry wt.) and D. candida (0.25% dry wt.). In spite of considerable efforts, attempts to establish stable cell suspension cultures producing large amounts of tropane alkaloids, particularly the medicinally used (-)-hyoscyamine, (• (atropine) and (-)-scopolamine, have as yet not been successful. However more promising results have been reported from hairy roots induced by the bacterium A g r o b a c t e r i u m rhizogenes on several genera and species w i t h i n the Solanaceae (e.g. Kamada et al. 1986; Mano et al. 1986; Jung and Tepfer 1987). In the course of our investigation of tropane alkaloids produced by in vitro hairy root cultures of a Datura candida hybrid, we have recently reported a high yield of scopolamine (Christen et al. 1989). The alkaloid yield (scopolamine + hyoscyamine) obtained after one m o n t h of culture was 0.68% dry wt. It was 1.6 and 2.6 times the amount found in the aerial parts and in the roots of the plant from which the culture was developed. Scopolamine was the principal alkaloid and the scopolamine/hyoscyamine ratio of ca. 5:1 (17.3% of the alkaloid mixture) makes these hairy root cultures worthy of consideration for further

Offprint requests to." P. Christen

Extraction of alkaloids The lyophilized hairy roots, harvested after 1 month of culture in liquid MS medium, were powdered and extracted twice w i t h 0.2M sulphuric acid for 2 hrs. After centrJfugation, the supernatant was made alkaline with 1 M NaOH (pH 12) and extracted with CHCI 3. The CHCI~ extracts were dried with anhydrous ha^SO. J z and the solvent evaporated under reduced pressure. Gas-liquid chromatography and mass spectrometry GLC analysis of the alkaloid extract was carried out using a Perkin-Elmer 8700 gas chromatograph fitted with a N,P detector. A 15m x 0.239 i.d. fusedsilica capillary column coated with the methylsilicone phase DB-I (film thickness 0.25 ~m) (J&W, Scientific) was used with h e l i u m as carrier gas at 0.5 bar pressure. Volume injected: i ~i. Conditions: isothermal at 35~ 2 min; 35-260~ 10~ isothermal at 260~ The injector insert was packed with silylated glass wool, the injector temperature was 300~ The detector temperature was 300~ Injection mode: splitless. GC-MS analysis was performed with the same column using a H e w l e t t - P a c k a r d 589A gas chromatograph in combination with the mass spectrometer VG-12250. Conditions: isothermal at 35~ 2 min; 35300~ 30~ isothermal at 3000C; ionizationenergy 70eV. The injector temperature was 180~ Chemical ionization mass spectra were recorded on the same instrument using ammonia as reagent gas. The different constituents were identified by comparison of their retention times w i t h those of authentic compounds and of their mass spectral data with those published (Lounasmaa 1988). An estimate of the relative percentage of each alkaloid was carried out by computerized peak area integration. RESULTS AND DISCUSSION

102 Table 1. A l k a l o i d s identified and their percentage yields calculated from total a l k a l o i d content of hairy roots of the Datura candida hybrid.

I0O

95

Rt (nfin) I

Tropine +'b

M+ Con

9o

(%)

85 80.

140

10.14

141

13.8

2 Pseudotropine b

10.36

14I

2.9

7S.

3 Oscine"'b

11.45

155

1.5

70-

12.23

183

2.3

65,

13.30

199

4.1

60-

6 N-Methylpyrrolidinylhygrine b

14.76

224

0.6

55.

7 3-Hydroxy-6-propionyloxytropane.

14.89

213

2.9

50-

8 3-Hydroxy+6+butyryloxytropane (or 3-1 lydroxy-6-isobutryloxytropane)

16.80

227

1.1

4 3ct-Aceto•

",b

5 3-Acetoxy-6-hydroxytropane b

9 3~+tigloxyloxytropane (tigloidineY,b

HsG2C1

~

5

~

98

45. 40.

16.88

223

2.8

10 3- Hydroxy-6-(2-methylbutyryloxy-) tropane b (or 34 iydroxy-6-(isovaleryloxy-)tropane

17.70

241

1.1

11 3-Tigloyloxy-6-hydroxyt ropane b

18.96

239

2.2

25

12 3u-Hydrox y-6~-tigloyloxytropa ne'j'

19.07

239

1.9

20

13 3ct-Tigloyloxy-6[~-acetoxytropa ne',l"

19.6

281

0.9

14 Hyoscyamine ''b

20.28

289

3.4

15 6-Hydroxyapoatropine

20.65

287

0.7

16 Apohyoscyamine *,b

21.16

271

5.3

17 Scopolamine *'b

21.40

303

17.3

18 6-iso-l~,-hydroxyhyoscyamine*,b

22.06

305

4+8

19 Aposcopolamine *'~'

22.18

285

3.4

35. 3o

82

140 $

213 1

o...

9

+- . . .

'o . . . .

16

2

. . . . .

~'6o ~

Fig. 1 Mass spectrum of 3-hydroxy-6-propionytoxytroparte (7)

"Direct comparison with authentic sample 100-

bComparison with published rns data 9S 9O

Table 2. Alkaloid structure of hairy roots of the Datura candida hybrid P.,

R:

-OCOCHC

1

-OH

H

2

-OH

H

4

-OCOCH,

H

5

OCOCH,

OH

OH

OCOC,H,

OH

OCOC~H,

OCOCC,H, ]1 CH, OCOCC,H+ u CH2 -OCOCHC

H

H,C CH, 10

OH

OCOC.H,

11

OCOC=CH

OH

OCOCHC j-I,

-OH0.00

I

-OCOC =CH

OCOCHC,,H, I

I

-OCOCH

19

H3C

Ss

R=

42

H3C

5o

H

q

45 4o

-6,7-

3s

-epoxy

ao-

12

25!

-OH

20~ 15"

CH=OH

H,C CH, 13

r.-~5co

65 6o

OH

t

-OCOC =CH

96 OH

70

3 t-.R1

CH=OH

H,C CH, 12

H

CH~OH

8

-OCOC=CH

7a

1

7

9

~H,

T556~

so

: -OH 3

R~

R!

as

-6,7-epoxy

CH2OH

+

~ - ~~ 7/ \

+;

~m3

10"

Lla'

5. 0

. . . .

-...

, ~OO

l'i~i" l ,,, 20O

30@

Fig. 2. Mass spectrum of alkaloid (g) H+C CH,

The alkaloids identified from the hairy roots of the D. candida hybrid are given in Table i. A total of 19 alkaloids comprising 73% of all the peaks detected was recognized of w h i c h 17 alkaloids are known compounds. The structures of all the alkaloids detected are given in Table 2. Scopolamine (17) is the main compound accounting for ca. 17% of the alkaloid mixture. (See e x p l a n a t i o n above, Table i). The second most abundant compound is tropine (I) (13.8%), widely distributed in the Solanaceae. Both major alkaloids have been identified previously in each of the parent plants. Oscine (3), the more stable geometric isomer produced by spontaneous rearrangement of scepine has been detected p r e v i o u s l y in another tree datura, D. sanguinea (Evans and Major 1968).

3-Acetoxy-6-hydroxytropane (5) has not been reported from species of the Solanaceae although it has been isolated from the leaves of Peripentadenia mearsii, Euphorbiaceae (Johns e t a l . 1971). The mass spectrum of (5) is in agreement with the suggested structure and the fragmentation pattern confirms the nature and positions of the substituents; thus, signals at m/z = 155 (M + - C~H40), 140 (M + - C?H302), 122 (subsequent loss of waterJ and 94 (base peaR) are consistent w i t h attachment of the ester group at C-3 (acetoxy substitution at C-6 would have shown a strong signal at m/z = 113) (Lounasmaa 1988, Johns et al. 1971). The structure of alkaloid (6), w i t h a molecular ion at m/z = 224 and a base peak at m/z - 84, is readily deduced by comparison of its mass spectrum with that of N-methylpyrrolidinylhygrine, identified in Datura innoxia by Witte et al. (1987).

103 The electron impact mass spectrum of compound (7) (Fig. i) exhibits a molecular ion peak at m/z = 213 corresponding to the formula C.I H.^NO^. Further $ I~ J indication of the molecular welght comes from the chemical ionization mass spectrum w h i c h displays the (M+H) + peak at m/z = 214. The fragmentation pattern is characteristic of a dihydroxytropane derivative. + The occurrence of fragment ion peaks at + m/z = 156 (M - C~Hr0) and 140 (M - C~H~0~) suggests J a J L the presence of propanoic acid ester. T~e base peak at m/z = 113 together w i t h a prominent signal at m/z 96 confirm the attachment of the ester function at C-6 and a free hydroxyl group at C-3 of the tropane nucleus. On the basis of this data, alkaloid (7) corresponds to 3-hydroxy-6-propionyloxytropane. However, with the data available, it was not possible to assign the stereochemical orientation of the 2 substituents. Propionyl esters are, as yet, not commonly known in the tropane series. 3~-Propionyloxytropane has been reported from Datura innoxia (Witte et al. 1971) and from a species of Bruguiera (Loder et al. 1969); Beresford and Woolley (1974a) isolated 6 $ p r o p i o n y l o x y - 3 ~ - t i g l o y l o x y t r o p a n e from Datura innoxia. The mass spectr~/m of compound (8) (Fig. 2) presents a fragmentation pattern corresponding to the well established characteristic fragmentation of a monoester of tropane-3,6-diol. The molecular ion peak at ~/z = 227 corresponds to the elemental composition C H NO The base peak at m/z = 113, together 12. 21 3" + wlth signals at m/z = 156 (M - 71), 140 (M + - 87) suggest the esterifying acid as C H 0 . As in the 482 case of alkaloid (7), the strong peak at m/z = 96 confirms the attachment of the ester group on the carbon 6 and the free hydroxyl group at C-3 of the tropane nucleus. The alkaloid (8) is therefore a dihydroxytropane esterified with either n-butyric acid or isobutyric acid on carbon 6. However, with the data available and in the absence of NMR spectral data, it is not possible to define w h i c h isomer (nbutyryl or isobutyryl) is attached to the carbon 6. 3 ~ - I s o b u t y r y l o x y - 6 ~ - h y d r o x y t r o p a n e as well as 3~-nbutyryloxytropane have been identified as components of the aerial parts of two subspecies of Anthocercis alhicans by Evans and Ramsay (1981). The mass spectrum of alkaloid (9) exhibits the f r a g mentation pattern of a 3-substituted tropane nucleus possessing a tigloyl moies and in this respect, it is identical with the mass spectrum of the authentic material tigloidine (3-%igloyl ester of ~-tropine). The mass spectrum of alkaloid (i0) is characteristic of a disubstituted tropane nucleus and gives the molecular formula C I ~ HNO . The base peak at m/z = 113 indicates that ~ e i ~ r e ~ hydroxy is at the C-3 position. The fragmentation is identical w i t h that described by Beresford and W o o l l e y (1974b) for 3~hydr oxy-6~- (2-met hylbutyryloxy- )t ropane. In the absence of NMR data, it is not possible to define which acid (2-methylbutanoic acid or isovaleric acid) is attached to the carbon 6. There is no phytochemical preference for either, as esters of both these acids occur naturally w i t h tropine derivatives. However, the presence in the mixture of a 6$-tigloyl ester (12), the dehydro derivative of (I0), suggests it is rather 3 - h y d r o x y - 6 - ( 2 - m e t h y l b u t y r y l o x y - ) t r o p a n e than 3-hydroxy-6-(isovaleryloxy-)tropane. T h e alkaloids (Ii) and (12) are two tigloyl derivatives. Both have been reported ot occur in the D. candida cultivar parent (El-Imam and Evans 1989). They have the same molecular weight but their fragmentation patterns differ. The base peak of (ii) is at m/z = 94 suggesting that the alkaloid is a dihydroxytropane derivative having

the ester function attached to the carbon 3. An ion peak at m/z = 140 (M + - 99) is assigned to the loss of a tigloyl moiety and at m/z = 122 to a subsequent loss of water and hence alkaloid (ii) is identified as 3tigloyloxy-6-hydroxytropane. The base peak in the mass spectrum of (12) at m/z = I13, together with a prominent signal at m/z = 96 confirm the attachment o f the tigloyl moiety at C-6 and the free hydroxy at C-3' of the tropane nucleus. The retention time and the fragmentation pattern is identical to those of the corresponding referefice compound and thus the alkaloid (12) is 3~-hydroxy-6~-tigloyloxytr0Pane. This compound has been identified previously (Evans and Griffin 1963) as a minor component of the alkaloid mixture of Datura cornigera leaves. The mass spectrum of alkaloid (13) gives a molecular ion of 281 with principal fragments consistent w i t h a disubstituted tropane-diol possessing acetyl and tigloyl moieties. The alkaloid is identified as 3~tigloyloxy-6~-acetoxytropane by comparison with authentic material. Hyoscyamine (14) is one of the major fied in the mixture. This alkaloid ributed within the Solanaceae and is interest from the medicinal point of

alkaloids identiis widely distof particular view.

The alkaloid (15) has been identified as 6-hydroxyapoatropine by comparison w i t h literature data (Hartmann et al. 1986). It shows a molecular ion peak at m/z 287 corresponding to the molecular formula C H 0 From the base peak at m/z = 94, it is 17 21 3". assumed that the alkaloid is a dihydroxytropane derivative. 6-Hydroxy-apoatropine has been isolated previously from Atropa belladonna (Hartmann et al. 1986). Two other ape- derivatives, apohyoscyamine (16) and aposcopolamine (19) have been identified from the hairy root cultures. The presence of 3 ape- compounds in the alkaloidal mixture is not surprising since there is an easy thermal loss of water from tropic acid derivatives leading to the ape compounds. It is well known that scopolamine is fermed~in the plant by epoxidation from hyoscyamine. This latter compound is first converted to 6~-hydroxyhyoscyamine by a 2-oxoglutarate-dependent oxygenase then to scopolamine by a 6B-hydroxyhyoscyamine epoxidase. 6~-Eydroxyhyoscyamine (18) has been unambiguously identified in the alkaloid mixture. Thus it can be assumed that the development of the whole plant is not necessary to the biosynthesis of scopolamine, the hairy roots having a sufficiently developed enzymatic system to carry out the epoxidation. Our results demonstrate that the hairy root cultures serve as an interesting alternative for the production of secondary metabolites. Examination of Table i indicates that the alkaloids produced by the hybrid are typical of those found in many species of Datura. The transformed roots produce tropane alkaloids w h i c h do not differ significantly from those produced by the parent plants. Scopolamine (17) w h i c h has been identified as the principal alkaloid in the non-infected plants, is also the major alkaloid of the hairy roots. Application of gas-liquid c h r o m a t o g r a p h y in combination w i t h the highly sensitive nitrogen detector is a convenient method for the analysis of crude alkaloid mixtures. Gas chromatography combined with mass spectrometry indicates the presence of two hitherto unreported alkaloids.

104 ACKNOWLEDGEMENTS The authors are indebted to Mr G.L. Langley and Mr M.E. Harrison (University of London) for performing the GC-MS analysis. This work was supported by a grant from The Royal Society under the European Science Exchange Programme to P.C.

REFERENCES Beresford, P.J., Woolley, J.G. (1974a) Phytochemistry 13~ 1249-1251 Beresford, P.J., Woolley, J.G. (1974b) Phytochemistry 13, 2511-2513 Christen, P., Roberts, M.F., Phillipson, J.D., Evans W.C. (1989) Plant Cell Reports 8, 75-77 Ei-Dabbas, S.W., Evans, W.C. (1982) Planta Med. 44, 184-185 El-Imam, Y.M.A. and Evans, W.C. (1989) Fitoterapia, in press

Evans, W.C., Griffin, W.J. (1963) J. Chem. Soc. 43484350 Evans, W.C., Major, V.A. (1968) J. Chem. Soc. (C) 2775-2778 Evans, W.C., ~Ramsey, K.P.A. (1981) Phytochemistry 20, 497-499 Hartmann, T., Witte, L~ Oprach, F. and Toppel, G. (1986) Planta Med. 52, 390-395 Johns, S.R., Lamberton, J.A., Sioumis, A.A. (1971) Aust. J. Chem. 24, 2399-2403 Jung, G. and Tepfer, D. (1987) Plant Science 50, 145151 Kamada, H., 0kamura, N., Satake, M., Harada, H. and Shimomura, K. (1986) Plant Cell Reports 5, 239-242 Loder, J.W., Russell, G.B. (1969) Aust. J. Chem. 22, 1271-1275 Lounasmaa, M. (1988) In: The Alkaloids, Vol. 33 (Brossi A., ed.), and literature cited therein, Academic Press, New York Mano, Y., Nabeshima, S., Matsui, C. and Ohkawa, H. (1986) A~ric. Biol. Chem. 50, 2715-2722 Safford, W.E. (1921) J. Wash. Acad. Sci. ii, 173-189 Wettstein, R. (1895) In: Die nat~rlichen Pflanzenfamilien, IV(36): 4 (Engler, A., Prantl, K., eds.) Englemann, Leipzig Witte, L., M~ller, K., Arfmann, H.A. (1987) Planta Med. 53, 192-197