Passage of the fern ptaquiloside into bovine milk Me Alonso-Amelot, U Castillo, F de Jongh

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Me Alonso-Amelot, U Castillo, F de Jongh. Passage of the bracken fern carcinogen ptaquiloside into bovine milk. Le Lait, INRA Editions, 1993, 73 (3), pp.323-332. ￿hal-00929344￿

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Original article

Passage of the bracken fern carcinogen ptaquiloside into bovine milk

ME Alonso-Amelot, U Castillo, F De Jongh

Laboratorio de Oulmlca Ecologica, Departamento de Ouimce, Facultad de Ciencias, Universldad de Los Andes, Mérida 5101, Venezuela

(Received 9 October 1992; accepted 19 March 1993)

Summary - Ptaquiloside has been discovered for the first time in milk samples 01 a bovine fed a dietary complement (6 kg fresh weighVday) of freshly-eut bracken fronds. This was equivalent to ca 216 mg/day pure ptaquiloside. The compound was found in a quantity of no less than 0.11 mg/I in milk. This was determined by the appearance of pterosin 6 - the decomposition product of ptaquilo- side - in prepurified milk sampi es treated with milg 1I1kllii (pH 8.0 and 11.5). The lack of base treat- ment led to the absence of pterosin B. A mi!k j}rilpurlflcatlon protocol based on meth,anol-acetonitrile precipitations in succession, hexllne and dlchloromethane extractions, alkaline treatment, reextrac- tion and silica gel microcolumn chromatography followed by reverse-phase HPLC was developed for the detection 01 ptaquiloside. bracken 1Pterldlum aqulllnum 1mllk 1ptaqulloslde 1human cancer 1HPLC

Résumé - Passage du carcinogène ptaquiloslde de la fougère Bracken dans le lait de vache. Le ptaquiloside fut découvert pour la première fois dans le lait de bovins dont l'alimentation avait été supplémentée avec des frondes de fougère fraîchement coupées (6 kg de matériel trais par jour). Cette quantité équivaut à 216 mgljour de ptaquiloside pur. Pas moins de 0, 11 mg/I de ce composé a été détecté dans le lait. Cette détermination a été réalisée en mesurant la ptérosine B produite par la décomposition du ptaquiloside après traitement du lait prépurifié en milieu légèrement basique (pH 8,0 et 11,5). En l'absence de traitement alcalin, aucune production de piérosine B n'a été observée. Afin de détecter le ptaquiloside, nous avons développé un protocole de prépurification du lait basé sur des précipitations successives au méthanol-acétonitrile, extractions à l'hexane et au dichloromé- thane, traitement alcalin, réextraction et chromatographie sur microcolonne de gel de silice, le tout suivi par l'analyse par HPLC en phase inverse. fougère / Pteridium aquillnum / lait / ptaquiloside / cancer humain / HPLÇ 324 MEAlonso-Amelotet al

INTRODUCTION Of major concern is the possible link be- tween bracken infestation and the appear- ance of esophageal and in Bracken fern is con- individuais living in these areas. This situa- sidered 1 of the 5 most successful organ- tion was first proposed by Evans et al isms of the plant kingdom (Page, 1976, (1971), then reported in Costa Rica 1982; Harper, 1977; Fletcher and (Villalobos-Salazar, 1985, 1987; Villalobos- Kirkwood, 1979). It grows on every conti- Salazar et al, 1989a), and suggested for nent and at every latitude except for the some areas of Wales (Galpin and Smith, polar ice-caps (Tryon, 1941), with an ex- 1986) and the Andean states of western tensive coverage of agricultural land (Fen- Venezuela (Alonso-Amelot and Castillo, wick, 1988). The widespread clearance of unpublished observations). The actual forests in the tropics by ranchers and mine mechanism of this connection has never prospectors is rapidly expanding the en- been established since bracken is not con- croachment of bracken on hillsides, moun- sidered, apart from rare occasions, as be- tain grasslands and cool Andean valleys of ing suitable for human consumption the formerly bracken-free pastureland (Hodge, 1973). However, there is sufficient (Gliessman, 1978; Ortega, 1990). evidence to believe that milk may provide ln addition, the range of chemicals with that link. On the one hand, bracken con- biological impact in this fern is of major ec- tains ptaquiloside, a glucoside norsesqui- onomic importance. In addition to the terpene (Van Der Hoeven et al, 1983; cyanogenic glycosides and tannins Niwa et al, 1983) (see fig 1) of strong car- (Cooper-Driver and Swain, 1976) and the cinogenic potential (Hirono et al, 1987). cytotoxin 1-indanone derivatives Once separated from the , the pta- pterosins- (fig 1) (Saito et al, 1975), there quiloside aglycone becomes a potent elec- is the pro-histaminic activity and mutage- trophilic dienone that reacts nicity of braxin 1A (Salto et al, 1990), and stronqly towards various weak nucleo- the thiamine-destroying compounds dacti- philes including water, amines and other lifric acid (Fukuoka, 1982) and thiaminase very mild bases (Matoba et al, 1987). Aiso (Evans, 1976; McCleary and Chick, 1977) ptaquiloside must be capable of passing which cause coordination disorders and through the cell and nucleus membranes, death in horses and mules (Diniz et al, as it brings about chrornosomal aberration 1984). Still other compounds appear asso- (Matsuoka et al, 1989) via irreversible ciated with damage to the bone-marrow damage to uncoiled DNA (Ojika et al, tissue (Evans, 1986), severe leucopenia, 1989). thrombocytopenia (Philp and Gowdey, 1967), excess oral mucus breathing diffi- cultY and death in cattle (Evans, 1976), bright blindness in sheep (Barnett and Watson, 1970), heart failure in pigs (Evans et al, 1972; Harding, 1972), and bovine en- H~~ zootic hematuria (Rosenberger, 1971), a H~OH chronic condition of the urinary tract that OH invariably leads to death. The combination ptaqulloslde pterosln B of its wide-spread growth and aggressive chemistry makes bracken a most severe Fig 1. Structureof ptaquilosideandpterosinB. problem for the animal farming industry. Structure du ptaquiloside et de la ptérosine B. Bracken carcinogen ptaquiloside in milk 325

On the other hand, various canceraus mass. The suspension was centriluged, the vol- tumors have been induced in rats and ume of the liquid phase corrected to 300 ml by mice given oral doses of milk obtained evaporation at 36 "C, and 20 9 sodium chloride was added. This solution was extracted sucees- from cows fed with dietary complements of sively with hexane (3 x 50 ml) and dichlorome- bracken Iern (Evans et al, 1972; Pamukcu thane (6 x 25 ml) (extract A). To the aqueous et al, 1978; Villalobos-Salazar et al, layer was added 15 ml of an aqueous sodium 1989b). Earlier results have also indicated hydroxide (0.003 N) solution and allowed ta that calves experienced lowered bone mar- stand at 36 "C for 2 h with stirring. The mixture row activity when allowed to suck their was then extracted with dichloromethane (6 x 25 ml) (extract B) to draw out the pterosin B de- bracken-fed mother cow's milk (Evans, rived from ptaquiloside in the milk sample. 1986). Ali this evidence points toward the pres- ence of deleterious bracken metabolites in Microcolumn prepurification bovine milk. Some toxic compounds in ani- mai feed such as aflatoxins and some pes- The modified procedure of Alonso-Amelot et al ticides have been known for 50me time (1992a) was used. Extracts A and B were sub- either directly or after derivatization by mitted to the same protocol: the dichlorome- body enzymes to pass into the milk in bo- thane solutions were evaporated over silica gel vines. Bracken metabolites may be no ex- (100 mg) at room temperature in vacuo, and the ception. However, in spite of these advanc- solid residue was transferred to a microcolumn prepared from a Pasteur pipette lilled with 300 es, 50 far the chemical analysis of milk has mg silica gel 70-230 mesh. The column was been poor in providing direct evidence of eluted with 1) 2.5 ml hexane-- 9:1 secondary bracken compounds. It has and 2) 3.0 ml hexane--ethyl acetate1:4. Only been considered that to tormally demon- the second fraction contained pterosin B. This strate the existence of a link between eluent was evaporated in a current of dry nitro- bracken and human cancer, the detection gen gas, and redissolved in HPLC grade metha- nol (2.0 ml). of the bracken carcinogen 1 in milk is re- quired. It is the purpose of this paper to re- port on the tirst detection of ptaquiloside in HPLC analysis milk tram cattle exposed to bracken fern.

A LOC Milton Roy HPLC apparatus composed MATE RIALS AND METHODS of a Constametric Il pump, a Rheodyne 20-1-11 loop injector and a Waters 490E multichannel UV-VIS detector tuned at 260 nm connected to a radial 0.5 x 10 cm C-18 column was used. The Milk extraction procedure optimum separation conditions were as follows: solvent: methanol-water 65:35 in the isocratic mode, flow: 1.0 ml/min, at room temperature. The entire sequence was carried out at 36 "C to The pterosin B peak appeared at Rt = 10'10". increase the rate of protein and Iipid precipita- tion. A milk sample (2 1)was warmed to 36 oC and methanol (4 1)was added with stirring for 30 min (5 h at 36°C). The suspension was filtered Cow conditioning and milk sampling through cheesecloth, then filter paper and finally centrifuged (3 000 rpm, r = 38 cm). The super- A 3-year old 100% Holstein female in its 4th natant was evaporated in vacuo at 36 oC to 300 milking month was selected from a herd and ml and acetonitrile (1 1)was added in portions maintained on a Kikuyu (Pennisetum clandesti- while the deposited solids were gradually re- num) plot (altitude 1 900 m) and commercial ani- moved to prevent the formation of a sticky mai feed concentrate at a rate of 2 kg day. The 326 ME Alonso-Amelot et al positive heallh condition of the animal was mon- Conversely, any pterosin B appearing in itored for 4 months before the bracken-feeding the liquid chromatograms of milk extracts experiment was starled. Freshly-cut bracken treated with mild base alter precipitation fronds (6 kg) were then administered orally in and solvent extraction should come exclu- an admixture with fresh Kikuyu grass and mo- lasses every morning for 7 consecutive d. Alter sively from the decomposition of ptaquilo- the morning milking was collected (24 1daily av- side. Another potential pterosin B precur- erage),2 1sampies were separated and imme- sor present in bracken is the pterosin diately taken to the laboratory for analysis as' glucoside pteroside B. When an authentic described above. sampie of this compound was added to a milk sample which was then subjected to the purification sequence including the RESUL TS AND DISCUSSION base treatment step at a higher tempera- ture (50 oC), it remained totally unchanged Ptaquiloside is a very unstable compound. and no detectable pterosin B was pro- Once isolated from the plant it decompos- duced. This analytical strategy is depicted es quickly into pterosin B under mild acid, in figure 3. base or heat (Saito et al, 1989). Any The bovine specimens for milking were clean-up procedure used to detect this distributed into 2 groups. In the first group compound in milk must avoid these condi- a herd of 4 Holstein cows were kept in a 2- tions in order to preserve its molecular in- ha plot that was heavily infested with tegrity. In particular, commonly-used rea- bracken growth. Alter 24 months 3 of the gents for milk analysis such as acetic, animais had developed enzootic hematu- sulfuric and hydrochloric acids must not be ria. At their medium lactation period, milk utilised. The direct analysis of ptaquiloside samples were taken from the animais once in a complex matrix such as milk is there- a wk for 3 wk and analyzed for the con- fore not practical. Nevertheless, a milk pre- tents of pterosin B naturally occurring in purification sequence was designed to de- the fern, and for ptaquiloside via its base fat and deproteinise milk samples under transformation into pterosin B following the neutral conditions. lt was based on the analytical sequence described above. precipitation of insoluble material via meth- None of these samples showed signs of anol and acetonitrile, followed by extrac- natural or induced pterosin B in the HPLC tion with hexane and dichloromethane as chromatograms down to a detection limit of described in figure 2 (see also the Materi- 0.4 ng per injection. ais and Methods section). The rernaining ln the second group, one healthy 3- aqueous phase was then analyzed by year-old Holstein cow in her 4th month of HPLC after silica gel microcolumn prepuri- lactation was given oral complements in fication (Alonso-Amelot et al, 1992a). This the order of 6 kg fresh weight (850 9 DM) strategy ensured that any pterosin B that of freshly-cut bracken fronds of intermedi- was to be detected at this point would not ate growth (ca 40-50 days from emer- come from ptaquiloside decomposition, gence) every 24 h in addition to its normal but from the ingested bracken that con- Kikuyu grass diet and animal-feed concen- tained pterosin B as a natural metabolite. trate. The ptaquiloside content of the Indeed, a ptaquiloside-enriched sample fronds was estimated by HPLC (Alonso- obtained from the purification of a water Amelot et al, 1992b) to be 0.250 ± 0.050 extract of bracken resisted the indicated mg per 9 vegetation biomass. This gave milk precipitation and microcolumn prepuri- an average of 216 mg/carcinogen/d added fication scheme without apparent change. to the diet. This treatment was extended Bracken carcinogen ptaquiloside in milk 327

MeOH (41, 30 DÜn, 36°C)

SOLIDS I-----...L-----I • FILTRATE '----,------'

Evaporate (36° C) MeCN (11, 1 h) WASTE Centrifuge

FILTRATE

Hexane (3x50 ml) WASTE AQ.LAVER 1---..L.---lORG LAYER~---J

NaCI (20g) DCM (6x25 ml)

AQ LAYER I--....L...-----l ORG LAYER Extract "A" NaOH(36° C, 2h) DCM (6x25 ml) ,....------, ORGLAYER AQ LAYER Extract "B"

J1COlumn HPLC prepurification

Fig 2. Milk purification sequence for the detection of ptaquiloside, the bracken lem carcinogen. Séquence de purification pour la détection du ptaquiloside, le carcinogène de la fougère.

for 7 consecutive d and then discontinued. under the toxicity level (as yet undeter- Milk samples were taken daily in the early mined) of the cow in question. morning and 7 d after the bracken treat- Ali the milk sampies fram this animal ment had been ceased. During the treat- that were taken after the first 48 h of brack- ment there was no net reduction of the av- en feeding did not contain any detectable erage volume of milk produced (24 I/d), nor natural pterasin, but the extracts treated was there any indication of sickness or dis- with mild base at pH 11.5 showed a dis- turbance in the animal. Hence the amounts tinct pterosin B peak in the liquid chroma- of bracken metabolites administered were tograms. This is shown in figure 4 where 328 ME Alonso-Amelot et al

Adjust pH=1I (No OH)

<§~3) (PTAQ) . PTER

MEDIUM POLAR ORGANICS

PTAQ = PTAQUILDSIDE PTER = PTEROSIN B

Fig 3. Conceptual purification strategy for the detection of ptaquiloside in milk. Concept de la séquence de purification pour la détection du ptaquiloside dans le lait.

trace A corresponds to the milk of the un- ln order to further assess the presence of treated animal, while after bracken feeding ptaquiloside in the milk, the medium of a pre- and treatment with base (trace B) a dis- purified bracken-fed cow's milk extract taken tinct pterosin B appears. In the absence of trom the 3rd d of the experimental feeding base this signal did not become apparent. had its pH adjusted to 8.0 and was warmed Traces C and D correspond to a prepuri- in a constant-temperature bath 38.0 ± 0.5°C fied methanolic extract of P aquilinum to promote slow and controlled transforma- fronds utilised in the feeding assay and of tion of the contained ptaquiloside into ptero- a pure pterosin B standard respectively. sin B. Aliquots were drawn out every 10 min, The identity of this compound in the prepurified by the microcolumn method and chromatograms was determined by vari- the concentration of pterosin B thereby pro- ous methods, including comparison of re- duced determined by HPLC. The ptaquilo- tention times, co-injection and peak en rich- side-pterosin conversion was assessed by ment with pterosin B standards. In following the height of the pterosin B peak. It addition, the UV spectrum of this peak ob- was found to increase smoothly and asymp- tained with the aid of the multichannel tomatically to completion during the first 60 spectrophotometric detector employed min of warming. A parallel experiment using was identical to that of an authentic sam- a ptaquiloside-enriched milk sam pie under pie, as iIIustrated in figure 5. It is c1ear that the same pH and temperature conditions, the only way for pterosin B to appear after displayed a similar behaviour was observed, the base treatment of the milk extract must as shown by the data in figure 6. Hence the have been the base-induced decomposi- hypothesis that ptaquiloside was present in tion of ptaquiloside originally present in the the milk of the bracken-fed cow was fully milk. demonstrated. Bracken carcinogen ptaquiloside in milk 329

That the herd of 4 cows exposed to vol- side excreted through their milk may have untary feeding of bracken fern did not been minimal. possibly under the detection show signs of ptaquiloside in the milk can limit of the present method. Second, in the be explained on 2 grounds. Flrst, although voluntary feeding situation there was no it was c1earthat the animais did in fact eat precise record of the last time that the limited amounts of fern since they devel- cows had actually eaten bracken. lt could oped enzootie hematuria only attributable weil be that, ptaquiloside being an unsta- to fem intoxication, their feeding on this ble chemical, its mean residence time in plant may have been only casual or under the rumen couId be short, particularly un- stress when other grasses became scarce. der the rather high temperatures of the ru- Therefore. the concentration of ptaquilo- men mass (40-42 oC). In tact, a milk sam- pie from the bracken-force-fed cow obtained 7 d after the last meal including bracken did not show any détectable pta- A B quiloside-derived pterosin B in the ex- tracts. Hence, animais exposed to bracken

c o

1 1 Rt 0 5 10 15 min. Fig 4. HPLC chromatograms of milk and brac- ken frond fractions: A) milk extract A. B) Milk fraction treated with dilute base (extract B) sho- wing a distinct pterosin B peak. C) Water- 220 240 260 280 300 }20 }40 dichloromethane extract of bracken fronds used WAVELEN31H ( n m ) for the feeding experiment. D) Pterosin B stan- -dard. For specifie HPLC conditions see text. Fig 5. Comparison of the UV spectra of pterosin Chromatogrammes de HPLC des fractions de B peak obtained in the HPLC trace after base lait et frondes de fougère. A) Extrait A du lait. 8) treatment. Solid line: spectrum of pterosin stan- Fraction du lait traité avec une base diluée (ex- dard. Broken line: spectrum of HPLC peak attri- trait B) qui montre clairement un pic de ptéro- buted to pterosin B. sine B. C) Extrait d'eau-dichlorométhane des Comparaison des spectres UV du pic de ptéro- frondes de fougère utilisées dans l'expérience sine B obtenu par HPLC après traitement alca- de l'alimentation. D) Standard de ptérosine B. lin. Trait continu: spectre de ptérosine standard. Pour les conditions spécifiques de HPLC, voir le Trait pointillé: spectre du pic de HPLC attribué texte. à la ptérosine B. 330 ME Alonso-Amelot et al

excreted into the milk, which represents l00~------'" 1.2% of the total ingested carcinogen. 01

CONCLUSIONS i -//-----'------~ The exposure of dairy cattle to bracken fern is not only of economic concern owing ..1 to disease and death of livestock, but the discovery of the potent carcinogenic princi-

o 5 la 15 20 Z6 30 35 40 45 50 66 60 65 70 pie of Pteridium aquilinum in milk reported

TDŒ (loin) here, in addition to the evidence of carcino- genicity of the milk from animais that feed Fig 6. Relative % conversion of ptaquiloside on bracken, extends the bracken problem into pterosin B. Curve (1): in a prepurified milk sample of a bracken-fed bovine. Curve (2) : in a to the public health domain. This requires healthy cow's milk contaminated with plant- a review of bracken control policies in ail derived ptaquiloside. Both runs at 38 ± 0.5°C. countries with bracken-infested cattle graz- Pourcentage relatif à 38,0 ± D,SoCde conver- ing areas, the development of more effi- sion de ptaquiJoside en ptérosine B. Courbe A : cient growth control or eradication technol- dans un échantillon prépurifié de lait chez un bovin alimenté avec des fougères. Courbe B : ogy of P aquilinum, and the design of dans un lait provenant d'une vache saine conta- practical analytical methodology to detect miné avec le ptaquiloside de la plante. ptaquiloside in milk processing plants.

voluntary feeding together with abundant ACKNOWLEDGMENTS alternative grasses may eat the fern at ir- regular intervals and insufficiently small This investigation was financially supported by amounts for ptaquiloside to appear in the the Consejo Nacional de Investigaciones milk in significant quantity. Cientfficas y Technol6gicas, CONICIT of Cara- cas, Venezuela (grant No PC-064), Consejo de This hypothesis required a more quanti- Oesarrollo Cientifico, Humanistico y Tec- tative view of ptaquiloside content in bovine nol6gico, COCHT, of Universitad de los Andes, milk. There is no presumption that the ex- Mérida, Venezuela (grant n° C-526-91) and Fun- traction method presented here is quantita- daci6n Polar of Caracas, Venezuela. tive, since ptaquiloside losses from copre- cipitation and adsorption in the precipitates was very difficult to estimate. So only a lim- REFERENCES ited assessment of the proportion of ptaqui- loside relative to the ingested material can Alonso-Amelot ME, Pérez-Mena M, Calcagno be advanced at this time. The concentra- MP, Jaimes-Espinoza R (1992a) Quantitation tions of observed pterosin B were calculat- of pterosins A and B, and ptaquiloside, the ed against a calibration curve by standard main carcinogen of Pteridium aquilinum (L procedures. When these values were trans- Kuhn), by high pressure Iiquid chromatogra- phy. Phytochem Anal 3, 160-164 lated in terms of ptaquiloside, it was found to beno less than 0.11 mg/I fresh milk. In Alonso-Amelot ME, Pérez-Mena M, Calcagno terms of the 24 I/d produced by the test ani- MP, Jaimes-Espinoza R, Castillo U (1992b) Ontogenie variation of biologically active me- mai, this becomes 2.60 mgld ptaquiloside tabolites of Pteridium aquilinum (L Kuhn), Sraeken eareinogenptaquiloside in milk 331

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