Toxicity of Palicourea marcgravii: Combined Effects of Fluoroacetate, N-methyltyramine and 2-Methyltetrahydro-ß-carboline Wilhelm Kemmerling Chemisches Institut der Tierärztlichen Hochschule Hannover, Bischofsholer Damm 15, 30173 Hannover, Bundesrepublik Deutschland Z. Naturforsch. 51c, 59-64 (1996); received November 7/Dezember 4, 1995 Palicourea marcgravii, N-methyltyramine, 2-Methyltetrahydro-ß-carboline, co-Fluorofatty Acid, Fluoroacetate Feeding experiments carried out with cattle and horses could prove the toxic effects of P. marcgravii (Rubiaceae) in all cases. The typical symptoms of “sudden death”, however, are observed in ruminants only. This difference could not be explained so far. Apart from fluoroacetate, two more substances also have influence the toxic effects and have been isolated from P. marcgravii for the first time: N-methyltyramine and 2-methyltetra- hydro-ß-carboline (2-Me THBC). Structure elucidation of these compounds is mainly accom­ plished by “H-NMR, I 3C-NMR and MS techniques. Due to the small quantity of fluoroacetate (5.4 jig/g plant), the main toxic effect obviously lies in the two discovered substances. In contrast to the slow death of horses (monogastriers), the “sudden death syndrome” of cattle (ruminants) can be explained as a result of the higher resorbility of these two substances in the gastro-intestinal system. Given orally, both substances influence the monoamine oxidase type A (MAO-A): N- methyltyramine acts as a competitive substrate, and 2-Me THBC is one of the most effective MAO-A-inhibitors. Thus, the decomposition of the specific MAO-A-substrates noradrenaline and adrenaline as well as of N-methyltyramine itself is inhibited. The a- and ß-receptors of the sympathetic system are stimulated more strongly, which leads to a drastic rise in blood pressure and thereby to a more rapid distribution of fluoroacetate in the body. This results in a reinforced input of fluoroacetate in the cells of especially active organs of the body (heart etc.). Thus, even smaller quantities of fluoroacetate are lethal.

Introduction Thereby the citrate cycle is blocked (Metzler, P. marcgravii (Rubiaceae) is a poisonous plant 1977). which is endemic in the Amazonian area. The The quantities of fluoroacetate are relatively plant causes the “sudden death” of cattle (Döber- low, amounting to 5.4 ^ig per g dried plant (Krebs, einer and Tokarnia, 1986), which is described as Kemmerling and Habermehl, 1994). Subcutaneous injection of sodium fluoroacetate yields a LD 50 follows: it takes only 1 - 1 0 min from the appear­ ance of the first symptoms until death takes place. value of 200 ^ig/kg in rats (Gribble, 1973). There are signs of cardiac insufficiency such as Experimental poisoning of horses (Tokarnia et positive venous pulse and muscle tremor. The ani­ al., 1993) does not lead to the symptoms of “sud­ mals lie down or fall on their belly or side. den death” as in the case of cattle. Main symptoms Tachypnoe, rowing movements with their legs, are nervous manifestations as well as intensive opisthotonus and finally death occur. sweating, restlessness, abrupt involuntary move­ Until now, fluoroacetate has been considered to ments of the head or the whole body. The clinical be the only cause. The mechanism is well-known: course of the poisoning ranged from 10 to 43 In the organism fluoroacetate is transferred to flu- hours. It may be concluded that more substances orocitrate which is irreversibly bound to aconitase. must be responsible for the toxic effects.

Material and Methods

Reprint requests to Dr. Wilhelm Kemmerling, Widu- The mass spectra were taken on a Finnigan kindweg 15, 34431 Marsberg. spectrometer MAT 312. Measurements were

0939-5075/96/0100-59 $ 06.00 © 1996 Verlag der Zeitschrift für Naturforschung. All rights reserved. D 60 W. Kemmerling • Poisonous Effects of Palicourea marcgravii taken with an ionization potential of 70 eV. The The combined diethylether phases were reex­ signal intensities were given as percentages of the tracted twice with 300 ml fresh 1 n H 2 S 0 4 each. basal peaks. The temperature is given in degrees The ether phases were set aside and the acid aque­ centigrade. ous phases were combined. The 'H-NMR spectra were taken on a Bruker The acid aqueous phases (1.4 1 approx.) were spectrometer AM 300 in combination with tetra- stirred with 5 g of zinc powder in a glass beaker methylsilane as internal standard. CDC1 3 or for 24 hours with a magnetic stirring device. CD 3 OD respectively served as solvent. The signals The surplus of zink powder was removed by fil­ were characterized by their multiplicity (s = singu- tering, and the acid phase was adjusted to pH 12 let, d = doublet, t = triplet), coupling pattern (/ in with N H 3 (conc.). 15 g NaCl were dissolved per 100 Herz) and integration. ml extract. The alkaline extract was extracted four The 13C-NMR spectra were taken on a Bruker times with 700 ml CH 2 C12. spectrometer AM 300 using tetramethylsilane as The combined CH 2Cl2-phases were dried over internal standard. CDC1 3 or CD3OD respectively Na2 S 0 4 and the solvent was removed in a rotation served as solvent. Measurements were taken ac­ evaporator. The extraction yielded 1.4 g dark red- cording to the DEPT-method. The chemical shifts brown, thick alkaloid crude extract. are given as 6 -values in ppm. For the analytic thin-layer chromatography, pre­ Enrichment o f the substances from the fabricated foils by MERCK (Kieselgel 60 F 254) alkaloid crude extract were used. Dragendorff-reagent was used as a spray rea­ The alkaloid crude extract (1.4 g) was chromato­ gent for the thin-layer chromatography. graphed on a silica gel column (diameter 45 mm, The column chromatography was performed 140 g silica gel), with CHCI 3 : MeOH : NH 3 (conc.) with silica gel (Flashgel) by Baker, grain diametre 80 : 10 : 1. The fractions in the range of RF 0.23 (N- 0.02-0.036 mm, with low pressure (“flash”). The methyltyramine) and RF 0.68 (2-Me THBC) were solvent mixtures are given with the data. collected separately. The thin-layer control was The solvents were applied dried and distilled. performed by Dragendorff spray reagent. The al­ kaloids showed an intensive yellow colour on TL. Plant material This process yielded crude extracts of 0.62 g N- The plants (leaves) were collected in Brazil, methyltyramine and 0.21 g 2-Me THBC. State of Para. The collecting date wasn't given. The leaves were dried and ground. Isolation of N-methyltyramine The crude extract of N-methyltyramine (0.62 g) Extraction of the leaves from P. marcgravii was chromatographed on a silica gel column (dia­ 400 g leaves were extracted with 4 litres of pet- m eter 25 mm, 40 g silica gel) with CHC1 3 : M eO H rol-ether (four days) and subsequently 4 litres of 80 : 10. It was eluated until all nonalkaloid sub­ methylene chloride (four days), by aid of a heating stances were removed (TLC control). Subse­ device, a 6 litre round beaker with 2 0 0 0 ml soxhlet quently N-methyltyramine was eluated with and reflux cooler. The PE and CH 2 C12 extracts CHCI3 : MeOH : NH 3 (conc.) 80 : 10 : 1. This pro­ gained were put aside. The plant material was cess yielded 520 mg crystalline substance. dried (removing rests of CH 2 C12) and then ex­ tracted with 4 litres of methanol for four days. In Isolation of 2-Me THBC this way, 1 2 0 0 g leaves were extracted altogether. The methanol was removed by evaporation and The crude extract of 2-Me TH BC (0.21 g) was 160 g of brown, thick extract were yielded. chromatographed on a silica gel column (diameter 25 mm, 20 g silica gel) with CHCI3 : MeOH 80 : 5 Production of alkaloid crude extract until all non-alkaloid substances were removed 160 g M eO H -extract were suspended in 1000 ml (TLC control). Subsequently, 2-Me TH BC was elu­

1 n H 2 S 0 4 (with ultra sound). Extraction was per­ ated with CHCI3 : MeOH : NH 3 (conc.) 80 : 10 : 1. formed twice with 500 ml diethylether each. This procedure yielded 51 mg crystalline substance. W. Kemmerling • Poisonous Effects of Palicourea marcgravii 61

All in all the yields of the alkaloids were 42.5 MS, 110°C: M+ = 186 (20.5), 143 (100), 128 (5), (uig 2-Me THBC and 433.3 jig N-methyltyramine 116 (10.2), 115 (15.4), 77 (7) (see Fig. 2). per g dried leaves. The isolation was performed several times. It could be observed that the amount of N-methyl- tyramine in the plant material was swaying. The N-Methyltyramine highest amount was 433.3 ^g. 13C-NMR (see Figs 1 and 3); Spectroscopic data ■H-NMR, 300 MHz, CDC13: 7.0 (d, 2H-2,6, J = 9 Hz), 6.7 (d, 2H-3,5, J = 9 Hz), 2.75 (t, 2H, J = 6 2-Me THBC Hz) and 2.85 (t, 2H, J = 6 Hz; -CH 2 -CH 2-), 2.4 (s, 13C-NMR (see Figs 1 and 2); 3H; N-CH3), CD3OD two exchangeable H (-OH, *H-NMR, 300 MHz, CDC13: 7.47 (d, 1H-Ha, J = -N H -CH 3); 8 Hz), 7.3 (d, 1H-Hb, 7 = 8 Hz), 7.12 (m, 2H -H C),MS, 120°C: M+=151 (100), 135 (5.1), 121 (13.1), 3.57 (s, 2H, A r-CH 2-N (-), 2.81 (m, 4H, 120 (11.4), 108 (38), 107 (59.3), 91 (16.3), 77 (47.8) A r-C H 2 -CH 2 -N 0 , 2.48 (s, 3H, N -CH 3); (see Fig. 3).

C H 3 35! H— N I a C H 2 54 257

ß C H 2 35.560 21.25 52.15 131.270 130.572 130.572 Fig. 1. 13C-NMR N-methyltyramine. : 6 ' 2-Methyltetrahydro-ß-carboline 116.464 116.464 (2-Me THBC). The numbers next to the 157.177 C-atoms show the chemical shift in ppm (75 MHz, DEPT, 2-Me THBC in OH CDC13, N-methyltyramine in CD 3OD).

- i - H 1

—

100 80 PPM Fig. 2. Spectra of 2- methyltetrahydro-ß- carboline Mass Spectrum (110°C) 1 3C-NMR (75 MHz, DEPT, Solvent CDCI 3) (2-Me THBC). 62 W. Kemmerling • Poisonous Effects of Palicourea marcgravii

-9-H

Solv

120 140 160 160 140 120 100 MASS UNIT Fig. 3. Spectra of Mass Spectrum ( 120°C) 13C-NMR (75 MHz, DEPT, Solvent CD 3OD) N-methyltyramine.

Results and Discussion nonalkaloidal compounds can be eluated, whereas The plant material is freed from unpolar parts the alkaloids stay bound. by the extraction with petrol-ether and methylene Afterwards the alkaloids are isolated in pure chloride. Afterwards, extraction is continued with form with a solvent containing ammonia. methanol. The extracts gained by this procedure N-methyltyramine causes, among other effects, also contain the alkaloids. positive venous pulse and tachypnoe (guinea pigs) The enrichment of the two alkaloids is based on and “wobbling legs” (sheep and goats). These acid-base separation. In the acid medium they ex­ symptoms found by Evans et al. (1979) are the ist as cations showing a high polarity. same like those described in cattle by Tokarnia The MeOH extract of the plants suspended in et al. (1986). P. marcgravii also contains 2-Me THBC in addi­ 1 N H 2 S 0 4 can be separated without any difficulty from the more unpolar parts of the extract by aid tion to N-methyltyramine. Both alkaloids have of diethylether. The alkaloids stay in the acid enormous effects on the physiology of an animal aqueous phase. The reduction with zinc sets the organism and potentiate the poisonous effect protoned alkaloids free again from the partly (Fig. 4). formed N-oxides. The isolated 2-Me THBC is one of the most ef­ Now the pH is adjusted to 12 approximately, by fective MAO-A inhibitors (Meller et al., 1977), i.e. the decomposition of adrenaline and noradrena­ means of NH 3 (conc.). The N-methylated amino groups of the alkaloids are then uncharged. In ad­ line is inhibited. The MAO catalyzes the oxidative dition, the enrichment of the aqueous phase with desamination of primary and secondary amines. NaCl leads to a higher volatility of the substances. There are two types of MAO: Type A and type B Therefore, they can be extracted with methylene have different substrate specifity. The neurotrans­ chloride. mitters adrenaline and noradrenaline are specific The isolation of the substances from the alkaloid type A substrates. This has been proved by Singer crude extract must be carried out in two steps by et al. (1979) with enzyme models. column chromatography. The two alkaloid frac­ The second isolated substance, N-methyltyra­ tions of the first column chromatography also con­ mine, is also a MAO-A substrate (Singer et al., tain nonalkaloidal substances (apart from the al­ 1979). It competes with adrenaline and noradrena­ kaloids) with the same /?F-value. line for the active centre of the MAO-A. Thus, the Therefore in the second column chromatogra­ activity of the MAO-A is “blocked” in two ways: phy no ammonia is used for isolation purposes. 1) The MAO-A inhibitor 2-Me THBC lowers Because of the weak acidity of the silica gel the the activity. W. Kemmerling • Poisonous Effects of Palicourea marcgravii 63

Activation of a- and ß- Process of the Pal.marc. (feed) Receptors of the Sym-_ Citrate Cycle in the with pathetic System Cells of Heart, • N-Methyltyramine lood Vessels • 2-Me THBC Synthesis ot \ e • Fluoroacetate N-Methyloctopamine ©

Digestive Processes Constriction of 0 Heart Inhibition of the Blood Vessels Frequency r Decomposition of Animal Organism with MAO-A Substrates: Adrenaline, N-Methyltyramine Noradrenaline 0 2-Me THBC ^Synthesis of Fatty Acids: Fluoroacetate 0 integration of Fluoroacetate in Adipositas Tissue ® (“Decontamination”) Rise in Blood Pressure

0 Distribution of 0 Infiltration of Fluoroacetate Fluoroacetate ^ esp. in the Citrate Cycle of _ in the Body Cells of Particulary Active Organs______

Meaning of the Arrows: ------® ► the more ... the more ------^ ► the more ... the less the less... the less Fig. 4. Pattern of the physiology of the poisonous effect.

2) N-methyltyramine serves as a competitive The rise in blood pressure causes an increase in substrate. the energy demand in the cells of heart and blood Both effects result in a strong rise of the concen­ vessels. The adrenergic stimulation of the citrate trations of the MAO-A substrates adrenaline and cycle is opposed by the inhibition caused by fluor­ noradrenaline in the blood. This leads to a oacetate. Under these conditions the latter is dis­ stronger stimulation of the a- and ß-receptors of tributed rapidly in the body and transformed to the sympathetic system and thereby to a dramatic aconitase-blocking fluorocitrate. This preferably rise in blood pressure and decrease of the diges­ happens in places of the highest consumption of tive activity. Thus, N-methyltyramine works as an indirect sympathomimeticum. This is proved by HN — CH3 HN— CH3 HN — CH3 tests performed with heart cells culture (Kemmer­ c h 2 c h 2 c h 2 ling, 1995). c h 2 CH-OH CH-OH But it is also conceivable that it works as a direct I I sympathomimeticum. N-methyltyramine was trans­ formed from dopamine-ß-hydroxylase (DBH) to fl *^^1 DBH N-methyloctopamine with 25 % of the dopamine - n rS activity (Fujita et al., 1971) (see Fig. 5). T ~ / T N-methyloctopamine differs from adrenaline VOH ' OH OH only in one hydroxyl group in position 3, and it N-Methyltyramine N-Methyloctopamine Adrenaline possibly has the same effect. Fig. 5. Reaction of dopamine-ß-hydroxylase (DBH). 64 W. Kemmerling • Poisonous Effects of Palicourea marcgravii energy, i.e. for example in the heart. Whether ized. i.e. they are more unpolar and therefore re­ death is caused by high blood pressure, inhibition sorption is better. The presence of alkaloids is re­ of the citrate cycle or a combination of both, de­ sponsible for the lethal effect of even smaller pends on the respective doses of the substances in amounts of fluoroacetate in cattle. the plant and their resorbility in the gastrointesti­ The strong acid pH in the stomach of horses is nal system of the grazing animals. responsible for a protonisation of the alkaloids The isolated alkaloids allow to explain the dif­ which are therefore resorbed to a lesser degree. ferences in the poisonous effect on ruminants That is why death is delayed. (cattle) and monogastriers (horses) for the first A fourth substance group participating in poi­ time. In this way, cattle die of “sudden death” (To- sonous effect are oo-fluorofatty acids. This could be karnia, Döbereiner, 1986) whereas horses die after proved by a new extraction method followed by a 10-43 hours (Tokarnia et al., 1993). W hen con­ 19 F-NMR-examination (Kemmerling, 1995). sidering similar doses, this difference can only be explained by the different resorbility in the gastro­ intestinal systems. Acknowledgements The ruminant system causes a better digestion Thanks are conveyed to Dr. Weisser, Zentrum of the food. Furthermore in the rumen, reticulum Biochemie, Medizinische Hochschule Hannover, and omasum the alkaloids are mostly deproton- for fruitful discussions.

Döbereiner J. and Tokarnia C.H. (1986), Poisoning of Krebs W.C., Kemmerling W. and Habermehl G. (1994), cattle by Palicourea marcgravii (Rubiaceae) in Bra­ Qualitative and quantitative determination of fluoro- sil. Pesq. Vet. Bras. 6, 73-93. acetic acid in Arrabidea bilabiata and Palicourea Evans C.S., Bell E.A. and Johnson E.S. (1979), N- marcgravii by 19F-NMR spectroscopy. Toxicon 32, methyltyramine, a biologically active amine in acacia 909-913. seeds. Phytochemistry 18, 2022-2023. Meller E.. Friedmann E., Schweitzer J.W. and Friedhoff Fujita T. and Ban T. (1971), Structure-activity study of A.J. (1977), Tetrahydro-ß-carbolines: Specific inhibi­ phenethylamines as substrates of biosynthetic en­ tors of type A monoamine oxidase in rat brain. J. zymes of sympathetic transmitters. J. Medic. Chem. Neurochem. 28. 995-1000. 14/2. 148-152. Metzler D. (1977), Biochemistry. The chemistry of the Gribble G. (1973), Fluoroacetate Toxicity. J. Chem. living cell. Acad. Press, 528 Educat. Vol. 50, Num. 7, 460-462. Singer T.P., Von Korff R.W. and Murphy D.L. (1979), Kemmerling W. (1995), „Sudden Death“: Charakteri­ In: Monoamine Oxidase: Structure, Function and Al­ sierung der verursachenden pflanzlichen Toxine und tered Functions. Academic Press, 116-117, 405. Klärung ihrer Wirkweise. Dissertationsschrift Uni­ Tokarnia C.H., Costa E.R. and Diomedes J. (1993), Ex­ versität Hannover. perimental poisoning of horses by Palicourea marc­ Kemmerling W. (1995), Palicourea marcgravii: „Gift­ gravii (Rubiaceae). Pesq. Vet. Bras. 13, 67-72. cocktail“ gegen Freßfeinde. Biologie in unserer Zeit. Verlag Chemie, 69469 Weinheim, Nr. 5, 307-313.