Phytochemisfry, Vol. 35. No. I. pp. 107-I 10. 1994 Printed in Great Britain.

0031 !M22,?M $6.00+0.00 0 1993 Pergamon Press Ltd

TROPANE ALKALOID PATTERNS IN PLANTS AND HAIRY ROOTS OF HYOSCYAMUS ALBUS

KATJA DOERK-SCHMITZ, LUDGER WIITE* and A. WILHELM ALFERMANNT

Institut fiir Entwicklungs- und Molekularbiologie der F’tlanzen, Heinrich-Heine-Universitit Diisseldorf, Universit%ts.straDe I, D-40225 Dtisseldorf, Germany; *Institut fiir pharmazeutische Biologic, Technische Universitlt Braunschwei&

MendelssohnstraBe I, D-38106 Braunschwei& Germany

(Receiued in revised form 17 June 1993)

IN HONOUR OF PROFESSOR 0. H. VOLK’S NINETIETH BIRTHDAY

Key Word Index-Hyoscyamus albus; Solanaceae; GC-MS; hairy roots; plant organs; tropane alkaloids.

Abstract-Hyoscyamus albus is a solanaceous plant forming tropane alkaloids. The roots as the site of biosynthesis show a rich alkaloid spectrum of at least 26 compounds, whereas the alkaloid composition of the aerial parts is less complex. Some of the alkaloids, i.e. 3-propionyloxytropane, 3-isobutyryloxytropane, 3j?-tigloyloxynortropane and 6- hydroxy-3-phenyl-acetoxytropane are described as components of H. albus for the first time. Three hitherto unknown compounds were tentatively identified as N-methylpyrrolidinyl-cuscohygrine, occurring in two isomeric forms, and as 3a-(p-hydroxyphenyl)lactoyloxytropane (4’-hydroxylittorine). Compared with plant roots, hairy roots show a rather similar but not identical alkaloid pattern. The low proportion of scopolamine in hairy roots is especially remarkable.

INTRODUCTION

The tropane alkaloids hyoscyamine and scopolamine are widely used in medicine, e.g. in ophthalmology, in the treatment of gastrointestinal spasm, in the treatment of organophosphate poisoning and in anaesthesia. Trppane alkaloids are formed by several solanaceous speci&. The most important tropane alkaloid-producing plants be- long to the genera Datura, Hyoscyamus, Atropa and Duboisia. In Romeike’s excellent work, the roots were shown to be the site of tropane alkaloid biosynthesis [ 1 J. Therefore it is possible to obtain these alkaloids not only from the field-grown plant, but also from cultured hairy roots which emerge from the transformation of plant cells by Agrobacterium rhizogenes. Here we report on the alkaloid patterns of roots, leaves, stems and fruits of H. albus L. plants and hairy root cultures.

RESULTS AND DISCUSSION

Alkaloid pattern of Hyoscyamus albus

The alkaloid patterns detected in roots, leaves, stems and fruits of the intact plant, as well as in hairy root cultures are shown in Table 1. Some of the alkaloids listed are, to our knowledge, hitherto unknown for H. albus. Newly detected alkaloids are 3b-tigloyloxynor-

tAuthor to whom correspondence should be addressed.

tropane, which was previously described for H. pusillus [2], and for H. gyiirfyi [3], 3-propionyloxytropane, 3-isobutyryloxytropane and a hydroxy-3-phenylacetoxy- tropane. The position of the hydroxyl group at the tropane moiety of the latter alkaloid can be assigned to a 6-position from the fragment ion at m/z 23 1 [M - 44]+. Whether it is (3R,6Rh6J-hydroxy-3a-phenylacetoxy- tropane (=i’/?, if numbered clockwise proceeding from the 1R bridge carbon of hyoscyamine) or (3S,6S)-6/l-hy- droxy-3rx-phenylacetoxytropane (= 68) cannot be de- duced from the GC-MS data, since both compounds are enantiomers and do not separate on an optically inactive GC-column. The problem applies for the hydroxy apoa- tropine as well. A (3R,6R)-6/?-hydroxy-3z-phenyl- acetoxytropane was isolated from Erythroxylum hyper- icijdium by Al-Said et al. [4].

The 3S,6S-( = 68) and the 3R,6R-( = 7/I?) hydroxyhyos- cyamines are diastereomers because of the asymmetric C- atom of the tropic acid moiety. However, if racemization of tropic acid takes place, separation also occurs with only one isomeric form at the 6-position. Therefore it cannot be excluded that there is only one 6-hydroxy- hyoscyamine in our samples that forms two peaks due to partial racemization of tropic acid. Bachmann showed that racemization of 6/?-hydroxyhyoscyamine does take place [Bachmann, P., personal communication].

3-Propionyloxytropane most probably occurs as the a- and /?-isomers, the substance with the retention index 1414 possibly being the latter. In the case of 3-isobutyryl- oxytropane the a- and fi-isomers do not separate under

107

IO8 K. DOERK-SCHMITZ et al.

Table 1. Alkaloid patterns of plant organs and hairy roots of Hyoscyamus albus

Alkaloid RI Plant root Stem Leaf Fruit Hairy roots

Hygrine 1060

Tropinone 1154

Tropine 1167

Pseudotropine 1185

Oscine 1255 Scopine 1285

3x-Acetoxytropane 1305 3p-Acetoxytropane 1317 3a-Propionyloxytropane 1405 3/?-Propionyloxytropane 1414 3-lsobutyryloxytropane 1454 N-Methylpyrrolidinyl-hygrine A 1567

N-Methylpyrrolidinyl-hygrine B 1578 3/l-Tigloyloxynortropane 1635 3a-Tigloyloxytropane 1645

Cuscohygrine 1650

Tigloidine 1655 3wPhenylacetoxytropane 1936 Apoatropine 2024 6-Hydroxy-3-phenylacetoxytropane 2115

Aposcopolamine 2130 N-Methylpyrrolidinyl-cuscohygrine At 2165 Norhyoscyamine 2167 N-Methylpyrrolidinyl-cuscohygrine Bt 2176 Littorine 2176 Hyoscyamine 2176 6Hydroxyapoatropine 2200 Norscopolamine 2282 Scopolamine 2290 4’-Hydroxylittorinet 2315 7B-Hydroxyhyoscyamine 2335

6jI-Hydroxyhyoscyamine 2355 cis-Feruloyloxytropane 2685 truns-Feruloyloxytropane 2733

+ + + + + + + + + + + + + + + + + + + + + + + + + + +

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.

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--

-. .._ + - +

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+ + + _ + + + + + +

*The detection of N-methylpyrrolidinyl-hygrine and N-methylpyrrolidinyl-cuscohygrine in the stems is very uncertain.

tThe identification of these alkaloids is based on the MS data only.

the conditions used [Bachmann, P., personal communic- ation], so it remains unclear whether both forms are present.

Moreover, scopine and oscine were identified for the first time in roots and fruits of H. u/bus, the latter being formed from scopine by rearrangement of the epoxide group under acid or basic conditions [S]. Their occur- rence, however, is likely to be due to degradation of scopolamine during the extraction procedure. The exist- ena of norscopolamine in stems, leaves and fruits is probably due to demethylation of scopolamine in ageing parts of the plant as it is known for norhyoscyamine from Atropa belludonna [6].

Three new alkaloids were also detected. Two of them were tentatively identified as isomers of N-methylpyrroli- dinylcuscohygrine. The structures were assigned from the molecular ion at m/z 307, a fragmentation pattern very similar to that of the corresponding hygrine derivat- ives and the retention time distances of the two isomers

also corresponding to those of N-methylpyrrolidinyl- hygrines. As for the hygrine derivatives, the presence of two GC-peaks can be explained by the occurrence of different R- and S-configurations at the bridge C-atoms [7J leading to four possible isomers. The number of isomers is not easily determined, particularly as the stereochemistry of cuscohygrine itself is not fully estab- lished [8]. The third alkaloid has a molecular ion at m/z 305, corresponding to a hydroxylated hyoscyamine or littorine derivative. The mass-spectrum is not consistent with a hydroxyl group in the tropane moiety, therefore a hydroxylation in the p-position of the organic acid moiety is assumed. The occurrence of an ion at m/z 107, that might be due to a p-cresolic fragment, supports this idea. Nevertheless, an o- or m-position of the hydroxyl group is also possible. A small ion at m/z 142, which was found to be characteristic of littorine [93, suggests that the base is 4’-hydroxylittorine (3a-(p-hydroxyphenyl)lactoyloxy- tropane).

Tropane alkaloids in Hyoscyamus albus 109

Alkaloid distribution in plants

The alkaloid composition of the plant roots was found to be more complex than that of the aerial parts. This coincides with results obtained for A. belladonna [6]. Datum innoxia [fl and Duboisia myoporoides [9]. Esters with acid compounds other than tropic acid and phenyl- lactic acid seem to be limited to the roots. N-meth- ylpyrrolidinyl-hygrine and N-methylpyrrolidinyl-cusco- hygrine were undetectable in leaves and fruits, but there were hints of their occurrence in stems. For A. belladonna [a], D. imoxia [fl and D. myoporoides [9], the accumu- lation of hygrine and cuscohygrine is described to be root specific. Ghani et al. [lo] reported the same for the Occurrence of cuscohygrine and littorine in H. albus plants, but hygrine was not detected by these authors. The H. albus plants investigated here contained all three alkaloids in the aerial parts as well as in the roots. The only exception was that cuscohygrine could not be detec- ted in fruits. The quantities of these alkaloids, however, were very small in the upper parts compared with the roots as shown for littorine in Table 2.

Quantitative investigations revealed that in H. olbus plants the roots are not only the site of alkaloid biosyn- thesis, but also an important site of storage. The contents calculated on a dry weight basis are much higher in the roots than in the other organs. This is shown for hyoscya- mine in Table 3. The only alkaloid with comparable levels in roots and green parts is scopolamine. Our results are in agreement with the findings of Parr et al. [2], who conclude from data obtained from hairy roots and petioles of Hyoscyamus species that in this genus the green organs play only a minor part in alkaloid storage.

Alkaloids in hairy roots

The hairy roots of H. albus show an alkaloid pattern very similar, but not identical, to that of the plant roots

Table 2. Distribution of littorine in a Hyoscyamus albus plant. The littorine content is calculated on a dry wt basis

Roots L&WCs

Stems Fruits

Veg. growth (%)

0.068 0.002 0.004

Flower stage

WI

0.13 0.002 0.008

Fruit stage

(%)

0.073 0.001 0.007 Traces

Table 3. Distribution of hyoscyamine in a Hyoscyamus albus plant. The data are calculated on a dry wt basis

Roots L4XVeS

Stems Fruits

Veg. growth

W)

0.21 0.033 0.03

Flower stage

WI

0.27 0.11 0.055

Fruit stage

W)

0.24 0.007 0.03 1 0.11

(for details see Table 1). The differences, however, con- cern only trace compounds.

The quantitative difkrences of the alkaloids calculated on a dry weight basis are more remarkable in the way that the hairy roots greatly surpass the roots of the intact plant with reference to their alkaloid content. This could be due to the fact that in the case of the intact plant the alkaloids are transported to the upper parts. The greater share of immature tissue might also be a reason as discussed in ref. [l 11. As an example, it is mentioned that hyoscya- mine and littorine, the major alkaloids of both the transformed and the plant roots, are five to lO-Told higher in hairy roots at the stationary phase than in plant roots (data not shown).

It should be noted that in contrast to other hairy root cultures of H. albw [f 123, the transformed roots invest- igated here form rather low levels of scopolamine (cu 0.01% of dry weight or less), although the parent plants synthesize considerable amounts of this alkaloid (data not shown). The reason for the poor scopolamine biosyn- thesis has still to be elucidated.

EXPERIMENTAL.

Plants. Seeds of H. albus were obtained from the Zentralinstitut fiir Pflanzengenetik- und Kulturpflanzen- forschung (Gatersleben, Germany). Sterile seedlings were potted and cultivated in a greenhouse. The plants were harvested at the stage of vegetative growth, at the flower stage and at the time of fruit formation. Roots, stems and leaves were freezedried separately.

Extraction of alkaloids. The freeze-dried plant material was powdered and extracted with 0.1 N H,SO,. For the hairy roots fresh material was used. After 2 hr the cell debris was removed. The supematant liquid was made alkaline with 25% NH, and applied to an Extrelut column. The alkaloids were eluted by using a mixt. of CH,CI,-MeOH (17:3) and the extracts were evapd to dryness.

Hairy roots. Transformed roots were induced by co- cultivation of H. albus kaf disks with the supervirulent Agrobucteriutn strain R 1601 [13] and cultivated in liquid MS-medium as described previously [14]. For quantitat- ive measurements, hairy roots were used which were obtained from those plants used for the other investiga- tions reported here. Alkaloid levels were followed throughout a 4-week culture period.

Anulysis ofalk.aloids. The alkaloid spectra were invest- igated by GC-MS as descrii previously [14]. The identities of the alkaloids were confirmed by comparing the measured data with those of authentic compounds, or with data obtained from the literature [6, 7, 93.

N-Methylpyrrolidinylcuscohygrine (RI 2165). EIMS 4OeV[m/z(%)]:307(1)[M]+,224(1),167(2),152(1),98 (2), 84 (100)~ 42 (8).

4’-Hydroxylittorine (RI 2315). EIMS 40 eV [m/z (%)I: 305(3)[M]+, 142(4), 140(3), 124(100X 107(4X83(10),82 (12), 42 (15).

For quantification an FID-detector was used and homatropine was chosen as int. standard. The analyses

110 K. DOERK-SCHMITZ~~ al.

were performed on a fused silica capillary column (30 m x 0.32 mm) coated with a DB 17 stationary phase and

connected to a 0.53 mm phenylsilicone deactivated pre- column. He was used as carrier gas at 2 ml min- ‘. Samples (0.5 ~1) were injected directly on to the column and run with the following temp. program: 92-130”. 2” min- ‘, 130-290”, 6” min- ‘. The temp. of the on- column injector exceeded the column temp. by 3” throughout the program. The detector temp. was set to 300”.

Acknowledgements-This research was financially sup- ported by the Fonds of Chemical Industry (Frankfurt, Germany) and by a grant from the Evangelisches Stu- dienwerk (Schwerte, Germany). The supply of seeds by the Zentralinstitut fur Pflanzengenetik- und Kulturpflan- zenforschung (Gatersleben, Germany), as well as the kind gift of Agrobaccerium strain R 1601 by Dr Pythoud (Bern, Switzerland) are gratefully acknowledged. Furthermore, we would like to thank Dr Shimomura (Tsukuba, Japan) for placing 6/?-hydroxyhyoscyamine and 7P-hydrox- yhyoscyamine at our disposal, Dr Evans (Nottingham, U.K.) for providing (3R,6R)-6P-hydroxy-3a-phenyl- acetoxytropane and Dr Bachmann (Braunschweig, Germany) for discussing the stereochemistry of the 6- hydroxy derivatives.

REFERENCES

1. Romeike, A. (1956) Flora 143, 67. 2. Parr, A. J., Payne, J., Eagles, J., Chapman, B. T.,

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Robins, R. J. and Rhodes, M. J. C. (1990) Phyto- chemistry 29, 2545. Doerk, K., Ionkova, I., Witte, L. and Alfermann, A. W. (1991) Poster presented at the IAPTC confer- ence, German section, Hamburg, 18-19th Sep- tember. Al-Said, S., Evans, W. C. and Grout, R. J. (1986) J. Chem. Sot. Perkin Trans I 957. Werner, G. and Schmidt, K.-H. (1967) Tetrahedron Letters 14, 1283. Hartmann, T., Witte, L., Oprach, F. and Toppel, G. (1986) Planta Med. 52, 390. Witte, L., Miiller, K. and Arfmann, H.-A. (1987) Planta Med. 53, 192. Southon, I. W. and Buckingham, J. (eds) (1989) in Dictionary of Alkaloids, p. 273. Chapman and Hall,

London. Bachmann, P. (1989) Ph.D. Thesis, University of Wiirzburg, Germany. Ghani, A., Evans, W. C. and Woolley, V. A. (1972) Bangladesh Pharm. J. 1, 12. Deans, S. G. and Kennedy, A. 1. (1991) Agro. Food Industry Hi-Tech 2, 11. Shimomura, K., Sauerwein, M. and Ishimaru, K. (1991) Phytochemistry 30, 2275. Pythoud, F., Sinkar, V. P., Nester, E. W. and Gordon, M. P. (1987) Biotechnology 5, 1323. Doerk, K., Witte, L. and Alfermann, A. W. (1991) Z. Naturforsch. 46c, 5 19.