Plants containing Pyrrolizidine Alkaloids

29 Pages·2016·1.46 MB·English

by  Helmut Wiedenfeld

Plants containing Pyrrolizidine Alkaloids - Toxicity and
Problems
Helmut Wiedenfeld
To cite this version:
Helmut Wiedenfeld. Plants containing Pyrrolizidine Alkaloids - Toxicity and Problems. Food Ad-
ditives and Contaminants, 2011, 28 (03), pp.282-292. <10.1080/19440049.2010.541288>. <hal-
00673672>
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Food Additives and Contaminants

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Plants containing Pyrrolizidine Alkaloids - Toxicity and
P
Problems

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Journal: Foodr Additives and Contaminants

Manuscript ID: TFAC-2010-149.R2
Manuscript Type: Special IsRsue
Date Submitted by the 15-Nov-2010 e
Author:
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Complete List of Authors: Wiedenfeld, Helmut; Universität Bonn, Pharmazeutisches Institut
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Chromatography - GC/MS, Risk assessment, Toxicology -
Methods/Techniques: e
cytotoxicity
Additives/Contaminants: Natural toxicants - alkaloids
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Food Types:

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Page 1 of 27 Food Additives and Contaminants
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Plants containing Pyrrolizidine Alkaloids - Toxicity and Problems
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6 Helmut Wiedenfeld
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Pharmaceutical Institute, University of Bonn, An der Immenburg 4, D- 53121 Bonn,
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Germany; phone: [phone]; fax: [phone]; E-mail: [email protected]
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15 Received 07 May 2010
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Abstract: o
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20 Pyrrolizidine alkaloidrs (PA) are toxic for human and lifestock. The PA undergo a metabolic
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22 toxication process in the liver which is the first target organ for PA poisoning. World-wide
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many episodes of human PA intoxications are well reported. In many cases the reason for
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25 these intoxications have beeen PA-contamination in food. The main tools for analyzing food
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and fodder on PA content aree based on GC- and HPLC-separation, followed by MS(-MS)
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detection. Actual incidents with toxic PA are the "Jacobaea vulgaris (syn. Senecio jacobaea)-
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30 problem" in Europe and the "Ageratum conyzoides-problem" in Ethiopia.
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33 Key Words: Pyrrolizidine alkaloids; poisoneing in humans; PA-analysis; PA in food
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35 v
36 i
37 Pyrrolizidine alkaloids (PAs) possessing a 1,2 double-bond in their base moiety
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(necine) are hepatotoxic, carcinogenic, genotoxic, teratogenic and sometimes
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40 pneumotoxic. PAs have been estimated to be pwresent in about 3% of all flowering
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42 plants [Smith, Culvenor, 1981]. PAs mainly occur in species of the plant families
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44 Asteraceae, Fabaceae and Boraginaceae. O
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Several reports in literature proof the fact that PA containing plants are hazardous for
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livestock [Gilruth, 1903, 1904; Bull, Dick, 1959]. Since a long time ago, it is also well
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49 established that humans can be affected by toxic PA, too [Willmott, Robertson, 1920;
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51 Steyn, 1933; Bourkser, 1947; Milenkhov, Kizhaikin, 1952; Tandon, Tandon, 1975;
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53 Mohabbat, Srivasta, et al., 1976; Chauvin, Dillon, et al., 1994; Mayer, Lüthy, 1993].
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Here, the main source for the intoxication was found to be PA contaminated grain
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and bread.
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58 The International Program on Chemical Safety (IPCS), a joint agency of WHO, FAO
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60 and ILO, stated that "consumption of contaminated grain or the use of PA-containing
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Food Additives and Contaminants Page 2 of 27
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plants as herbal medicine, beverages, or food by man, or grazing on contaminated
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5 pastures by animals, may cause acute or chronic disease" [IPCS, 1989].
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7 Besides this, also different medicinal plants were found to show a hazardous
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potential for humans [Roeder, 1995, 2000]. Many investigations on herbal
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preparations or extracts from medicinal plants were done and prove the toxic risk by
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12 the consumption of phytomedicines which contain PA plants. It can be assumed that
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14 so-called "bush-teas" are the reason for several liver diseases reported in Jamaica
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and several developing countries from Africa and West Indies [Bras et al., 1954,
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1957, 1961]. While PA poisoning is mainly a problem in developing countries
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because there the use of traditional medicine is common, within the last 25 years
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21 especially in industri alised countries the use of herbal medicine has become more
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23 and more common dPue to an increased interest of people in alternative medicine,
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25 hand in hand with a greateer influence of the "green wave".
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In addition, also several foeod products of animal origin can be a source for PA
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28 ingestion: Milk has been shrown to contain PA when milk-producing animals
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30 consume PA-containing plant material [Schoental, 1959; Dickinson, Cooke, et al.,
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32 1976; Dickinson, 1980; Johnson, Robertson, et al., 1978; Goeger, Cheeke, et al.,
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1982; Lüthy, Heim, et al., 1983; Candrian, Lüthy, et al., 1984; Molyneux, James,
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1990]. Human milk has also caused liver diseases in neonates and infants [Roulet,
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37 Laurini, et al., 1988].
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39 Honey was shown to be another source of PA exposure; here it seems that the
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contamination may be due to the plant pollen, which is rich in PAs, being transferred
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by bees into the honey [Deinzer, Thomson, et al., 1977; Culvenor, Edgar, et al.,
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44 1981; Roeder, 1995, 2000; Edgar, Roeder, et al., 2002; Beales, Betteridge, et al.,
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46 2004; Boppré, Colgate, et al., 2005; Betteridge, Cao, et al.n, 2005]. Eggs from poultry
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48 exposed to PAs in PA-contaminated grain were also shown to be a possible source
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of PA exposure for humans [Edgar, Smith, 1999].
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Recently it was shown in Germany that salads can sometimes be contaminated with
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53 PA-containing plants [BfR, 2007a]. It was found that, especially in supermarkets,
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55 ready-packed rocket salat and also salad mixtures have been contaminated by
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Senecio vulgaris, a typical weed of field-crops.
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While in several countries the use of PA containing plants or preparations from them
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60 are controlled by state regulations, nothing exists in case of regulating contaminated
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Page 3 of 27 Food Additives and Contaminants
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food and fodder. In the EU the EFSA recommended in its opinion [EFSA, 2007] that
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5 feed material which can be contaminated with PA should be monitored. More data
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7 are needed to assess human PA exposure from resulting from feed and carry-over
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into animal products. Meanwhile the so-called "zero-tolerance principle" can be
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applied; this principle is used in cases where no safe or tolerable level can be
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12 determined based on available, valid scientific data, or if insufficient toxicological
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14 data are available. Due to their genotoxic and carcinogenic potential this principle
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can be applied for PA in food and fodder [BfR, 2007b].
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Actually, in Middle Europe common groundsel (Senecio vulgaris, L.) and tansy
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19 ragwort (Jacobaea vulgaris, syn. Senecio jacobaea, L.) are of particular concern.
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21 Common groundsel, a common field-weed, has been found as a contaminant in
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23 salads and tansy ragwPort has been discussed recently on account of its extensive
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expansion into pastures and meadows which has led to a great number of
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intoxications in livestock (mainly horses), especially in Germany
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28 [http://www.jacobskreuzkraut.der; http://www.izn.niedersachsen.de/servlets/ download
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30 ?C=39412784&L=20]. As well as the hazard for grazing animals (i.e. direct toxicity),
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the possibility of contaminated hay and silage and transfer of PAs into foods such as
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33 milk and milk products is under investigeation and considered to become problematic.
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35 v
36 i
37 Toxification process of pyrrolizidine alkaloids
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PA produced by Senecio-species are ester alkaloids derived mainly from the necines
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retronecine and otonecine. They are carcinogenic, mutagenic, genotoxic, fetotoxic
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and teratogenic.
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44 PAs themselves show a more or less low acute toxicity but in vivo they undergo a 3-
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46 step metabolic toxication process in the liver, which is, ans a result, the first target
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organ for the toxicity.
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This toxication process is well investigated [Mattocks, 1968; Culvenor, Downing, et
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51 al., 1969; Jago, Edgar, et al., 1970; Mattocks, White, 1971a, 1971b; Culvenor, Edgar,
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53 et al., 1971; Mattocks, 1972; IPCS, 1989].
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After absorption an hydroxyl-group is introduced in the necine at position 3 or 8 by
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the cytochrome P-450 monoxogenase enzyme complex in the liver (Figure 1: IIa and
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58 IIb). These hydroxy-PAs (OHPAs) are unstable and undergo a rapid dehydration to
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60 the didehydropyrrolizidine alkaloids (DHPAlk; Figure 1: III). This dehydration results
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Food Additives and Contaminants Page 4 of 27
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in a second double-bond in the necine followed by spontaneous rearrangement to an
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5 aromatic pyrrole-system III.
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Figure 1: Enzymatic hydroxylation and didehydropyrrolizidine products
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12 PAs occur mainly as their N-oxides in the plants and these cannot be directly
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14 converted to the OHPA, but on oral ingestion they are reduced by the gut enzymes or
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the liver microsomes and NADH or NADPH to the free bases and therefore they
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show equal toxicity to that of the free bases [Mattocks, White, 1971a, 1971b; Powis,
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19 Ames, et al., 1979; Chou, Wang, et al., 2003; Wang, Yan, et al., 2005a; Wan, Yan, et
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21 al., 2005b; Wang, Ya n, et al., 2005c].
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23 Otonecine-type PAs (PFigure 1: Ib) are metabolised to the OHPAs [Culvenor, Edgar,
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et al., 1971; Lin, Cui, et al., 1998; Lin, Cui, et al., 2000]. These otonecine-PAs
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possess a methyl-function at the nitrogen and a quasi keto-function at the bridge-
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28 carbon 8. After hydroxylation ofr the N-methyl-group it is lost as formaldehyde leaving
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30 a NH-function which undergoes condensation with the C8 keto group to produce
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product IIb (Figure 1) which spontaneously dehydrates to the DHPAlk III.
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33 The metabolites III are able to generaete stabilized carbonium ions (Figure 2: IV and
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35 VI) by loss of hydroxy groups or ester fuvnctions as hydroxyl or acid anions. These
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37 carbonium ions can react rapidly with nucleophiles (Figure 2: VII).
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Figure 2: DHPAlk and carbonium ion building
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44 In vivo the metabolites IV and VI react rapidly with nucleophilic mercapto, hydroxyl
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46 and amino groups on proteins and the amino groups of punrine and pyrimidine bases
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in nucleosides like DNA and RNA. These alkylated products show abnormal
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functions and in the case of DNA, mutations are possible. As this metabolic
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51 toxification takes place in the liver it is obvious that this organ is the first target for the
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53 intoxication and results in the liver damage leading to the veno-occlusive disease
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(VOD) in which the veins are narrowed.
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58 Detoxication of PA
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Page 5 of 27 Food Additives and Contaminants
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3 As well as the metabolic activation, detoxification of PA also occurs in vivo: hydrolysis
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5 of the ester bonds in PA from type Ia or Ib by esterases leads to necic acids and to
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7 the free necines. Both are non-toxic products and - on account of their higher water-
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solubility - can be renally excreted. The rate of hydrolysis is dependent on the level of
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steric hinderance of the ester linkages (see before); and it has been shown that the
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12 more highly branched the necic acids are the more resistance to hydrolysis
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14 [Culvenor, Edgar, et al., 1976; Mattocks, 1986]. This means, that macrocyclic
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diesters (Ia and Ib) with more complex acid moieties are more hazardous on account
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of their lower rate of hydrolytic detoxication.
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19 The N-oxides of PAs (the form occurring most commonly in plant sources ) are highly
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21 water soluble and can therefore be excreted renally. Besides their natural
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23 occurrence, N-oxidatioPn of PAs also takes place in the liver and can be seen as a
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detoxification process (Figure 2) [Mattocks, 1968; Jago, Edgar, et al., 1970;
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Mattocks, White 1971b; Williams, Reed, et al., 1989; Miranda, Chung, et al., 1991].
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28 However it has been shown thart the N-oxides - besides excretion - can be converted
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30 by dehydration or by acetylation followed by elimination of acetic acid to the DHPAlk
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(Figure 2: III) [Mattocks, 1986; Culvenor, Edgar, et al., 1970].
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35 PA toxicity in humans v
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37 PA poisoning of humans can progress from an acute to a sub-acute and finally to a
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39 chronic state [McLean, 1970; Peterson, Culvenor, 1983; IPCS, 1989; Huxtable, 1989;
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Prakash, Pereira, et al., 1999; Fu, Xia, et al., 2004; Stegelmeier, Edgar, et al., 1999].
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Acute poisoning is characterised by haemorrhagic necrosis, hepatomegaly and
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44 ascites; death is caused by liver failure due to necrosis and liver dysfunctions
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46 [Peterson, Culvenor, 1983; IPCS, 1989; Huxtable, 1989;n Prakash, Pereira, et al.,
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1999].
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Sub-acute levels are characterised by hepatomegaly and recurrent ascites;
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51 endothelial proliferation and medial hypertrophy leading to an occlusion of hepatic
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53 veins, resulting in the VOD which can be seen as a characteristic histological sign for
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PA poisoning [Peterson, Culvenor, 1983; IPCS, 1989; Huxtable, 1989; Prakash,
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Pereira, et al., 1999; Fu, Xia, et al., 2004]. The VOD causes centrilobular congestion,
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58 necrosis, fibrosis and liver cirrhosis, the end-stage of chronic PA intoxication.
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Food Additives and Contaminants Page 6 of 27
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A very important fact is that PA toxicity not only correlates with the amount and
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5 duration of uptake. It is shown that the susceptibility for PA intoxication is also
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7 dependent on gender (males seem to be more sensitive than females) and on age:
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children and especially neonates and foetuses show a much higher sensitivity than
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adults: In 2003 it was shown that the daily uptake of ~ 7 µg PA (from a herbal tea
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12 containing comfrey) during pregnancy did not show a toxic effect in the mother´s liver
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14 but damaged the foetal liver in such a way that the newborn child died after 2 days
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[Rasenack, Müller, et al., 2003].
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It has also been observed that cofactors can exacerbate the PA poisoning: liver
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19 damaging agents, bacterial or viral infections as well as medical drugs like
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21 barbiturates or meta ls like copper or mycotoxins like aflatoxins can increase the
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23 severity and likelihooPd of PA liver damage [Yee, Kinser, et al.,2000; Newberne,
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Rogers, 1973; White, Mattocks, et al., 1973; Tuchweber, Kovacs, et al., 1974; Lin,
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Liu, et al., 1974: Bull, Culvenor, et al., 1968].
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28 There is a large number of rerports in the literature about different liver diseases
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30 (mainly VOD) possibly connected with PA poisoning. But, on account of the fact that
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those reports mainly describe sub-chronic or chronic intoxications, in most cases the
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33 connection cannot be proven becausee there is often a long time period between the
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35 outbreak of the liver disease and a possibvle ingestion of PA-containing material.
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37 The following table therefore lists only those cases where the source of PAs was
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39 identified and the liver disease was therefore undoubtedly caused by PA intoxication:
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44 Table 1: human intoxications :
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46 Location and Affected Observed Source of PA n Lit.
47 year people damage l
48 South Africa, 11 adult Abdominal pain, Senecio illicifolius, S, Willmot, Robertson,
49 1920 people vomiting, burchelli y1920
50 cirrhosis
51 Jamaica, 1954 23 adults VOD Bush-teas with Bras, Brooks, et al.,
52 Crotalaria fulva 1961
53 South Africa, 15 children; VOD Bush-teas; Crotalaria Freiman,
54 1968 10 died sp.? Schmaman, et al.,
55 1968
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57 Venezuela, 5 years old VOD Crotalaria anagyroides, Grases, Beker, 1972
58 1969 girl C. pumila consumed as
59 infusion and as
60 vegetable soup
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Page 7 of 27 Food Additives and Contaminants
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Kuweit, 1970 Adults Liver carcinoma Heliotropium Macksad, Schoental,
4 ramosissimum et al., 1970
5 ("Ramram")?
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Jamaica, 1970 6 children VOD Bush-tea from Crotalaria Brooks, Miller, et al.,
7 and Senecio sp. 1970
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Iraq, 1970 9 children VOD food contaminated by a Al-Hasany,
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Senecio spec. Mohamed, 1970
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Afghanistan, 7200 VOD Wheat contaminated Mohabbat, Srivasta,
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1970-72 people with Heliotropium et al., 1976
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popovii, ssp. gillianum
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India, 1973 486 people VOD Cereals contaminated Tandon, Tandon, et
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with Crotalaria spp. al., 1976
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Ecuador, 1973 Woman VOD Herbal tea with Lyford, Vergara, et
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F Crotalaria juncea al., 1976
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India, 1973, 4 male Endemic ascites Millet contaminated with Krishnamachari,
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1975 opeople Crotalaria spp. Bhat, et al., 1977
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China, 1973, 2 adults VOD Gynura segetum Hou, Xia, et al., 1980
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1978
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India, 1974- 6 people VOD Heliotropium eichwaldii Datta, Khuroo, et al.,
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1977 1978
23 P
Martinique, 2 children VOD Bush-teas with Saint-Aimé , Ponsar,
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1975 Crotalaria retusa and/or et al., 1977
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Heliotropium sp.
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USA, 1976, 4 children Vein congestion Senecio longilobus Stillman, Huxtable, et
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1977 and necrosis of al., 1977
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liver
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UK, 1976 Woman VOD Maté (Paraguay tea) McGee, Patrick, et
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contaminated with PA of al., 1976
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Runknown origin
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USA, 1984 49 year old VOD Food supplement Ridker, Okhuma, et
33 woman econtainig Symphytum al., 1985
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spp. root
35 v
China, 1985 4 women VOD Herbal tea containig Culvenor, Edgar, et
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Heliotrpopium al., 1986
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lasiocarpum
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Switzerland, 59 years VOD Herbal tea consisting of Margalith, Heraief, et
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1985 old man Senecio spp. al., 1985
40 and 27 w
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years old
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son
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Switzerland, 5 days old VOD Herbal tea containing Roulet, Laurini, et al.,
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1986 baby Tussilago farfara 1988
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consumed during whole
46 n
pregnancy
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UK, 1986 13 years VOD Herbal tea containing Weston, Cooper, et
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old boy Symphytum spp. al., 1987
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Tadjikistan, 3906 Abdominal pain, Heliotropium Chauvin, Dillon, et
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1992, 1993 people hepatomegaly, lasiocarpum al., 1993
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ascites,
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alteration of
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consciousness
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Peru, 1994 38 year old VOD Herbal tea from Senecio Tomioka, Calvo, et
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woman tephrosioides al., 1995
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Spain, 1995 73 years VOD Senecio vulgaris Cansado, Valadés,
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old man et al.,. 1995
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Austria, 1995 18 month VOD Herbal tea with Sperl, Stuppner, et
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old boy Adenostyles alliariae al., 1995
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Argentina; 23 old VOD Herbal tea containing Vilar, Garcia, et al.,
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Food Additives and Contaminants Page 8 of 27
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1999 woman Senecio vulgaris 2000
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Germany, Fetus VOD Symphytum spp. Rasenack, Müller, et
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2002 al., 2003
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Afghanistan, 270 people VOD Wheat contaminated Kakar, et al., 2010
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2008 (50 with "charmac"
8
fatalities) (Heliotropium popovii,
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ssp. gillianum)
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14 PA determination
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Several methods can be used for the qualitative and quantitative determination of
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PAs in plant material or its preparations:
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19 TLC: Thin-layer chromatography (using the detection method of Dann / Mattocks
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21 [Dann, 1960; Mattoc ks, 1967]) is a quick, sure and easy method for a qualitative
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23 detection of PAs. UsinPg TLC in a densitometric way [Bartkowski, Wiedenfeld, et al.,
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1997], it is also possible to have quantitative results; the detection limit is about 1 - 10
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µg.
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28 LC-MS: Different HPLC methords are described. Obviously, this is the mostly used
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30 analytical method for the determination of PAs. A great benefit is the fact that PA-N-
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oxides can be analyzed as well as the free bases. Depending on the equipment, the
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33 detection limit is less than 0.1 µg per inejection.
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35 GC-MS: also in the case of GC a lot of mevthods are described. A GC problem is that
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37 N-oxides have to be reduced to the free bases before being analyzed. This reduction
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procedure is described intensively and doesn´t seem to be a limiting factor. The
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detection limit is similar to that in LC.
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All three methods can only be used accurately in case reference material is available.
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44 The reason is that the single PAs show different detector responses in the respective
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46 analyzing methods. n
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A further interesting - but unfortunately very specific - quantification method is the
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enzyme linked imuno sorbent assay (ELISA). Via a PA hemisuccinate and bovine
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51 serum albumine, enzyme linked antigenes (AG-E) can be synthesized and used for
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53 the production of antibodies (polyclonal as well as monoclonal) in rats [Röder,
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Pflüger, 1986, 1995]. This method is very sensitive and needs no complicated
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cleanup procedure. Unfortunately, it is sensitive only for a concrete PA structure; that
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58 means, antibodies produced by a retrorsine-AG-E can only be used for the analysis
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