Uptake of host plant alkaloids by root parasitic Pedicularis species

Phytochemwy, Vol 29, No 6, pp 1811-1814, 1990 Prmted m Great Brltam

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0031-9422/90 $300+000 1990 PergamonPress plc

UPTAKE OF HOST PLANT ALKALOIDS BY ROOT PARASITIC PEDICULARIS SPECIES MARILYN J. SCHNEIDER*

and

FRANK R. STERMITZ~

Chemtstry Department, Wellesley College, Wellesley, MA 02181, U S A, tDepartment of Chemtstry, Colorado State Umverstty, Fort Collins, CO 80523, U S.A (Recezvedzn rewed form 8 November 1989) Key Word Index-Pedzcularzs, Scrophulanaceae, alkalotds

Pzcea engelnzannzz; Thermopszs;Seneczo; Lupznus,parasmsm;

Abstract-Five species of Pedicularis were examined for evidence of alkaloid transfer from host plants. Pedicularis~ groenlandica and P. bracteosa were found to take up the pyrrolizidine alkaloid seneciomne from the host Seneczo trzangularis. Pedicularzs crenulata contained anagyrme from Its host Thermopszs montana and P. grayi contamed Nmethylcytisine from its Thermopszs divarzcarpa host Pedicularzs racemosa contained quinolizidmes from a Lupinus argenteus hybrid In addition, P bracteosa was found to contam pimdmol, taken up from the host Pzcea engelmanniz

INTRODUCTION

Pedicularis (Scrophulariaceae) is a very large genus of ca 400 species of chlorophyll-containing hemiparasitic herbs. Some 60 species occur in the New World. Pedicuha-is, as root parasites, have the potential to take up secondary metabolites, such as alkaloids, from their hosts. The phenomenon of alkaloid transfer from host plant to parasite has been observed for several species of the related genus Castilleja [l]. Recently, Pedzcularzs semibarbata was found to take up quinolizidine alkaloids from its host Lupinusfulcratus [Z]. Alkaloid transfer from a legume host might account for the reported isolation of N-methylcytisine (3) from Pedicularis algae [3] and P. dolichorrhiza [4]. Pedicularzs zs sometimes used m herbal teas or medicines under the names ‘betony’ or ‘wood betony’ [S], although these names are also used for Stachys. It has been stated [S] that large quantities of Pedicularzs could be harmful and that “the potency of the various species is variable” Secondary metabolite transfer from different, perhaps even toxic, host plants could account for the harmful effects and variabihty. The object of the present research was to determine whether alkaloid transfer is a general phenomenon in Pedzcularis by investigating possible alkaloid uptake from additional host plants. RESULTS AND DISCUSSION

In order to study alkalotd transfer to Pedzcularzs, montane sites were chosen where alkaloid-containing species and Pedicularzs were found growing in close association. Potential host/parasite plant combinations were identified and leaf samples were subjected to a field test for alkaloids In this manner five host/parasite systems were identified (Table 1). In each case, individual Pedicularis plants not growmg near alkalotd-containmg *Author to whom correspondence should be addressed

plants were examined and found to be free of alkaloids. At Cameron Pass, in north central Colorado, Seneczo triangularis grows together with P. bracteosa and P. groenlandica along the edge of a swampy area. Seneczo trzangularzs contains the pyrrolizidine alkaloid senecionine 1, present as its N-oxide. Senecionme and/or senecionme Noxide were found m the parasitic P. bracteosa and P. groenlandica samples. These results represent the first report of pyrrolizidine alkaloid transfer to a parasitic Pedicularis. At Michigan Hill, in central Colorado, Thermopszs montana grows with P. crenulata in a sedge meadow, while T. divaricarpa and P. grayi are found in a nearby aspen grove. Both T. Montana and T dzvaricarpa contam quinohzidine alkaloids. Alkaloids previously reported from T montana mclude N-methylcytisine, cytisme, CItsolupanme, thermopsine, anagyrme and hydroxylupanine, [6,7] although the amounts of each alkaloid may vary throughout the growing season [6] The major alkaloid found m T. montana above ground and root extracts was anagyrine 2 The major alkaloid in the parasitic P crenulata was also found to be anagyrme. Alkaloids previously reported from T. divarzcarpa include anagyrme, baptifohne, cytisine, N-methylcytisme, rhombtfoline (GC-MS evidence) and N-formylcytisine [8] In our study, the major alkaloids found in above ground parts of T. dzuarzcarpa were N-methylcytistne (3), rhombifoline (4), thermopsme (5) and anagyrme (2) The only major alkalotd in T diuaricarpa roots was Nmethylcytisine (3), matching the 3 isolated from the parasitic P. grayz. This finding suggests that only alkaloids present in the roots may be transferred to the parasitic Pedicularzs. A similar result was obtained in a related greenhouse study of Castilleja zntegra parasitism of Oxytropzs serzcea The mdohzidine alkalotd swainsonine, present only in the above ground parts of 0. sericea, was not transferred to parasitic Castzlleja. At Lost Trail Pass, in western Montana, a Lupznus argenteus hybrid grows with P. racemosa The Lupinus

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M J SCHNEIDER and F R STERMITZ

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1 Pedlcularrsfiost plant combmatlons

Table Pedzcularls sp

Host plant

MaJor alkaloid

P hracteosa P. groenlandlca P crenulata P gray P racemosa

Seneclo trlangularls Seneclo trlangularls Thermopsrs montana T dwarrcarpa Luprnuy argenteus hybrid

Senecionme (1) Senecronme (1) Anagyrme (2) N-Methylcytisme (3) Tetrahydrorhombtfohne Lupanine (or stereoisomers)

a mtxture of several alkaloids, with tetrahydrorhombifolme and lupanine (or their stereoisomers) as maJor components. Parasitic P racemosa plants appear to contain the same alkaloids. A sixth host/parasite system was discovered with P bracteosa which had been collected under Engelmann spruce (Ptcea engelmannir) The pmtdmol, 6, was isolated from Pedicularis bracteosa and characterized by its ‘H and ’ 3C NMR and mass spectrum. Outstanding characteristics of the proton NMR of thts compound m&de two methyl doublets at 6 1 04 and 1 16, and three downfield proton signals at 62 60,2.95 and 4 13 A 13C DEPT NMR experiment revealed the presence of three methine, four methylene, and two methyl carbons. COSY and HETCOR experiments established the connectivity and assignments for key sections of the molecule The above informatton together wtth M, (157) determined from the ammoma chemtcal ionizatton mass spectrum established the structure of the alkaloid as the pimdmol, 6 Alkaloid 6 1s known as a synthetic intermediate [9], but has not prevtously been isolated as a natural product Investtgation of spruce needles, wood/bark and root samples mdtcated that 6 was present in the trees as well. A few alkalotds such as pmidme 7 have been isolated from Ptnus species [lo], but this 1s the first report of an alkaloid isolated from Pzcea Picea engelmannii also contams other alkalotds, but the pmtdmol6 1s clearly the maJor alkaloid transferred to Pedularzs bracteosa The tdenttttes of these alkalotds as well as the absolute configuratton for 6

wtll be reported elsewhere It 1s interesting to note that in one indtvtdual plant collected next to S. triungularw and along the edge of a lme of Ptcea engelmannzl, the crude alkaloid proton NMR showed the presence of senectonine, but also the two sharp doublets at 61.04 and 1 16 charactertsttc of 6. The occurrence of both senectonine and 6 suggests that a smgle Pedtcularu plant can parasitize two different alkaloid-containing hosts Thts work clearly indicates that uptake of alkalotds by Pedlcularu 1s a fairly widespread phenomenon, the ecologtcal import of which 1s currently under study It 1s highly likely that the prevtous reports [3, 41 of qumohztdme alkaloids m Pedzculurts arise through root parasitism on a qumohztdme-contammg host. In terms of herbal tea use of Pedvxdarzs, It should be noted that senectonme has very recently [l l] been tmphcated m a case of hepatic veno-occlustve disease, anagyrme has been tmphcated as a teratogen [ 121 and a number of ptpertdme alkaloids have a variety of potent btologtcal acttvtty [13].

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EXPERIMENTAL ‘H and ‘%NMR Bruker ACE-300 MHz GC-MS PerkmElmer Sigma 3 gas chromatograph and VG MM16 mass spectrometer with a VG datasystem 2000 The mass spectrometer was operated m the EI mode at 70eV GC column J & W DB-5 30 meter capillary column, InJector temp 320”, column temp program 180” (3 mm), heating rate 5” mu- ’ to 300”, He carrier gas Plant materml Plant collections are listed m Table 2 Identlfications were by D H Wdken (Department of Biology, Colorado State Umverslty), except for the Lupmus argenteusx L caudatus hybrid, which was identified by D Dunn (Department of BIOlogy, Umverslty of Mlssourl, Columbia) This taxon has also been designated [14] L x alpestrls Alkaloidfield test A small plant leaf was crushed m a mixture of 3 2 CHCI,-MeOH (1 ml) and 1% NaHCO, (1 ml) The organic phase was dried by filtration through Na,SO, and evapd TLC (slhca gel, 3 2 CHCI,-MeOH) and iodoplatmate spray reagent were used to detect alkaloids General alkalord rsolatums Unless otherwlse stated, above ground plant parts were used A dried and ground plant sample

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Uptake of alkaloids by root parasites

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Table 2. Pedicularis and host plant collections

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Species

Population

Voucher No.

P. bracteosa Pennel P. groenlandica Retz. P. crenulata Benth. P. grayi A. Nels. P. racemosa Dougl. ex Hook Senecio triangularis Hook Thermopsis montana Nutt. ex T. & G. T. divaricarpa A. Nels. Picea engelmanni Parry Lupinus argenteus Pursh x L. caudatus Kell. (or L. x alpestris A. Nels.)

Cameron Pass Cameron Pass Michigan Hill Michigan Hill Lost Trail Pass Cameron Pass Michigan Hill Michigan Hill Cameron Pass

csu CSU CSU CSU FRS CSU CSU CSU CSU

Lost Trail Pass

FRS 364

7

was extracted with MeOH at room temp. for one to several days. After evapn of the MeOH, the residue was taken up in 1 M HCI and extracted with hexane. The aq. phase was made basic (K&O,) and extracted with CHCI,. Drying (Na,SO,) and evapn of the CHCI, layer gave a crude alkaloid mixture. Isolation of senecionine from Pedicularis bracteosa and P. groenlandica. The general alkaloid isolation procedure was followed except that the aq. HCI phase, before basification, was treated with Zn dust for one day and then filtered. One P. bracteosa plant (1.2 g dry wt) gave 9 mg of crude alkaloid. Two P. groenlandica plants (1.6 g dry wt) gave 4 mg of crude alkaloid. This sample was further purified by analytical TLC (silica gel, development and elution with CHCl,-MeOH 3 : 2 to give 2 mg of purified alkaloid. ‘H NMR spectra of alkaloid samples from each plant matched that of authentic senecionine. Isolation of alkaloids from Pedicularis crenulata and P. grayi. The general alkaloid isolation procedure was followed. Two P. crenulata plants (1.77 g dry wt) gave 4.5 mg of crude alkaloid mixture. One P. grayi plant (21.6 g dry wt) gave 26 mg of crude alkaloids. The alkaloid mixtures obtained were subjected to GCMS to identify major components. GC-MS of the major component of P. crenulata and T. montana matched that for authentic anagyrine in R, and fragmentation pattern. GC-MS of the major component of P. grayi and T. divaricarpa roots matched that for authentic N-methylcytisine in R,s and fragmentation pattern. Isolation ofalkaloidsfrom Pedicularis racemosa and Lupinus argenteus hybrid. The general alkaloid isolation procedure was followed. L. argenteus hybrid (5.41 g dry wt) yielded 90 mg of crude alkaloids. P. racemosa (5.21 g dry wt) yielded 9.5 mg crude alkaloids. The alkaloid mixtures obtained were analysed by GCMS. The two major alkaloidal components of P. racemosa matched those of L. argenteus hybrid in R,s time and fragmentation pattern. Lupinus component A (R, 10.67 tetrahydrorhombifoline or stereoisomer) EIMS m/z 70 eV (rel. int.): 246 (8). 207 (95), 112 (38), 108 (19), 96 (8), 95 (20), 80 (21), 58 (100). Lupinus component B (retention time 12.6, lupanine or stereoisomer) EIMS m/z 70 eV (rel. int) 248 (44), 247 (32), 150 (39), 149 (47), 136 (lOO), 134 (36). Pedicularis component A (R, 10.53) EIMS m/z 70 eV (rel. int.) 246 (3), 207 (lOO), 112 (30), 108 (14), 58 (lOO),55 (19). (The peak at 246 in Lupinus and Pedicularis samples may be

17640 44136 17677 17676 363 8788 17678 17821 8789

due to the concomitant elution of an additional, unidentified alkaloid). Pedicularis component B (R, 12.47) EIMs m/z 70 eV (rel. int.) 248 (47), 247 (35), 150 (39), 149 (49), 136 (IOO),134 (30), 98 (28). The above data for components A and B compared favorably with literature EIMS values for tetrahydrorhombifoline and lupanine, respectively [lS]. Isolation of6from Pedicularis bracteosa. Dried and ground P. bracteosa (59.2 g) was extracted with MeOH at room temp. for 4 weeks. After evapn of the MeOH, the residue was taken up in Hz0 and extracted with Et,0 and then CHCI,. The aq. phase was made basic (K&O,) and then extracted repeatedly with CHCI,. Evapn of the CHCI, extracts gave crude 6 (111 mg) as a solid which appeared 295% pure by ‘HNMR. Alternatively, purification by vacuum liquid chromatography (l*C silica gel; H,O-MeOH gradient) prior to acid/base extraction gave near colourless 6, [a] h* = - 17” (CHCI,; c 0.99). ‘HNMR (CDCI,) 61.0 (lH, m, H-3), 1.04 (3H, d, J=6.3 Hz, H-7), 1.16 (3H, d, J ~6.1 Hz, H-lo), 1.35 (2H, m, H-3, H-4), 1.47 (lH, ddd, 5=14.4, 9.1, 3.3 Hz, H-8), 1.49 (IH, m, H-5), 1.60, (lH, ddd, 5=14.4, 5.6, 3.2 Hz, H-8), 1,60(1H,ddd,H-3), 1,85(1H,m,H-4), 2.60(1H,dqd, 5=11.0,6.2,2.6 Hz, H-2),2.95 (lH,dtd,J=8.1, 5.4,2.7 Hz, H-6), 4.13 (lH, qdd, J=9.2, 6.1, 3.1 Hz, H-9); ‘%NMR (CDCI,) 664.94 (C-9), 54.86 (C-6), 52.45 (C-2), 43.84 (C-8), 33.83 (C-3), 30.41 (C-5), 24.62 (C-4), 23.59 (C-lo), 23.03 (C-7). CIMS (NH,) m/z (rel. int.) 158 (lOO),98 (23), 159 (lo), 138 (9.5), 156 (9); EIMS [MI+ 157.1458, talc. for C9H,,N0 157.1462. This material could be recrystallized from EtOAc to give mp 70.5-72”, ref. [9] for (+)-6: 8c81”. Acknowledgements-This work was supported by grant CHE8521382 from the National Science Foundation and as a part of the USDA Science Education Administration Western Regional Research Project W-122 in cooperation with the Colorado State University Experiment Station. Wellesley College is acknowledged for partial salary support. The high resolution mass spectrum was obtained by the Midwest Center for Mass Spectrometry, University of Nebraska (NSF grant CHE-8211164). We thank R. J. Molyneux (USDA Pacific West Regional Research Center) for swainsonine analyses of Oxytropis and Castilleja samples.

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M J. SCHNEIDERand F R STERMITZ Am. Chm. SOL 77, 6.3.6.!.

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