Toxicooi, 1976, VoL 14, pp . 33-64. Per~smon Prag. Printed is Cimat Hrihin.

DUBOISIA MYOPOROIDES: NATIVE ANTIDOTE AGAINST CIGUATERA POISONING EvA liiJFVA, * G. Loisoxj~ and B. Ho1.MSrsnT* "Department of Toxicology, Swedish Medical Research Council, Karolinska Institutet, 5-104 Ol Stockhohn 60, Sweden tSouth Pacific Commission, Post Box No . D5, Noumea Cedex, Naw Caledonia (Acceptedfor publfcaüon 28 August 1975)

Eve Dvt+ve, G. Lolsox and B. Hoi.usffrtTnT . Dubofsia myoporofdes : native antidote against ciguateaa poisoning. Toxicon 14, 35--64, 1976.-The leaves of Dubofsia myoporofdes collected in New Caledonia and preserved in alcohol up to the time of analysis contained the following alkaloids: nicotine, nonnicotine, atropine and scopolamine . Various minor alkaloids reported by other authors in Australian D . nryoporoldes were not found in the plant material . The analyses were carried out with gas chromatography-mass spectrometry . The different results with New Caledonian and Australian D . myoporofdes may be due to a difference in species, as suggested by V~~~~ (Etude des tensors en scopolamine of hyoecyamine den Duboisfa ntyoporoides R. Br. cultivés en conditions art~cielles. Thesis, University of Paris, 1972). Different conditions with regard to location, soil andclimate do not seem sufficient explanation for the discrepancies. A review of the literature of ciguatera poisoning reveals that the manifestations only partly resemble those of anticholinestaase poisoning. Nevertheless, a mixture of atropine and PAM has been suggested for the treatment of the entity . In this context it is interesting to note that the natives of New Caledonia have already arrived at a powerful mixture of trepans and pyridinum alkaloids as an antidote .

of the species Duboisia found in Australia have been reviewed by BAItNARD (1952) . Of the three species known, Dttboisia myoporoides commands special interest because the natives of New Caledonia are reported to use it as an antidote in ciguatera poisoning (JAOQuiss, 1945; BARRAU, 195 . A number of chemical analytical investigations of D. myoporoldes have been carried out in this century. D. myoporoldes has been reported to wntain a large number of alkaloids, as seen from Table 1 . In view of the importance of ciguatera poisoning, it would seem to be relevant to reinvestigate the alkaloid content of D. myoporoides. Such a reinvestigation is presented in this paper together with a discussion of the entity of ciguatera poisoning. PLAxTS

MATERIALS AND METHODS

D . myoporoldes leaves were collected in July,1972, in New Caledonia. A voucher specimen is kept in

the herbarium of Orstom Noumea, undea No . 360. The fresh leaves wea+e immediately put in alcohol and shipped by air to the Department of Toxicology, Karolinska Institutet, Stockholm. Anabasino and 1norniootine were obtained from K do K, Hollywood, California. Pelletierine tannate was kindly given to ua by Docent Lembitu Reio of the Department of Chemistry, Univerity of Stockholm. The proceduro recommended by the Pltarnracopoea Nordics (1964) for the isolation of alkaloids, was followed. The powdec+ed plant material (20 g) was treated with chloroform (30 ml), to which a 1 M solution of sodium carbonate (10 mn was adckd and stirred with diatomaceous earth (10 g). The mixture was packed in a glass column (17 x 600 mm), the alkaloids eluted with 300 ml chloroform (flow rate 1 ml per min). The elutee was passed through another column of the same siu containing a mixture of diatomaceous earth (15 g) and 1 M phosphoric acid (2 ml). The alkaloids were eluted with chloroform saturated with ammonia (230 ml) and pas .~ed through a cohmm (17 x 80 mm) containing aluminium oxide (10 g) (flow SS TOXlCON 1976 Yol. I~

56

EVA DUFVA, G. LOISON and B. HOLMSTEDT

C4

~

q g

a~ ~.g

~

p,

â

g .~ g

~

q

r,

r,

ôâ

., ô ô

û °af

7Y)XfCON 1976 Vol. If

.,

~~

~ ..

..

..

Duboisia and Ciguatera

57

rate 1 ml per min). The chloroform extract was dried with anhydrous sodium sulphate, filtered and evaporated to dryness. In another experiment, the dried non-powdered leaves (12 g) we1+e treated with water (120 ml) at 50° for 24 hr. The water phase was filtered off and prepared for analysis, according to F1sx et aJ. (1955) . Ges chromatographic analysis was performed with a Varian Model 2100 apparatus, equipped with a hydrogen Same ionization decoction system. The stationary phase was 3~ 5E-30 (ultraphase) on a column support of Gas Carom Q 100-120 mesh. The dimension of the glass column were 2"25 x 3"2 mm, i.d . The carrion gas (nitrogen) flow was 25-30 ml per min. The igjector and detector temperature was 250° . The column eemperaturo was at first programmed from 70 to 275° at 4° per min and then at shorter intervals (e.g . fmm 90 to 120, to make sure that no peaks were lost. In quantitation of the alkaloids, the column temperature was kept at 110° for the nicotine alkaloids and 220° for the tropane alkaloids. Quantitation was carried out by comparison with peak heights of reference compounds iAjected under the same conditions . The principles of the technique for gas chromatography-mass spectrometry (GC-MS) have bcen deacrfbod earlier (Ho1~srEVr and I,nanox~r, 196'n. The MS work was carried out with an LKB 9000 gas chromatograph-mass spaKromcter (LKB-Produkten, Bromma, Sweden). The ion source temperature was 270°, the electron energy was 70 eV and the electron ionization current 60 pA. Tho column packing was 3 5E-30 (ultraphase) on 100-120 ash Gas Chrom Q. Column temperatures were similar to those for the GC analysis . With a computer i(PDP-12, Digital Equipment Corp ., Maynard, Mass., U.S .A .), interfaced with the T.KR 9000, the spectra from the mass spectrometer wero recorded on tape and plotted. Mass fragmontography was carried out with the same equipment, using the set-up and the program descrl~ed by E1.ß et al. (1973) . The computer is interfaced with the mass spectrometer and is set to adjust the aeoelerating voltage so that m/o 140, 148, 154, 162 are brought into focus and recorded . The mass fragmeatograms were obtained with temperature programming as described under GC. Synthetic mixture

3 G

Duboisia myoporoides

6

5 4

Fix. l. Gas c111eoaew~roox,~ : (agoolensa ca~utr arBEn S mm per min) . Peak 1 nicotine RT = 2"S cm . Peak 2 norniootine RT = 3"55 cm . Peak 3 anabasine RT = S"1 cxn. Peak 4 apoatropine RT = 11 "2 cm. Peak S atropine RT = 11'8 cm. Peak 6 scopolamine RT = 13"45 cm. The column temperature was set isothermally (110 until after the anabasine peak, subsequently the temperature was programmed at 4° per min. When the atropine peak came out the temperature was 220°. TOXICON 1976 Yol. 1~

SS

EVA DUFVA, G. LOLSON and H. HOLMSTEDT RFCULTS

The retention times with GC of D. myoporoides extract and a synthetic mixture are given in Fig. 1 . In the Dttboisia extract, five peaks are distinctly visible, with the retention times corresponding to nicotine, nornicotine, apoatropine, atropine and scopolamine. The identity of these components was ascertained by the recording of mass spectra from reference compounds and compounds in the extract . Mass spectra of peak 6 and a reference compound are reproduced in Fig. 2. 100 80

138

Scopolamine

HsH-c,H,3,,~c~H7 oH

°~ Vis°" ~ Y~ LHtFHC~H2

94

303M'

154

60 40

I

20

60 80 100 120 140 160 180 200 220 240 260280 300 m/°_ Duboisic mlroporoides Peck 6 100 80 60 40 20 i1 60 80 100 120 140 ~160 I80 200 220 240 260 280 300 m/e

FIC .

2. Lower panel : mass spectrum of compound in e~uent from peak 6 of alkaloid fraction . Upper panel: mass spectrum of reference compound. Conditions : see Experimental .

Since isopelletierine has been reported to occur in the leaves of D. myoporoides and Wu.xnvsox, 195, examination was made to ascertain the presence of this compound in the mixture. No gas chromatographic or mass spectrometric evidence for the occurrence of this compound was obtained . Anabasine, also reported to be present in D. myoporoides (see Table 1), has a different retention time from that of nicotine. Although it has the same base peak and molecular ion, the rest of the mass spectrum is distinctly different (Fig. 3). No evidence for its presence was obained. Ions specific for each individual compound were selected for recording in Fig. 4. This experiment gives added confirmation of identity of the alkaloids. The amount of the (MORTII~SIIt

TAH>I~

2. Drszxmvrcox oF wixwroms nv w wwr~e >~awer (1/10) wxn n~

Alkaloids Nicotine Nornicotine Atropine Scopolamine TOXICON 197tf Yol. I~

T~

i awvps or DrtbnisJa myoporoides

mg/100 g dry plant 300 22 20 159

~ 60 4 4 32

mg/100 ml water 48 4 9 20

39 S 11 25

DubotrJa

and Ciguatera

84

loo

59

Anatasine

60 -` m

so ao IOS ~o

20 BO

16

100

120 140 160

m/e

84 Nlcotlne

loo

m

133

0

60

`c

119

eo+ i 601

1

40''

162M~

133 161J

20~ 60

loo

80

100 120 140160

m/e

Dùboisia myoporädes I peak I

so so

ô o: ao 20

so so roo

120

m /e

lao Iso

FYci. 3. Lower panel : mass spectrum of compound in ~u~t from peak 1 of alkaloid fraction . Middle panel : mass spectrum of nicotine. Upper panel : mass spectrum of anatmsine. Conditions : see Experimental .

various alkaloids in dried leaves is presented in Table 2. Since a water infusion of D. myoporoides is used for the treatment of ciguatera poisoning, such an infusion was prepared and the alkaloid contents of the water phase analysed. These results are also presented in Table 2. DISCUSSION

In BARNARD'S (1952) review, detat~s of both the historical development and early chemistry are given. It appears that between 1872 and 1912 about ten analyses of D. myoporoides alkaloids were carried out, in the beginning using bioassays and later with traditional methods of alkaloid analysis. The impression is a confused one but contains reports of the occurrence of, e.g. the following compounds : atropine, nicotine, hyoscyamine and scopolamine . Modem work on the chemistry of D. myoporoides began with BnxcErit et al. (1937), TOXlCON 1976 Vol. l~

60

EVA DUFVA, G . L01SON and B. HOLMSTEDT Duboisia myoporoides 162 Nicotine m/e Nornicotine m/e Atropine m/e Scopolaminem/e

100 9080-

162 148

154

140 154

148

70~ 60~ in

50 ao30

140

20 10 0 1 0

1

-T - T- . . T

100 200 300 400 S00 600 700 B00 900 1000 Retention time

Sample number : 5

FYG . 4. MASS FRAGI~NTOGRAlI WITH SfiLECI'8D IONS REPRHSSNTATIVE OF BACH SINOIX COMPOIII~ .

who in a series of papers between 1937 and 1940 tried to define the individual alkaloids in D. myoporoides mixture (Table 1). They reported the absence of hyoscyamine but the presence of some previously unknown alkaloids to which they gave new names. Between 1940 and the analysis by MoRTli~x (1957), a dozen analyses were done using various chemical techniques, mostly a combination of chromatography and mixed-melting-point determinations of derivatives. It was established by BOTTOMLBY and MOItT>i~lt (1953) that D. myoporoides contained both tropane and pyridinium alkaloids. A large number of components, some of them new, were reported by BOTTOMLEY and MOItTn~t (1954). MOItTIhIEIt (1957) and MOxTn~lt and WILxuasox (1957) reported the occurrence of isopelletierine in the alkaloid mixture . This was further substantiated by a study of its structure by MoxTn~x (1957) . Another alkaloid also reported by MOxT~t and Wu irnvsox (1957) was anabasine. Theirs is the only report of the occurrence of this compound in Duboisüa. In our own analyses of the material from New Caledonia, preserved in alcohol, we could find no evidence whatsoever for the presence of either isopelletierine or anabasine. The mass spectrum of anabasine has similarities to, but no identity with, that of nicotine. It is recognized "that the alkaloid content can vary quantitatively and qualitatively in the species of Duboisia, due to location, ground conditions and time of the year" (BARGSR et al., 1937). Earlier, only one collection of D. myoporoides from New Caledonia seems to have been analysed, and then only after replanting in Canberra, Australia. VAILLANT (Thesis, University of Paris, 1972) carried out quantitation of tropane alkaloids by gas chromatography for Duboisia of both Australian and New Caledonian origins. The leaves were obtained both in the field and from plants grown in the phytotron under controlled experimental conditions . The range of values are given in Table 1. Simultaneous TLC showed that the Australian samples contained components not present in the New Caledonian material. In the latter, spots with Rf values of those of nicotine and nornicotine were found. No further identification or quantification of these components was attempted. TOX/CON 19715 Yol . l~

Duboisia

and Ciguatera

61

However, from his analytical results and based upon preliminary morphological examination, Vaillant suggests that the Australian and New Caledonian plant are two different species. This may well turn out to be the case . Our own studies on D. myoporoides from New Caledonia, preserved in alcohol, confirm the results of Vaillant . In addition, we have quantitated the pyridinium alkaloids which occur in greater quantities than the tropane alkaloids . By modern techniques, such as GGMS and mass fragmentography no trace of the additional alkaloids reported in the Australian material were found. One wonders about the validity of some early experiments with nicotine and Duboisia extracts. Farly attempts to study the pharmacodynamics of poorly documented material seem to indicate the occurrence of several components with both nicotine-like and anticholinergic action (ItINGBIt and Muxx~,L, 1879). Ciguaterapoisoning The incubation period for ciguatera intoxication varies with the individual, the species of fish and the quantity of toxic flesh ingested . Signs and symptoms usually occur within 4 hr ; the interval may be as short as 30 min in severe cases but may also last as long as 20 hr. The first symptom to appear is paresthesia with prickling about the lips, tongue and nose, together with a tingling sensation in the extremities . At the same time a state of malaise accompanied by perspiration appears. Nausea and digestive distress may occur concomitantly. The first findings last approximately 2 hr . A severe case of ciguatera poisoning presents a complex clinical picture. It is essentially a toxic polyneuritis in association, to a greater or lesser extent, with three other types of disturbance : gastrointestinal, cardiovascular and nervous (pain) . General symptoms are malaise, asthenia and chills frequently appear and there have been reports of oliguria and haematuria (RUS3SLL, 1971 ; BAGNLS, 1973). In the rare fatal cases, death apparently results from respiratory paralysis ; in experimental animals this respiratory paralysis seems to be of central origin, as the diaphragmatic twitch produced by direct stimulation of the phrenic nerve remains at control levels at the time of complete respiratory failure (lta nvat, 1972). According to Gxu.L and Crnxac (1911), le Dantec in the 19th century compared the symptoms of ciguatera poisoning to those provoked by the eating of the fly agaric . At this time, of course, nothing was known about the cholinergic nervous system and the observation passed largely unobserved . According to Li (196, ciguatoxin is an anticholinesterase which causes death through asphyxiation . Li's arguments were based on measurements of in vitro acetylcholinesterase determinations with a partially purified ciguatoxin . A crucial experiment like the determination of cholinesterase activity in the brain of a control group and a group exposed to ciguatoxin was conspicuously lacking. When such an experiment was performed by RAYIVSR et al. (1969), with a partially purified preparation from the liver and flesh of the moray eel, no in vivo anticholinesterase activity was recorded . A look at the comparative findings as shown in Table 3 also indicates that an anticholinesterase action is unlikely. Many characteristic muscarine-like and nicotine-like findings of anticholinesterases are lacking in descriptions of ciguatera poisoning. The lack of bronchial constriction and secretion of the salivary glands, mydriasis and other manifestations cannot be reconc~7ed with anticholinesterase poisoning. If indeed ciguatera is a single disease entity, is uncertain. Thus Oxu~o et al. (196 reported the following. "1'he musca,rinic signs and symptoms shown by our patient included profuse diaphoresis, nausea, vomiting, diarrhea, abdominal cramps, and respiratory wheezing. The nicotinic 7Y1X1CON I976 Yo(. I~

62

EVA DUFVA, G. LOISON and B. HOLMSTEDT T~1B 3

Symptoms and signa of poisoning with Symptoms and signs of poisoning with ciguatoxin (from RvssB.t, 1971 ; Coor~e, anticholinesterases following systemic 1964 ; Ono, 1965 ; Bxaxrs, 1973) absorption (from Hoi~sr$ux, 1959) Bronchial tree Feeling of enlarged air passages and more Tightness in chest, with prolonged wheezing free breathing,+ or respiratory wheezing .t eApiration suggestive of bronchooonstriction or increased secretion, dyspnea, slight pain in chest, increased bronchial secretion, cough. Gastrointestinal Onset early. Nausea is rapidly followed by Anorexia, nausea, vomiting, abdominal vomiting, then repeated episodes of abund- cramps, epigastric and subeternal tightness ant, watery diarrhea accompanied by (? cardiospasm) with 'heartburn' and erucabdominal cxamp and colic. These generally tation, diarrhea, tenesmus, involuntary subside within 24 hr, leaving the patient defecation . asthenic and dehydrated . Sweat glands Initial cold skin and perspiration . Increased sweating . Salivary glands Normal except for salivation during nausea. Increased salivation . Lscrimal glands No change. Increased lacrimation . Heart Slow pulse rate : between 35 and 50 beatsper Bradycardia . minute, ofton irregular. Mutlied heartbeats, reduced blood pressure, electrocardiography shows disturbance in rhythm. Pupils Mydriasis. Slight miasis, occasionally unequal, later more marked miosis. Ciliary body No change . Blurring of vision . Bladdea Oocasionel oliguria and hematuria. Frequency, involuntary micturition. SkeletoArthralgia localized mainly in the larger Easy fatigue, mild weakness muscular muscular joints : knee, ankle, shoulder, elbow. twitching, fasciculations, cramps, generalsystem Myalgia affecting, in particular, thigh and ized weakness, including muscles of respiraleg muscles. tion, with dyspnea and cyanosis. Nervous Pareathesie, sensitivity to cold objects, with Giddiness; tension; anxiety; jitteriness; system painful tingling of the extremities on contact restlessness ; emotional lability ; excessive with cold . Persistent pruritus . This may be dreaming ; insomnia ; nightmares ; headache ; severe and followed by peeling off of the tremor ; apathy ; withdrawal and depression ; skin . Superficial hyperesthesia with sen- bursts of slow waves of elevated voltage in sations of burning or electric discharge. EEG especially on overventilation ; drowsiMore rarely, paresis localised in the lower ness ; difficulty in concentrating; slowness of limbs with reduced knee and ankle r+etiexes. recall ; confusion ; slanted speech ; ataxia ; geaeralizod weakness ; coma, with absence of retibxes Cheyne-Stokes respiration ; convulsions ; depression of respiratory and circulatory centres with dyspnea, cyanosis, and fall in blood pressure . + Coore~r (1964).

1 Oxmnro (196~.

manifestations included muscle fasciculations, weakness, and absent deep tendon reflexes. The central nervous system manifestations included restlessness, stupor, and finally coma." No valid plasma cholinesterase determinations were made because of lack of preexposure values but the patient tolerated large amounts of atropine . Modem pharmacological studies on isolated atria of rats and rabbits suggest primarily cholinemimetic actions of low ciguatoxin concentration but make clear that direct effects on heart-muscles follow at only slightly higher levels ~RAYNER, 1972). One of the toxico logical actions of ciguatoxin appears to be related to its direct effects on excitable membranes, rather than to its reported anticholinesterase properties in vitro. The toxin has been found to increase the permeability of frog skin to sodium ions moving down a concentration TOXICON 1976 Vol. 14

Duboisla and Ciguateaa

63

gradient and to produce a tetrodotoxin-sensitive depolarization of frog muscle membranes (RAYNER, 1972).

The in vitro experiments by LI (196 led to the use in ciguatera poisoning of two wellknown antidotes for anticholinesterases : atropine and pyridiniumaldoxime methiodide (PAM). They have apparently been used with only moderate success in the therapy of some cases of ciguatera intoxication (Lolsox, 1970). In others, the administration of atropine was beneficial and the use of PAM as an antidote had a nearly fatal outcome (0>~o et al., 196 .

It is interesting to note that the natives of New Caledonia had already used the mixture of pyridinium and tropane alkaloids as an antidote against the poisoning. As evident from Table 2, a water infusion of the leaves of D. myoporoides, among other components, contains a powerful mixture of nicotine and scopolamine. We estimate that two mouthfuls would equal roughly 50 mg of nicotine and 20 mg of scopolamine. The some 20 other plants used as antidotes (BAGNL4, 1973 ; Rna~tr, 1973) have not yet been examined for active components . The contributions of native medicine should never be overlooked or minimized. It would seem wise in future experiments on ciguatera poisons) and poisoning, to include studies on the other alkaloids in Duboisia, as well as to study the other plants used as antidotes. Ackxowledgemexts~upported by the Swedish Medical Research Council B75-04X-4041-03 ; B75-04X199-11A ; B75-70E-3743-03 ; H73-40Y-2375-08K ; the National Institute of Mental Health Grant MH 12007; the Wellenberg Foundation ; the Tri-Centennial Fund of the Bank of Sweden 68/53 :1 and by funds from the Karolinska Institute. REFERENCF.S Bwar~ns, R. (1973) Icluyosarcotoxbre dans le Pacifique Sud. Monographie de la Commission du Pacifique Sud. South Pacific Commission . Bwaass:, G., Mwiern~t, W. F. and Mnn~r.., W. (1937) The minor alkaloids of Duboisia myoporoides . Part lI . J. clam. Soc. 1820. BARNARD, C. and Fanv~coas, H. (1945) Drug plant investigations. J. Coax. scient. irrd. Res., Aunt. 18, 277. Bwruawxn, C. (1952) Dubolalas of Australia. Ecox. Bot. 6 (1), 3. Bwxtswu, J. (1957) Le Duboisia myoporoldes R. Br. plante médicinale de la Nouvelle-Calédonie. J. Agric. trop . Bot. appl. 4 (9/10), 453. Bo'rrosu.EV, W. and MoxTn~x, P. I. (1954) Variation in the main alkaloids of Duboisia myoporoides R. Br . and D. lelchhardtli F. Muell. Aust . J. appl. Scl. S, 255. Coorat, M. J. (1964) Ciguatera and other marine poisoning in the Gilbert Islands. Pacific Sci. 18 (4), 411 . Er tcna, K., P18urou, L., Am tioRO, U ., Hoz.~r~r, B. and Ln~roo~r, J:E. (1973) Computer-controlled mass fragmentography with digital signal Processing. J. Chrotnarogr. 81, 47 . Frsx, M. S., Joxivsox, N. M. and Hoxrmla, B. C. (1955) l?iptadenia alkaloids. Irrdole bases of P. peregrine (L.) Benth. and related spaies. J. Ara. them. Soc. T1, 5892 . GRALL, C. and Cr.wnwC, A. (1911) Traité de Pathologie Exotique, Clfxiqui et T7rérapeutique : V. Intoxications et Empolsowxmexts BéribErl, pp . 283-294. Paris : Librarie J:B. BailliLne et Fils. Huts, L., Borroao ar, W. and MOnT~x, P. I. (1953) Oaurrence of nicotine together with hyoscine in D. myoporoides R. Br . Nature, Load. 4349, 435. HoL~srenr, B. (1939) Pharmacology of organophosphorus cholinesterase inhibitors. Pharmac. Rev. 2, 567. Ho~u~+T, B. and LII"rDaxmv, J:E. (1967) Chemical constituents and pharmacology of South American snuffs . Proc. Symposium In San FYancisco, Cali£, January 28-30, pp . 339-373. JwcQues, C. (1945) Duboisla myoporotdes. Rewe Agricole, Chambre d'Agriculture, Nouvelle-Calédonie, Janvier, pp. 5408-5409. Lt, K. M. mis, University of Hong Kong, 1966) Ichtyosarcotoxins in fishes of the Pacific Ocean with special reference to the mechanism of action . Reprinted in Poisonous and Yenoraous Marine Axinurls ojthe World, Vol. III, (Hwix~s±"n, H. W., Ed .) . Washington D.C . : U.S. Government Printing Ofiioe, 1970. Lorsox, G. (1970) Ichtyosarcotoxisme, Mars 10-19. Manilla : Org. mood. Santé. TOXICON 1976 Vol. I4

64

EVA DLJFVA, G. LOISON and B. HOLMSTEDT

M~erna, W. F. and Mrrcmna, W. (1940) The minoralkaloids of Dubolsia myoporoides . Part III, Valeroidine. J. darn . Soc. 1155 . Mox~, P. I. (1957) A note on Duboiria myoporoldas from the Acacia plateau. Aust. J. Sci. 20, 87 . Mottrm~, P. I. and Wn.an~ox, S. (1957) The oocurrenoe of nicotine, anabasine and iso-pelletierine in Duboisla nryoporoldes . J. them . Soc. 3, 3967. Osmmto, M. M., K~rux, J. P. and IvY, A. C., Jx. (1965) Ciguatera 9sh poisoning with cholinesterase inhibition. Hawaii med. J. 24(5), 354. Pharmacopoea Nordics (1964) Editio Suscita, Apotekarsacietetens fôrlag, Stockholm, Sweden, Vol. I, pp. 74-75. R~oanu, J. (1973) Les Plantes Médicinales de la Nouvelle-Calédonie. Travaux et documents de L'O.R.S .T.O.M . No. 23, Paris. RAYNHR, M. D. (1972) Mode of action of ciguatoxin. Fedn Proc. Fedn Am . Socs exp. Biol. 31, 1139 . RsYx~t, M. D., B~.ow, M. H. and Kos~, T. I. (1969) Marine toxins from the PaciSc-ciguatozin . J. FYsle. Res. Bd Can. 26, 2208 . Rnva~t, S. and Momma, W. (1879) Pituri . J. Plrysiol., Load. l, 377. Rvssmt,, F. E. (1971) Pharmacology of toxins of marine organisms. In : International Encyclopedia et Pharmacology and Therapy, Section 71, Chapter 15, pp. 3-114 (Rove, H., Ed .). Oxford : Pergamon Press.

TOXICON 1976 Yol. II