Calystegine distribution in some solanaceous species

Calystegine distribution in some solanaceous species

Khalid Bekkouchea, Youssef Daalib, Samir Cherkaouib, Jean-Luc Veutheyb,Philippe Christenb,*

aLaboratory of Medicinal Plants and Phytochemistry, Department of Biology, Faculty of Sciences-Semlalia, PO Box 2390, Marrakech, MoroccobLaboratory of Pharmaceutical Analytical Chemistry, University of Geneva, Bd d’Yvoy 20, 1211 Geneva 4, Switzerland

Received 29 January 2001; received in revised form 7 June 2001

Abstract

The distribution of eight calystegines (A3, A5, B1, B2, B3, B4, C1 and N1) and their content was investigated by gas chromato-graphy coupled to mass spectrometry (GC–MS) in Datura metel, Atropa belladonna, Hyoscyamus albus, Mandragora autumnalis,

Solanum sodomaeum, Withania somnifera, Withania frutescens and Brunfelsia nitida. The most frequently encountered calystegineswere A3, B1, B2 and B3, while distribution of N1 and C1 was more limited. In all the investigated samples, calystegines A5 and B4were never detected. This report focuses for the first time on calystegines in Withania and Brunfelsia genera and in Mandragora

autumnalis and Solanum sodomaeum species. # 2001 Published by Elsevier Science Ltd.

Keywords: Atropa; Brunfelsia; Datura; Hyoscyamus; Mandragora; Solanum; Withania; Solanaceae; GC–MS; Calystegines

1. Introduction

In the last 10 years, a large number of nitrogen-con-taining polyhydroxylated heterocyclic compounds havebeen isolated from plants (Nash et al., 1996). Thesenatural products are competitive inhibitors of variousglycosidases. The most efficient compounds are used totreat various diseases including diabetes, cancer, andviral infections (Jacob, 1995). Furthermore, these com-pounds exhibit additional activities, such as immuno-modulatory properties and inhibition of glycolipidsynthesis (Jacob, 1995). Among these metabolites, anew class of nortropane polyhydroxylated alkaloids,called calystegines, has recently been isolated from dif-ferent species belonging to the Solanaceae, Con-volvulaceae and Moraceae (Tepfer et al., 1988;Goldmann et al., 1990; Nash et al., 1993; Asano et al.,1994a,b; Schimming et al., 1998). Calystegines havebeen suggested to be nutritional mediators in the plantrhizosphere (plant-bacteria relationship) (Tepfer et al.,1988). Moreover, they possess glycosidase inhibiting

properties (Asano et al., 1995, 1997a,b) and an allelo-pathic activity (Goldmann et al., 1996). To date, 14calystegines have been isolated (Asano et al., 1997a,b).From a biosynthetic point of view, it is assumed thatcalystegines originate from the tropane alkaloid path-way (Drager et al., 1994) although the exact details havenot yet been confirmed.Most calystegines have been isolated in several sola-

naceous species and found to occur in the followinggenera: Atropa (Tepfer et al., 1988; Molyneux et al.,1993; Drager et al. 1994, 1995; Drager, 1995; Nash etal., 1997), Datura (Nash et al., 1993), Lycium (Asano etal., 1997b), Physalis (Asano et al., 1995), Hyoscyamus(Drager et al., 1994; Asano et al., 1996b), Mandragora(Drager et al., 1995), Scopolia (Asano et al., 1996a),Duboisia (Kato et al., 1997), Solanum (Nash et al, 1993;Drager et al., 1995; Keiner and Drager, 2000) andNicandra (Griffiths et al., 1996). In contrast, and to ourknowledge, the occurrence of calystegines in Brunfelsiaand Withania genera has not been reported. This paperdiscusses calystegine distribution in 8 solanaceous spe-cies belonging to 7 different genera. Particular attentionis given to the occurrence of calystegines A3, A5, B1,B2, B3, B4, C1 and N1 (Fig. 1), which were identifiedby GC–MS, with authentic samples as referencematerial.

0031-9422/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.

PI I : S0031-9422(01 )00283-7

Phytochemistry 58 (2001) 455–462

www.elsevier.com/locate/phytochem

* Correspondence author. Tel.: +41-22-702-65-61; fax: +41-22-

781-51-93.

E-mail address: [email protected] (P. Christen).

2. Results and discussion

Fig. 2 illustrates a typical gas chromatogram of asilylated calystegine standard mixture composed ofcalystegines A3, A5, B1, B2, B3, B4, C1 and N1 withoctadecane as internal standard. The method allowed agood separation of the investigated compounds in lessthan 19 min . As expected, calystegines A (3 OH groups)eluted faster than calystegines B (4 OH groups) andcalystegines C (5 OH groups). Indeed, since TMS deri-

vatization occurs on the hydroxy groups, interaction ofderivatized calystegines with the hydrophobic stationaryphase increases with the number of hydroxy groups.The calystegine profile and the amount of individualalkaloid in each species are reported in Table 1 andFig. 3. Calystegines were identified by comparing theirretention times and mass spectra with those of silylatedreference compounds, although no statement can bemade on the absolute configuration of the alkaloids, noreven on their enantiomeric purity. Important qualitativeand quantitative variations were observed among thespecies. The absence of reference material precluded theidentification of other calystegines. None of the investi-gated calystegines were detected in Datura metel L.Tetrahydroxylated calystegine B2 was present in allother species and its content varied significantly (from19.1 to 110.9 mg/g dry wt.). The most frequentlyencountered calystegines were B2, B3, A3 and B1, whilecalystegines B4 and A5 were never observed.

Fig. 1. Structures of investigated calystegines.

Fig. 2. Typical GC–MS chromatogram of a mixture of silylated standard calystegines with octadecane as internal standard.

Table 1

Distribution of calystegines in the investigated Solanaceae species

Plant species Organ Calystegines

A3 A5 B1 B2 B3 B4 C1 N1

Atropa belladonna Aerial parts + – + + + – – +

Hyoscyamus albus Leaves + – + + + – – +

Mandragora autumnalis Leaves – – – + + – – –

Mandragora autumnalis Roots + – + + + – – –

Solanum sodomaeum Leaves – – – + + – – –

Withania frutescens Leaves + – + + + – + +

Withania somnifera Leaves – – – + – – + –

Brunfelsia nitida Leaves + – + + + – + –

Datura metel Leaves – – – – – – – –

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In the aerial parts of Atropa belladonna L., the majorcompounds were A3, B2, B3 and B1. Calystegine B4,previously reported by Nash et al. (1997), was notidentified in our extract and calystegine N1 was detectedat a very low level. Calystegines N1 and B3 are reportedin this species for the first time.The presence of A3 and other calystegines of the B-

group in cultured roots of Hyoscyamus albus L. hasalready been reported elsewhere (Drager et al., 1994).However, to the best of our knowledge, N1 is reportedin this species for the first time. Calystegines B3 and B2were detected in leaves and roots of Mandragoraautumnalis Bertol. B1 and A3 were also observed in thelatter organ. Analogous studies (Drager et al., 1995)reported the presence of calystegines B1, B2 and A3 inleaves and roots of M. officinarum.Calystegines B2 and B3 were detected in the leaves of

Solanum sodomaeum L. which is consistent with similararticles reporting the presence of these metabolites inother Solanum species (Nash et al., 1993; Drager et al.,1995; Keiner and Drager, 2000).The calystegine profiles of the two Withania species

were found to be very different. Whilst calystegines B2and C1 were identified in W. somnifera L. only, thecalystegine spectrum of W. frutescens L. was morecomplex. All the investigated alkaloids, except A5 andB4, were found in the leaves of W. frutescens. This is thefirst time that calystegines are reported in the subtribe

Solaninae of the Solanaceae family. Calystegines A3, B1,B2, B3 and C1 were unambiguously identified in theleaves of Brunfelsia nitida Benth. The latter compoundwas in a high amount (357 mg/g dry wt.), and the mostabundant calystegine found in all the investigated spe-cies. To the best of our knowledge, this class of com-pounds has not been reported in this genus before.To date, calystegine N1 has been isolated from Hyos-

cyamus niger (Asano et al., 1996b) and Lycium chinense(Asano et al., 1997b) only. In our study, this calysteginewas found in three other species, A. belladonna, H. albusandW. frutescens. The distribution of pentahydroxlatedcalystegine C1 was more limited as this compound wasonly found in B. nitida, W. somnifera and W. frutescens.Previous studies have also reported the presence of C1 inH. niger (Asano et al., 1996b), Duboisia leichhardtii(Kato et al., 1997) and Capsicum sp. (Asano et al.,1997a).It is noteworthy that most chromatograms of the dif-

ferent investigated species showed additional uni-dentified compounds with a similar fragmentationpattern to that of calystegines. Fig. 4 shows the chro-matogram of H. albus leaf extract. Beside calysteginesA3, B1, B2, B3 and N1, some other peaks (X1 and X2)with specific calystegine MS profiles are also present. Asshown in Fig. 5, calystegine N1 (Fig. 5a) and uni-dentified compound X1 (Fig. 5b) exhibit similar MSpatterns. Beside fragment ions at m/z 390, 375 and 300,

Fig. 3. Calystegine content in eight Solanaceae species.

K. Bekkouche et al. / Phytochemistry 58 (2001) 455–462 457

corresponding to the molecular ion and to the loss of amethyl and dimethylsilyl groups, respectively, otherfragments at m/z 186 and 83, specific of the exocyclic N-group, are found. Indeed, only calystegines belonging tothe N-group exhibit such fragments which can bemainly attributed to the underivatized exocyclic aminogroup, as shown in the suggested fragmentation patternof calystegines N1 and X1 (Fig. 6). As a result, uni-dentified peak X1 may correspond to a new calystegineof the N-group, where the NH2 function is located onthe �1-pyrrolinium skeleton at a different position tothat of calystegine N1. Moreover, fragment m/z 217 istypical of polyhydroxy compounds, as already describedelsewhere (DeJongh et al., 1969).Compound X2 (Fig. 4) may correspond to a calyste-

gine with a particular hydroxylation pattern. Its massspectrum (Fig. 5C) clearly demonstrates that this com-pound does not belong to the N-group. To assess ourassumption, further studies are to be performed. Inparticular isolation and structure characterization ofthese compounds should be carried out.Before an overall chemotaxonomic conclusion

regarding the occurrence of calystegines throughout thisfamily can be drawn, further detailed phytochemicalstudies are required.

3. Experimental

3.1. Chemicals

Standard calystegines (A3, A5, B1, B2, B3, B4, C1andN1) were kindly donated by Dr. N. Asano (HokurikuUniversity, Kanazawa, Japan). All other chemicals andsolvents were of analytical grade.

3.2. Plant material

Roots or aerial parts from wild plants were collectedin different localities of Morocco for Mandragoraautumnalis, Datura metel, Hyoscyamus albus, Withaniasomnifera, Withania frutescens and Solanum sodo-maeum. Voucher specimens have been deposited in theHerbarium, Faculty of Sciences-Semlalia, Marrakech,Morocco. Seeds of Brunfelsia nitida and Atropa bella-donna were provided by the Fairchild Tropical Garden,Miami, Florida. Plants were grown at the StationFederale de Recherches Agronomiques, Centre desFougeres, in Conthey, Switzerland.

3.3. Extraction

Air-dried and powdered plant material (20 g) wasexhaustively extracted using Ultra-Turrax1 homo-genisation with water/methanol (80/20, v/v) for 30 min.After centrifugation, the supernatant was evaporated todryness and applied to a cation exchange column(Dowex 50W X8-400 H+ form). The column waswashed with water to remove non-binding con-taminants. Calystegines and bound compounds wereeluted with 2 N NH4OH. The concentrated residue wasapplied to an anion exchange column (Amberlite IRA-400 Cl� form) and eluted with water to obtain a purifiedplant extract.

3.4. GC–MS procedure

GC–MS experiments were carried out essentially asdescribed by Drager (1995) without further validationfrom our side. An HP 5890 series II gas chromato-graph and a HP 5972 mass selective detector (Hewlett-

Fig. 4. Calystegine chromatogram from Hyoscyamus albus leaf extract.

458 K. Bekkouche et al. / Phytochemistry 58 (2001) 455–462

Fig.5.MassspectraofcalysteginesN1(a),X1(b)andX2(c).

K. Bekkouche et al. / Phytochemistry 58 (2001) 455–462 459

Fig.5(continued).

460 K. Bekkouche et al. / Phytochemistry 58 (2001) 455–462

Packard) in the EI mode at 70 eV were used. Heliumwas used as carrier gas at a flow rate of 1 ml/min. Theinjection was performed in splitless mode at 250 �C andthe injected volume was 1 ml. The column was a HP5-MSfused silica capillary (30 m � 0.25 mm i.d.) coated witha phenyl-methyl silicone phase (film thickness 0.25 mm).

The temperature program was isothermal 100 �C for 5min, 100–270 �C at 10 �C min�1 and isothermal 270 �Cfor 5 min. GC–MS interface was heated at 280 �C. Full-scan mode was used and the run time was 25 min.

3.4.1. DerivatizationIn order to analyse calystegines by GC, a derivatiza-

tion procedure was applied to form trimethylsilyl (TMS)ether derivatives, according to the procedure describedelsewhere (Drager, 1995). The reference compounds orthe purified plant extract were dissolved in pyridine (1ml) and mixed with hexamethyldisilazane (HMDS) andtrimethylchlorosilane (10:1) to 2 ml and kept at 60 �Cfor 30 min. After evaporation to dryness under nitrogenat 50 �C, the residue was dissolved in heptane. Anoctadecane stock solution in heptane was added to thesamples as internal standard to a final concentration of100 mg/ml. The solutions were filtered through a 0.45mm microfilter and 1 ml of each solution was subjectedto GC–MS.

3.4.2. Calibration curves and quantitationCalibration curves, reporting peak area ratio as a

function of silylated calystegine concentration, wereestablished between 10 and 200 mg/ml, in the presence of100 mg/ml of octadecane as internal standard. No cali-bration curve was performed for calystegine N1 whichwas only available in a small amount. In the studiedconcentration range, linearity was demonstrated withcorrelation coefficients higher than 0.992 for all theselected compounds (Table 2). The limit of detection(LOD), defined as the lowest concentration of analytethat can be clearly detected, is estimated as three times

Fig. 5 (continued).

Fig. 6. Suggested fragmentation patterns of calystegines N1 and X1.

K. Bekkouche et al. / Phytochemistry 58 (2001) 455–462 461

the signal-to-noise ratio. LOD values were 2 mg/ml, forA3 and A5 and 1 mg/ml for all other standards giving alimit of quantification (LOQ) values of 6 mg/ml for A3

and A5 and 3 mg/ml for all other calystegines (Table 2).

Acknowledgements

The authors are grateful to Dr. N. Asano, Faculty ofPharmaceutical Sciences, Hokuriku University, Kana-zawa, Japan for his kind gift of calystegines, and K.B.acknowledges the Swiss Government (CommissionFederale des Bourses) for the award of a research fel-lowship during the development of this work.

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Table 2

Regression data for the calibration curves

Calystegines

A3 A5 B1 B2 B3 B4 C1

Range (mg/ml) 10–200 10–200 10–200 10–200 10–200 10–200 10–200Line y=0.4944+

0.0343 xy=�0.2159+0.0195 x

y=�0.2071+0.0344 x

y=�0.1795+0.0372 x

y=�0.28670.0364 x

y=�0.3910+0.0405 x y=0.0574+0.0318 x

Determinationcoefficient (r2)

0.9943 0.9927 0.9927 0.9979 0.9930 0.9942 0.9928

LOD (mg/ml) 2 2 1 1 1 1 1LOQ (mg/ml) 6 6 3 3 3 3 3

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