Accepted Manuscript

European medicinal and edible plants associated with subacute and chronic toxicity
part I: Plants with carcinogenic, teratogenic and endocrine-disrupting effects

Luka Kristanc, Samo Kreft

PII: S0278-6915(16)30113-2
DOI: 10.1016/j.fct.2016.04.007
Reference: FCT 8557

To appear in: Food and Chemical Toxicology

Received Date: 12 January 2016

Revised Date: 8 April 2016
Accepted Date: 10 April 2016

Please cite this article as: Kristanc, L., Kreft, S., European medicinal and edible plants associated with
subacute and chronic toxicity part I: Plants with carcinogenic, teratogenic and endocrine-disrupting
effects, Food and Chemical Toxicology (2016), doi: 10.1016/j.fct.2016.04.007.

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European Medicinal and Edible Plants Associated with Subacute and
Chronic Toxicity

Part I: Plants with Carcinogenic, Teratogenic and Endocrine-Disrupting Effects

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Authors: Luka Kristanc 1*, Samo Kreft 2

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Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000
Ljubljana, and Primary Healthcare of Gorenjska, ZD Kranj, Gosposvetska ulica 10, 4000
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Kranj, Slovenia. * Corresponding author.

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Faculty of Pharmacy, University of Ljubljana, Tržaška cesta 32, 1000 Ljubljana, Slovenia.
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Abstract

In recent decades, the use of herbal medicines and food products has been widely embraced in
many developed countries. These products are generally highly accepted by consumers who
often believe that “natural” equals “safe”. This is, however, an oversimplification because

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several botanicals have been found to contain toxic compounds in concentrations harmful to
human health. Acutely toxic plants are in most cases already recognised as dangerous as a

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result of their traditional use, but plants with subacute and chronic toxicity are difficult or
even impossible to detect by traditional use or by clinical research studies. In this review, we

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systematically address major issues including the carcinogenicity, teratogenicity and
endocrine-disrupting effects associated with the use of herbal preparations with a strong focus
on plant species that either grow natively or are cultivated in Europe. The basic information

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regarding the molecular mechanisms of the individual subtypes of plant-induced non-acute
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toxicity is given, which is followed by a discussion of the pathophysiological and clinical
characteristics. We describe the genotoxic and carcinogenic effects of alkenylbenzenes,
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pyrrolizidine alkaloids and bracken fern ptaquiloside, the teratogenicity issues regarding
anthraquinone glycosides and specific alkaloids, and discuss the human health concerns
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regarding the phytoestrogens and licorice consumption in detail.

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Key words: European plants; carcinogenic; teratogenic; alkenylbenzene; pyrrolizidine

alkaloid; phytoestrogen
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1. Introduction

The use of herbal medicinal products and phytonutrients is growing in popularity

around the world, with many people now using these products for self-medication and
culinaric purposes. It has been estimated that up to 80 % of the world’s population living
predominantly in developing countries use herbal medicines and traditional medical practices

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as a primary source of healthcare (Bodeker et al., 2005; Ekor, 2014). The high prevalence of
people seeking herbal therapy in developing countries, especially in rural areas, is associated

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with tradition, availability and low cost. In recent decades, the use of herbal remedies, which
are used in complementary or alternative approaches, has also been widely accepted in

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developed countries, including many European countries, especially the UK, France and
Germany (Braun et al., 2010; Calapai, 2008; Ekor, 2014). In contrast to its use in developing

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countries, phytotherapy has gained popularity in developed countries as a result of the
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widespread public belief that it is safe, balanced and nature-friendly. Furthermore, it is
associated with a healthy way of life and is viewed as a nonaggressive and holistic approach
to healing. Thus, people may spend a substantial amount of money for various over-the-
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counter remedies and food supplements of herbal origin with an intention to improve their
general well-being, appearance and health (Kong et al., 2003; WHO global atlas, 2005; WHO
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Monographs, 2002).
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In light of the global growth in interest in phytotherapy and phytonutrition, which

encompasses the use of exponentially increasing numbers of newly emerging herbal products,
the public health concerns regarding their safety are also increasing. In spite of the promising
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medicinal and nutritional potential of many herbal species, we must remain aware of the fact
that the majority of them have not been thoroughly studied in terms of their safety and that the
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adverse effects associated with their use have not been monitored in most cases as rigorously
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as conventional pharmaceuticals (Traditional Medicine Strategy, 2002). Although the

systematic control of the quality and safety of industrially produced herbal products has
undoubtedly been increased in Europe during recent decades, there is still room for
improvement (Raynor et al., 2011).
Plants with substantial acute toxicity are in general already recognised as dangerous
because of the historical incidents of poisonings. Subacute and chronic toxicity, on the other
hand, are difficult or even impossible to detect in traditional use or even when using clinical
research methods. Events that emerge only after long-term use or a lag time are not usually

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connected to a causative agent. These types of dangers can therefore go unnoticed even when
a plant is widely used.
Generally, the most poisonous plant substances are neurotoxins, which are followed
by cytotoxins and metabolic poisons that disturb the structural integrity and functions of the
internal organs, such as the liver, heart, kidneys, gastrointestinal system and lungs. In addition
to the inherent properties of toxic chemicals, it is important to keep in mind that dose is a

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crucial parameter. The idea that it is the dose that causes a poison to be toxic was recognized
by the first true toxicologist, Paracelsus, five centuries ago. Nevertheless, during the last few

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decades, it has become apparent that certain substances that originate in plants can cause
serious disturbances, including carcinogenesis, hormonal dysregulation, and the disruption of

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reproductive and developmental processes, even at very low doses which are below those
used for traditional toxicological studies (Vandenberg et al., 2012). Therefore, in contrast to
acute toxicity, the dose-effect relationship in cases of chronic toxicity is not always so

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straightforward.
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Another important aspect of the possible intoxications with herbal products and plant
food is their contamination or incorrect identification of plant in the product. A toxic Senecio
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species were, for instance, found as contaminant in rocket (Eruca sativa) salad (Wiedenfeld,
2011) and herbal teas (Rasenack, 2003; Schulz et al., 2015). Several dozens of Belgian
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women were intoxicated by herbal slimming pills contaminated (or possibly adulterated) with
Aristolochia species (Cosyns, 2003). Herbal medicinal products often contain toxic amounts
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of heavy metals or synthetic medicines (Saper et al., 2004; Au et al., 2000). Furthermore, in
folk medicine, people can incorrectly identify a plant and use a toxic one instead of medicinal
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or edible one (e.g. Veratrum album instead of Gentiana lutea (Verovnik, 1999)).
Adulterations, contaminations and misidentification of herbal products are by themselves
extensive topics and will not be covered in this review, nevertheless, they were dealt with to
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some extent elsewhere (Ekor, 2014; Farah et al., 2000).

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In this review, we systematically address the issues of carcinogenicity, teratogenicity

and the endocrine-disrupting effects that are associated with the use of herbal preparations.
Our focus is on plant species that either grow natively or are cultivated in Europe. We
sometimes widened our selection to species that are extensively used in Europe but not
cultivated there, including pepper and ginger. In the beginning of each chapter, some basic
concepts regarding the molecular mechanisms that have been implicated in individual
subtypes of plant-induced non-acute toxicity are described. This is followed by a discussion
about the pathophysiological, clinical and epidemiological characteristics of these substances.

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2. Genotoxic and carcinogenic plants

Herbal products are not always safe. On the contrary, some of them have been found
to contain substances that are potentially genotoxic or carcinogenic. In this respect, substances

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belonging to the groups of alkenylbenzenes and pyrrolizidine alkaloids are of special concern
to human health.

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Many secondary plant metabolites are known to attack DNA, either through covalent
binding (alkylation) or intercalation (Schmeller et al., 1997; Wink, 2000; Wink, 2008). Plant-

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derived alkylating agents, which will be more thoroughly presented in the following chapters,
include alkenylbenzenes, pyrrolizidine alkaloids, ptaquiloside, aristolochic acids and certain
furanocoumarins. If the alkylated DNA bases are not adequately repaired, or if the cell is not

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directed into apoptosis, the cell may proliferate, thereby fixing the mutation, which represents
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the first step of carcinogenesis. While plant-derived alkylating agents are generally
recognized as procarcinogenic substances, the relation between carcinogenesis and plant-
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derived intercalating compounds, such as β-carboline and certain other alkaloids, including
berberine and sanguinarine (contained in Berberis and Chelidonium), and anthraquinones
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(found for instance in Senna and Frangula), is not so straightforward (Lu et al., 2012;
Schmeller et al., 1997). It has been shown, that they are capable of inserting themselves
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between successive base pairs in DNA which can result in the disruption of DNA replication
and the occurrence of frameshift mutations (Schmeller et al., 1997; Wink, 2000). Although
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they can undoubtedly have a profound impact on the cell cycle, these plant-derived
intercalating agents have not been experimentally linked with carcinogenesis. Some of them
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have even been shown to have antineoplastic activity as a result of their antiproliferative and
proapoptotic effects (Lu et al., 2012; Wink, 2007).
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2.1 Alkenylbenzenes

Many alkenylbenzenes, i.e., estragole, eugenol, methyl eugenol, safrole, β-asarone and
myristicin, have been demonstrated to have genotoxic and carcinogenic properties (Figure 1
and Table 1), and the EU Scientific Committee on Food (EU-SCF) has suggested restrictions
for their use (Scientific Committee on Food, 2001a, 2001b, 2001c). They are present in a wide

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variety of plants, including nutmeg, cinnamon, basil, fennel, anise, tarragon and black pepper
(Table 1) (Scientific Committee on Food, 2001a, 2001b, 2001c). Alkenylbenzenes are used as
flavours and fragrances in many foods and food products, perfumes, soaps, aromatic oils and
detergents. Despite the existence of regulatory efforts in several countries (Van den Berg et
al., 2011), their use in food supplements that contain them in higher levels is not sufficiently
controlled. It has been namely shown, that the recommended use of some food supplements

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that contain high concentrations of alkenylbenzenes could lead to exposure levels that have
caused malignant tumours in experimental animals (Scientific Committee on Food, 2001a,

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2001b, 2001c; Van den Berg et al., 2011).

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Figure 1. The comparison of the chemical structures of the most common alkenylbenzenes.
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The molecules typically consist of a benzene ring substituted with a propenyl and a methoxy
groups.
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Nevertheless, the industrial expert panel from the Flavour and Extract Manufacturers’
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Association (FEMA) concluded that the consumption of flavours and the related exposure to
methyl eugenol and estragole is generally too low to pose a significant cancer risk in humans
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because it has been shown in rodent studies that the activating biotransformation pathway
does not play an important role when these drugs are used at low doses (Smith et al., 2002).
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Alkenylbenzenes must be metabolically activated to become genotoxic and to induce tumours

(Miller et al., 1983; Wiseman et al., 1985). The relevant bio-activating pathways of all
alkenylbenzenes are quite similar. In the most significant 1-hydroxylation pathway, 1-
hydroxy-derivatives (the proximate carcinogen) are formed, which are subsequently
transformed into ultimate carbocationic carcinogens through a process involving P450
cytochromes and sulfotransferases (Miller et al., 1983). In addition, highly reactive epoxide
metabolites have been detected in rodent livers following the exposure to alkenylbenzenes,

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but were found to be insignificant because they are rapidly converted to less toxic dihydrodiol
or glutathione conjugates (Guenthner and Luo, 2001; Luo and Guenthner, 1996).
It has been suggested that the results obtained in studies of herbal extracts may be
different from those obtained from isolated carcinogenic compounds (De Paula et al., 2007;
Jeurissen et al., 2008). This discrepancy could be associated with the complex mixture of
compounds present in the extracts that might influence either bio-activation (toxication)

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and/or detoxification pathways, the most significant of the later being glucuronidation and
binding with glutathione (Guenthner and Luo, 2001; Iyer et al., 2003). For instance, in HepG2

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human hepatoma cells the DNA alkylation mediated by the sulfotransferase-catalysed bio-
activation of estragol was supressed by a flavonoid substance nevadensin, present in the

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methanol basil extract (Alhusainy et al., 2010; Jeurissen et al., 2008). Therefore, the level of
the bio-activation of alkenylbenzenes could be significantly diminished when they are
consumed along with other herbal ingredients. The plants that are used in herbal medicine in

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Europe that contain genotoxic or carcinogenic alkenylbenzenes are listed in Table 1.
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Table 1. List of plants species containing alkenyl benzene compounds with genotoxic and/or
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carcinogenic properties.
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Plant Species Compound Genotoxic and/or Carcinogenic Activity Ref.

Foeniculum vulgare, Estragole Well established genotoxic (mutagenic and Drinkwater et al., 1976;
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clastogenic) and carcinogenic (especially EFSA, 2009a; Martins et

Ocimum basilicum,
hepatocarcinogenic) effects in various al., 2012; SCF, 2001
Artemisia dracunculus, rodent models. a,b,c; Swanson et al.,
1979
Pimpinella anisum
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Laurus nobilis, Eugenol Genotoxicity was indicated in a number of EFSA, 2009b; NTP,
in vitro assays (resulting from oxidative 1983; Swanson et al.,
Ocimum basilicum,
DNA damage). There is also modest 1979
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Geum rivale, evidence for genotoxic effects in in vivo

models (in mice).
Heracleum spp.
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Angelica archangelica, Isosafrole A weak hepatocarcinogenic effect was SCF, 2003a; Swanson et
found in rodents (a non-genotoxic al., 1979
Sassafras spp.
mechanism is suspected). In vitro systems
testing mutageniicty gave equivocal
results.

Petroselinum crispum, Apiol DNA adducts were detected in HepG2 Miller et al., 1983; Zhou
cells and laboratory animals (weaker et al., 2007
Anethum graveolens
effects than were observed for estragole
and safrole).

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Acorus calamus, Β-Asarone In vitro genotoxicity tests gave equivocal Cartus and Schrenk,
results. In vivo tests demonstrated an 2015; Cartus et al.,
Asarum europaeum
increased incidence of hepatic adenomas 2015; SCF, 2002
and carcinomas in mice and
leiomyosarcomas in rats.

Petroselinum crispum, Elemicin The reactive hydroxyl metabolites of Hasheminejad and

elemicin were found to bind covalently to Caldwell, 1994; Miller
Myristica fragrans
DNA and to induce hepatocarcinogenesis et al., 1983; Wiseman et
in rodents. al., 1987

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Myrrhis odorata Trans- Several in vitro and in vivo experiments Newberne, 1999;
Anethole showed that trans-anethole is not Swanson et al., 1979
genotoxic, but it was shown to induce

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hepatocellular carcinomas in rats.

Foeniculum vulgare, Methyl Methyl eugenol is mutagenic (can induce EFSA, 2009a; Zhou et

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eugenol DNA adducts through its 1-hydroxy- al., 2007
Ocimum basilicum,
metabolite) and has been associated with
Artemisia dracunculus, liver tumours in rodents.
Laurus nobilis,

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Zingiber officinale,
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Cymbopogon spp.

Petroselinum crispum, Myristicin Myristicin and its 1-hydroxy-metabolite EFSA, 2009a; Zhou et
have genotoxic and carcinogenic al., 2007
Anethum graveolens,
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(especially hepatocarcinogenic) effects in

Heracleum spp., various rodent models.
Pastinaca sativa,
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Myristica fragrans
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Ocimum basilicum, Safrole Safrole is a well-documented genotoxic EFSA, 2009a; Swanson

carcinogen. It was shown to be genotoxic et al., 1979
Piper nigrum,
and to have carcinogenic (especially
Myristica fragrans hepatocarcinogenic) effects in rodents.
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2.2 Pyrrolizidine Alkaloids

Pyrrolizidine alkaloids (PAs) are secondary metabolites found in thousands of plant

species from more than 12 unrelated botanical families that are distributed throughout many
geographical regions in the world (Roeder, 1995; Roeder, 2000; Smith and Culvenor, 1981).
It has been reported that approximately 3 % of the world’s flowering plants (roughly 6000
species) contain more than 600 PAs, roughly half of them exhibiting chronic toxicity (Smith
and Culvenor, 1981) (Figure 2). Although toxic PA derivatives were detected also in certain

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species of Convolvulaceae, Poaceae and Lamiaceae, they are primarily found in the members
of the following four plant families (Reimann, 2004; Smith and Culvenor, 1981):
• Asteraceae – in plants of the Senecioneae subtribe (24 genera, the genus Senecio is
prevalent) and the Eupatorieae subtribe (mainly in the genera Eupatorium and
Ageratum),
• Boraginaceae – in virtually all plant species of this family,

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• Fabaceae – in the subtribe Crotalarieae (not found in Europe), mainly in the genus
Crotalaria but also in the genera Chromolaena and Lotononis, and

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• Orchidaceae – for instance, in the genus Liparis.

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Figure 2. Some of the PAs which were characterized as mutagenic and/or carcinogenic in
animal studies. PAs are derived from esters of basic alcohols and share a common chemical
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structure that consists of a necine base moiety.

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In addition to the well described hepatotoxic effects of PAs, many of these substances
have also been shown to have genotoxic and carcinogenic properties (EFSA, 2011). In vitro
and in vivo studies demonstrated that PAs produce, upon metabolic activation, DNA adducts,
cross-linking and breaks, leading to gene mutations (most frequently G:C → T:A
transversions and tandem base substitutions) and chromosomal aberrations (Chen et al., 2010;
EFSA, 2011). Studies in experimental animals that were exposed to different PAs or their

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metabolites have demonstrated the development of cancers in the liver, lungs, intestines,
bladder, kidneys, pancreas, skin, brain and spinal cord, adrenal glands, blood (leukaemia), and
muscle (Chan et al., 2003; Chen et al., 2010; Hirono, 1993). It has been shown in both
experimental animals and human cell cultures that one of the multiple known genetic targets
of toxic PA metabolites is the gene TP53, which encodes the well-known tumour suppressor
protein p53 (Chen et al., 2010; Couet, 1993; Ji et al., 2005). It has been hypothesized that the

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carcinogenesis is particularly favoured by extended, low-level, and intermittent periods of
exposure to PAs since they have been found to be not only genotoxic but also antimitotic

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(Mattocks, 1968). Intermittent low-dose exposure to PAs, which is a most likely scenario in
the case of PA-contaminated herbal preparation or food use and is associated with the DNA

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injury and the inhibition of mitoses, would, namely, allow intervening periods of normal or
even increased mitotic activity, providing an opportunity for the tumours to evolve (McLean,
1970; Rai et al., 2008; Schoental, 1968). The importance of the antimitotic activity of PAs for

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the carcinogenic process has, however, not been thoroughly substantiated by the experimental
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studies.
A list of plants for which concerns have been raised regarding their PA levels is
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presented in Table 2. Some of them are foraged by animals, while others are weeds that grow
among grains that are harvested for human use. These can therefore contaminate the food
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supply. Several of the listed herbs have been used as medicinals for many centuries (the
principal medicinal genera that are currently in use are Senecio, Tussilago, Borago,
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Lithospermum, Heliotropium and Eupatorium).

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Table 2. Plant species and specific PAs that may have genotoxic and carcinogenic effects.
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Plant Species Compounds Genotoxicity and Carcinogenicity Ref.

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Tussilago farfara Senkirkine, Several in vitro genotoxicity studies have Chen et al., 2010;
tussilagine, provided weak positive results for senkirkine. An COC, 2008;
senecionine increased incidence of hepatic adenomas was EFSA, 2011; Fu
observed in male rats after senkirkine et al., 2004;
Petasites spp. Senkirkine, administration. The Committee On IARC, 1983;
Senecionine, Carcinogenicity (COC) and the IARC have
seneciphylline concluded that the present data are not sufficient
to ascertain the carcinogenicity of senkirkine.

Adenostyles spp. Senecionine, Senecionine was found to form DNA adducts and Chen et al., 2010;
seneciphylline, was positive in several genotoxicity assays, COC, 2008;
spartioidine, including UDS assays and a Wing spot test. In EFSA, 2011; Fu
integerrimine spite of a number of negative results for et al., 2004; Gree
senecionine in the Ames test, it is most likely et al., 1981; Mori

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Leucanthemum spp. Spartioidine, carcinogenic. et al., 1985;
integerrimine, Newberne and
retrosine, Genotoxicity was indicated in a number of in vitro Rogers, 1973
Jacobine, and in vivo tests of riddelliine and monocrotaline.
senecionine Riddelliine demonstrated carcinogenicity in
experimental animals and has been listed also as a
probable human carcinogen. In male rats that
Senecio spp. Riddelline, were given monocrotaline, liver cell carcinomas
floridanine, developed.
monocrotaline,
otosenine,

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senecionine

Symphytum spp. Symphytine, Genotoxic potential was identified for symphytine Chen et al., 2010;

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lycopsamine, in a Wing spot test. In experimental animals COC, 2008;
echimidine, exposed to symphytine, an increased incidence of EFSA, 2011; Fu
symglandine liver haemangiosarcomas and hepatic adenomas et al., 2004
was observed.

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Myosotis spp. Symphytine,
lasiocarpine,
myoscorpine

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Lycopsamine, Lycopsamine displayed no mutagenicity in Ames Chen et al., 2010;
intermedine, tests, but was found to be mutagenic in EFSA, 2011; Fu
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Eupatorium spp. supinine, Drosophila. Lycopsamine and its intermediates et al., 2004
rinderine, demonstrated only weak hepatic toxicity in rats.
echinatine
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Lithospermum spp. Lycopsamine,

intermedine,
lithosenine,
triangularine
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Cynoglossum Heliotrine, Many in vitro and an in vivo tests have Chen et al., 2010;
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officinale lasiocarpine, demonstrated the genotoxic potential of EFSA, 2011; Fu

cynoglossophine lasiocarpine and heliotrine (including clastogenic et al., 2004; NTP,
effects). Hepatic angiosarcomas were observed in 1978; Schoental,
Heliotropium spp. Lasiocarpine, a 2-year study of lasiocarpine in rats. In a study of 1975
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heliotrine, male rats that were exposed to heliotrine,

indicine increases in the incidence of pancreatic islet cell
heliosupine, tumours, transitory cell papillomas of the urinary
echinatine bladder and intestinal testicular tumours were
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observed.
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2.3 Ptaquiloside and Bracken Fern

Bracken fern (Pteridium aquilinum) is one of the most common fern species with a
wide geographical and ecological distribution. It is present in all continents, except in
Australia, from subtropic to subarctic areas (Taylor, 1990). Bracken fern is a very adaptable
plant and is capable of forming dense, rapidly expanding populations in course of the first
phases of the ecological succession in forest cleanings and other disturbed rural areas

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(Pakeman and Marrs, 1992). Its aggressive growth, charaterized by an extensive rhizome
system and rapidly growing fronds, sometimes enables it to be a dominant species in certain
plant communities, for instance in the understorey of pine and oak forests, in heather (Calluna
vulgaris) stands, acid grasslands and moors (Le Duc et al., 2003; Smith and Taylor, 1995).
Bracken contains different poisonous agents, including some cyanogen glycosides
(e.g., prunasin), two types of thermolabile thiaminases, a few thermostable thiamine

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antagonists (e.g., caffeic acid, astragalin and isoquercetin), and carcinogenic compounds (e.g.,
ptaquiloside and shikimic acid) (Alonso-Amelot and Oliveros 2000; Konishi and Ichijo, 1984;

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Meyer, 1989) (Figure 3). Ptaquiloside is certainly the major carcinogen in bracken (Smith and
Seawright, 1995). The distribution of ptaquiloside in a variety of ferns has been examined in

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Japan using chromatography and a modified Ames test (Saito et al., 1989). Nineteen of the 31
ferns that were tested were found to contain the potentially carcinogenic ptaquiloside and its
close analogues, in addition to Pteridium, and especially the ferns in the genera Pteris,

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Microlepia and Hypolepis (Saito et al., 1989).
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Figure 3. Some of the toxic compunds isolated from bracken fern, prunasin (a cyanogen
glycoside), ptaquiloside (the major carcinogen in bracken) and shikimic acid (a potential
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carcinogen-promoting agent).
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After the ingestion of the plant material or contaminated milk, both ptaquiloside and
its aglycone ptaquilosin are converted by the hepatic cytochrome P-450 enzymes into the
unstable dienone, which is regarded as the ultimate carcinogen (Kigoshi et al., 1993). The
dienone metabolite is strongly electrophilic, and it rapidly reacts with amino acids and
nucleotides, forming covalent adducts with DNA and causing DNA strand breaks (Ojika et
al., 1987; Ojika et al., 1989). Crude bracken extracts that were incubated with liver
microsomal systems have been found to be positive in Ames tests (Matsuoka et al., 1989).

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Furthermore, it was found out that ptaquiloside can cause chromatid exchanges in Chinese
hamster lung cells and unscheduled DNA synthesis in rat hepatocytes (Evans, 1986; Hirono et
al. 1984; Matsuoka et al., 1989; Mori et al., 1985; Saffhill et al., 1985). On the other hand,
shikimic acid and its metabolite cyclohexane carboxylate were not mutagenic in Ames tests,
and whether they are carcinogenic in experimental animals and humans remains controversial
(Jones et al., 1983). Hence, although shikimic acid is unlikely to be a cancer-initiating agent,

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it may act as a carcinogen-promoting agent (Jones et al., 1983).
Bracken carcinogenicity in animals had already been demonstrated fifty years ago

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(Evans and Mason, 1965). Many subsequent studies performed in either the field or the
laboratory have associated the ingestion of bracken or its extracts with diseases and cancers in

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many sites in different animal species, such as rats, hamsters, guinea pigs, cattle, sheep and
toads (Evans, 1987). The consumption of bracken was linked to a number of different,
generally well recognised syndromes, some of which are species specific (Smith, 2004): i)

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bovine enzootic haematuria and mixed tumours of the urinary bladder (in cattle), ii) acute
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haemorrhagic disease and upper alimentary carcinoma (in cattle and sheep), iii) thiamine
deficiency (in horses and sheep), and iv) retinal atrophy (in sheep). Three main routes of
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exposure to the toxic effects of bracken fern have been proposed in humans, i.e., consumption
of the plant, ingestion of milk from affected animals and inhalation of its spores.
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Bracken ferns have been part of human diets from antiquity. Prehistoric man is
thought to have regularly consumed bracken rhizomes in winter and the young fronds in the
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spring. During the First World War, the rhizomes of these plants were consumed by people in
Scotland (Vetter, 2009). Today, its fronds are traditionally eaten in Japan and some parts of
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South America, while its rhizomes are a part of the diet of Aboriginal populations and Maori
(Hirayama, 1979; Vetter, 2009). In certain parts of the world, especially in Russia, China and
Japan, bracken fern is even grown commercially for human use (Vetter, 2009). The usual
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procedure that is performed before eating the plant (boiling in water in the presence of
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different chemicals, such as soda ash) appears to render their components less harmful,
presumably by inactivating some of their toxic agents. Nevertheless, some carcinogenic
activity persists (Hirayama, 1979). In some countries (e.g., Japan, Brazil and Venezuela), a
close association was identified between the consumption of bracken fern and cancers of the
upper gastrointestinal tract (Alonso-Amelot and Avendano, 2002; Hirayama, 1979). It has
been found that the concentration of ptaquiloside in fronds increases with their maturation
process, while it is almost absent in rhizomes (Alonso-Amelot et al., 1992; Vetter, 2009).

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Ptaquiloside has been identified in the milk produced by bracken-fed cows, and the
concentrations at which it was found were observed to be roughly linearly dependent on the
dose that was ingested by the cow (Alonso-Amelot et al., 1996). The proportion of
ptaquiloside that was excreted with the milk was estimated to be as high as 8.6 % of the total
consumed substance (Alonso-Amelot et al., 1996). It is quite plausible that the ptaquiloside in
the milk of these animals is responsible for the association between bracken infestation and

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the incidence of gastric cancer in the populations of farmers that inhabit cattle-range areas in
Costa Rica and other countries where bracken growth is dense (Villalobos-Salazar, 1995).

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Indeed, tumours have been demonstrated in rats that were fed with the milk of cows that had
grazed bracken (Villalobos-Salazar, 1995). While this route of exposure may be relevant in

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small, geographically remote rural communities, the dilution resulting from the bulk
processing of milk is thought to reduce any risk from milk in most developed countries to
negligible levels (Vetter, 2009).

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Because spores have been found to be carcinogenic in mice, there is a theoretical risk
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that the inhalation and subsequent ingestion of spores could also be carcinogenic in humans.
To date, there have been no studies of the effects of exposure of animals to airborne spores,
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but studies have explored the effects of directly ingesting the spores (Povey et al., 1996). In
mice, spores administered by gavage produced DNA adducts (Wilson et al., 1998).
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Nevertheless, the significance of these adducts requires further investigation.

Recently, ecotoxicological studies investigating the concentration of bracken fern
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ptaquilosides in the soil of the bracken-populated areas were performed. The investigation
conducted in Denmark, has, for instance, detected the following concentrations: 0.008–40.6
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µg ptaquiloside/g dry soil in the autumn and 1.56–212 µg ptaquiloside/g dry soil in late winter
(Rasmussen et al., 2005). It has been established that the risk of ptaquiloside soil leaching
depends on many environmental factors, such as the soil pH (it is the highest in slightly acidic
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to neutral soil), the clay content and the rate of microbiological degradation (Rasmussen et al.,
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2005; Vetter, 2009). However, the health risk implications of the presence of ptaquiloside in
soil and in the groundwater for animals and humans are not well established and require
further ecotoxicological research.

2.4 Other carcinogenic plants

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The compendium of plants that are either wild-growing, cultivated or extensively
consumed in Europe that may have carcinogenic potential has not yet been exhausted. In the
following list (Table 3), an overview of the remainder of plants that might be of concern
because they have genotoxic and/or carcinogenic potential is provided. For the majority of the
selected botanical ingredients, carcinogenicity has been either reported or suspected.

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Table 3. List of plant species with potentially genotoxic and/or carcinogenic properties.

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Plant Species Compound Genotoxic and/or Carcinogenic Activity Ref.

Rubia tinctorum Lucidin, alizarin Lucidin and alizarin have genotoxic Blömeke et al.,

SC
properties (they were positive in a battery of 1992; EFSA,
short-term in vitro tests). Furthermore, 2009a; Westendorf
carcinogenic activity was demonstrated in
et al., 1988
rats (intestinal and liver tumours).

Galium odoratum, Coumarin

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The epoxide metabolites of coumarin Abraham et al.,
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produced in the liver of rats and mice were 2010; Bom et al.,
Melittis
shown to be carcinogenic, they caused 2003; NTP, 1993;
melissophyllum,
adenomas and carcinomas of the liver, bile
SCF, 1999
Verbascum spp., ducts and lungs, and adenomas in the
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kidneys. In contrary, hydroxy metabolites of

Anthoxanthum spp.,
coumarin produced in human liver are not
Melilotus spp., classified as carcinogens (Group 3 by
IARC).
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Cassia spp,
Lavandula spp.
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Aristolochia spp., Aristolochic Aristolochic acids are genotoxic and form Arlt et al., 2002;
acids DNA-adducts. They were indicated to be Chen et al., 2012;
Asarum spp.,
carcinogenic in rats, mice and rabbits, and Jelaković et al;
Bragantia spp. also in humans. Urothelial cancers have
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2012
namely been observed in patients suffering
from aristolochic acid nephropathy.
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Aloysia citriodora, Citral Several in vitro as well as in vivo tests for EFSA, 2009c;
Melissa officinalis, genotoxicity have shown negative results for NTP, 2003; Sinha
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citral (but positive results were found in et al., 2014

Cymbopogon spp. sister chromatid exchange assays and comet
tests). No evidence was found for the
carcinogenic activity of citral in rodents (but
lymphomas were induced in female mice).
Citral is considered safe for human use.

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Thymus spp., Myrcene No evidence of genotoxicity was found in in NTP, 2010
vitro and in vivo genotoxicity assays for
Humulus lupulus, myrcene. However, administration of
Cannabis spp., myrcene to rodents induced liver and kidney
tumours.
Petroselinum crispum

Citrus limon and 8- 8-Methoxypsoralen was demonstrated to NTP, 1989 and

Citrus reticulates Methoxypsoralen have genotoxic activity in several in vitro 2010
(essential oil and and in vivo assays. Carcinogenicity was

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(methoxsalen)
exocarp), detected in male rats. It is classified as Goup
1 carcinogen, but it has carcinogenic activity
Levisticum officinale,
only in combination with UV radiation.
Petroselinum crispum

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Mentha spp. Pulegone Mutagenicity assays of pulegone have Da Rocha et al.,
(especially M. provided negative results. Carcinogenic 2012; EMA, 2014;

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pulegium), activity was found in mice (liver tumours, NTP, 2011
osteomas, osteosarcomas) and in rats
Agastache spp. (urinary bladder tumours).

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Cycas spp. Cycasin Cycasin is a mutagenic and a carcinogenic Sieber et al., 1980
AN
agent, but only when it is deglucosylated to
its principal metabolite,
methylazoxymethanol (interestingly, in
adult mammals, the deglucosylation of
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cycasin is catalysed only by enzymes

present in the microflora of the gut). Long-
term administration of cycasin and
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methylazoxymethanol acetate by oral and

intraperitoneal routes has been shown to be
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hepatotoxic and carcinogenic in old-world

monkeys.
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3. Teratogenic plants
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In the case of pregnant women, the use of any drug or product, either natural or
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synthetic, must be especially seriously considered in light of the risk-benefit ratio. Many
substances can harm a developing embryo or foetus, especially when they are used during the
critical periods corresponding to organogenesis in the first trimester of pregnancy.
Nevertheless, throughout the world, many pregnant women consume a wide variety of herbs
and herbal products during pregnancy for many reasons that may be related or not to
pregnancy. For example, herbal products are used to treat nausea, vomiting, dyspepsia and
constipation, to induce abortion, to treat infections of the urinary tracts, to prepare for labour,

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etc. (Glover et al., 2003; Nordeng and Havnen, 2005). Plant products are still being routinely
administered to women during pregnancy and childbirth, especially in developing countries.
Plant toxins that can efficiently cross the placenta and that can induce congenital
malformations in a developing embryo or foetus when present at critical time periods during
gestation are called to have teratogenic effects (Keeler, 1984). Therefore, exposure to these
toxins in the prenatal stage can lead to teratogenesis, but many of them can also have a

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negative impact on the postnatal growth and maturation processes (developmental toxicity).
Many teratogenic mechanisms have been associated with the use of herbal products. These

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include the disruption of the microtubule assembly process, the folate antagonism, the
induction of oxidative stress, the specific receptor-mediated teratogenesis and DNA alkylation

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(Schmeller et al., 1997; Van et al., 2010; Wink, 2007 and 2008).

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3.1 Anthraquinone Glycosides
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Plant species containing anthraquinones (Figure 4), including senna glycosides or
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sennosides, cascarosides, emodine and rhein, are part of the formulations of numerous
medicinal products that are mainly used for the treatment of constipation. Anthranoid
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laxatives act by exciting peristalsis and increasing fluid secretion in the colon. Worldwide, the
most common such stimulant is made of sena species (Cassia angustifolia Vahl; Cassia senna
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L. – syn. Senna alexandrina Mill). However, many other plants containing anthraquinone
glycosides, such as those derived from Frangula alnus L., Rhamnus spp., Aloe spp. and
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Rheum spp., are often included (Roberts and Tyler, 1999).

C
AC

Figure 4. Chemical structures of two typical anthraquinone glycosides, found in

Cassia spp. (sennoside A) and Rheum spp. (cascaroside A).

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It is well known that constipation is very common in pregnant women. Although this
is not the topic of the current article, in which we focus mainly on the chronic toxicity issues,
it is appropriate to mention here that the use of laxatives containing anthraquinones might
potentially be dangerous because they can induce acute unwanted effects, such as uterine
contractions and increased blood flow to the uterus and its attachments, thereby increasing the
risk of foetal loss (Acs et al., 2009; Conover, 2003). The anthraquinones may even pass into

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breast milk to cause unwanted effects, such as spasms, in an infant (Acs et al., 2009).
The potential teratogenic effects of senna were recently examined in a case-control

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epidemiological study. No significant risk was found for more than 20 different congenital
abnormality groups in relation to treatment with senna during the second and third gestational

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month. It was also demonstrated, somewhat surprisingly, that the gestational age at delivery
was somewhat longer (by 0.2 week) and that the rate of preterm birth was lower (6.6 % vs.
9.2 %) in the newborn infants of mothers who had used senna during pregnancy (Acs et al.,

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2009).
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3.2 Alkaloids
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The use of the herbs mentioned in Table 4 should be avoided during pregnancy
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because abortifacient and/or teratogenic potential (demonstrated mainly in animal studies) has
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been linked to their alkaloid content (Figure 5). Many of these plants can also induce acute
(Lupinus spp., Veratrum spp., Conium spp., and the included members of the family
Solanaceae) or chronic (neurotoxicity: certain species in the genera Astragalus and
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Oxytropus; carcinogenicity: Cycas spp.; hepatotoxicity: certain PA-containing species)

toxicity.
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Table 4. List of plant species containing alkaloids that are potentially teratogenic.

Plant Species Active Constituents Responsible for Teratogenicity and their Ref.
Toxicity

Lupinus spp. Quinolizidine and piperidine alkaloids, such as lupinine Bunch et al., 1992;
(Figure 5), ammodendrine and anagyrine, which cause Keeler, 1983 and 1984;
congenital contracture-type skeletal malformations and cleft Panter et al., 2013
palates in foetuses (especially in cows, sheep and goats).

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Nicotiana spp. Piperidine alkaloids, such as anatabine (Figure 5) and Bunch et al., 1992;
anabasine, which cause congenital contracture-type skeletal Crowe and Swerczek,
malformations and cleft palates in foetuses (especially in 1974; Keeler, 1984;
pigs). Panter et al., 2013

Oxytropis spp. and The indolizidine alkaloid swainsonine, an alphamannosidase Bunch et al., 1992;
Astragalus spp. inhibitor that induces neurological defects of foetuses and Keeler, 1984; Lather et
(certain species of vasoconstriction in the placentae of sheep and cattle. al., 2011; Panter et al.,
these two genera, 2013
toxicity varies)

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Veratrum spp. Steroidal alkaloids, including veratridine (Figure 5), Incardona et al., 1998;
cyclopamine, cycloposine and jervine, which cause Keeler, 1984; Lather et

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craniofacial malformations, including cyclopia, in the al., 2011; Panter et al.,
foetuses of sheep, horses, goats and cows. 2013

Cycas spp. Quinolizidine and piperidine alkaloids. Lather et al., 2011

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Trigonella foenum- Alkaloids, such as trigonelline, that can cause a decrease in Araee et al., 2009;
graecum bone marrow cell proliferation and disrupt foetal Gaffield and Keeler,
development in rats and rabbits. 1994

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In Morocco, cases of pronounced congenital malformations,
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including hydrocephalus, anencephaly and spina bifida, were
linked to the consumption of fenugreek seeds during
pregnancy. However, this causality remains questionable
because fenugreek is widely used by people in Morocco and
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neighbouring countries.

Ruta graveolens Quinoline and quinolone alkaloids that induce changes in the De Freitas et al., 2005;
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formation of blastocysts, reducing the number and delaying Gutierrez-Pajares et al.,

the development of embryos in mice. Another study in mice 2003
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also showed no significant difference in the loss of embryos

before implantation between the treated and control groups,
but deaths were observed in the foetuses of treated females,
suggesting an embryotoxic effect.
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Conium maculatum Piperidine alkaloids, including coniine and γ-coniceine, Bunch et al., 1992;
which cause congenital contracture-type skeletal Keeler and Balls, 1978;
malformations, cleft palates, restricted foetal movements and Keller et al., 1980;
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arthrogrypotic limb deformities in calves. Panter et al., 2013

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Solanum spp. (green Glycoalkaloides, including solanidanes and spirosolanes, Gaffield and Keeler,
parts of plants and which are capable of inhibiting certain cholinesterase 1994
unripe fruits) enzymes and increasing the permeability of mitochondrial
membranes (thereby promoting apoptosis). These can cause
brain defects, spina bifida and cleft palate in the foetuses of
sheep and cattle.

Some PA-containing PAs (especially heliotrin) have been found to be teratogenic Edgar et al., 2011;
plant species, in rats (e.g., growth retardation, abnormal skeletal Wiedenfeld, 2011
especially development, and deficiencies in ossification). The exposure

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Heliotropium spp. of pregnant women to PAs through the consumption of
herbal teas, herbal medicines, and spices has been linked to
fatal hepatic sinusoidal obctruction syndrome (HSOS) in
human neonates.

Datura stramonium, The tropane alkaloid hyoscyamine, which causes deformities Lather et al., 2011;
in foals during foetal development. Panter et al., 2013
Mandragora
officinarum,
Hyosciamus spp.,

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Atropa belladonna

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M
D
TE
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Figure 5. Some of the alkaloids that were found to have teratogenic effects:
lupinine (from Lupinus spp.), anatabine (from Nicotiana spp.) and veratridine (from
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Veratrum spp.).
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3.3 Other Potentially Teratogenic plants

Other plant species in which the pharmacological potential to cause teratogenicity has
been described but not studied in as much detail as the previously mentioned plants are listed
in Table 5.

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Table 5. List of other potentially teratogenic plants that grow (or are available) in Europe.

Plant Species Active Constituents Responsible for Ref.

Teratogenicity and their Toxicity

Cannabis sativa Causes brain defects in the foetuses. Reece, 2009

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Sorghum bicolor Cyanogenic glycosides that can cause contracture-type Lather et al., 2011;
skeletal defects. Panter et al., 2013

Podophyllum spp. Absent finger anomalies, septal defects of the heart, Lather et al., 2011

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defects of the external ears and skin tags in human
foetuses.

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Prunus spp. (especially Cyanogenic glycosides that can cause contracture-type Panter et al., 2013
seeds of P. dulcis and P. skeletal defects and cleft palate.
armeniaca)

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Lathyrus spp. (L. cicera Osteolathyrogens, including γ-aminopropionitrile, which Panter et al., 2013
and L. odorata) cause congenital skeletal malformations in cows and
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sheep.
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4. Plants with Endocrine-Disrupting Potential

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4.1 Endocrine-Disrupting Chemicals with Plant Origins

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In recent decades, great concern has arisen regarding the potential human health risks
that may be associated with exposure to endocrine-disrupting chemicals (EDCs). Endocrine
disruptors are defined as exogenous natural or synthetic substances that alter the function of
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the endocrine system and that consequently produce adverse health effects in an organism or
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its descendants. EDCs influence homeostasis, development, and cell proliferation by

mimicking or inhibiting the actions of endogenous hormones or in other way altering the
function of the endocrine system (Choi et al., 2004; Cooper and Kavlock, 1997). EDCs may
interfere with the synthesis, storage, release, metabolism, transport, elimination, or receptor
binding of endogenous hormones (Cooper and Kavlock, 1997). Many EDCs, including
industrial chemicals, pesticides, environmental contaminants, heavy metals, and
phytoestrogens, have been associated with reproductive disorders in both men and women.
These include impaired fertility, decreases in sperm count and quality, cryptorchidism,

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hypospadias, miscarriages, endometriosis and irregularities in the menstrual cycle (Balabanic
et al., 2011).
In addition to the endocrine-disrupting potential of phytoestrogens, another well
studied type of endocrine imbalance that has been linked to plant substances is presented in
this review, i.e., pseudohyperaldosteronism, associated with the ingestion of products
containing licorice (Glycyrrhiza glabra).

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4.2 Phytoestrogens

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Phytoestrogens are plant secondary metabolites that are structurally and/or

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functionally similar to mammalian oestrogens or their active metabolites. The main groups of
phytoestrogen are the lignans, flavonoids (subgroups isoflavones, coumestans and

U
prenylflavonoids) and stilbenes (Limer and Speirs, 2004; Moon et al., 2006; Sirtoti et al.,
2005). Lignans are the main phytoestrogens in a normal diet, and they are found in many
AN
cereals, fruits and vegetables. They can be subdivided into enterolactones and enterodiols,
which have very weak oestrogenic properties (Limer and Speirs, 2004). Isoflavones are a
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subgroup of flavonoids that are most abundant in soybeans and other legumes but that are also
present in berries, wine, grains and nuts (Kurzer and Xu, 1997). They include, for example,
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genistein, daidzein and biochanin A (Figure 6). Isoflavones display variable oestrogenic
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activity, and they are considered to be natural SERMs (selective oestrogen receptor
modulators) (Zand et al., 2000). Isoflavones act as antioxidants in vitro, where they have been
shown to have antiproliferative activities (Fotsis et al., 1993; Peterson, 1995). Compared to
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studies on isoflavones, significantly fewer studies have been performed using coumestans,
prenylflavonoids and stilbenes. Coumestans are potent activators of oestrogen receptor (ER)
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signalling pathways, but they are not as common in the diet as other phytoestrogens (Kurzer
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and Xu, 1997; Limer and Speirs, 2004). The chemopreventive potential of the most common
stilbene, resveratrol, against breast cancer has been studied in rodent models (Limer and
Speirs, 2004).
Phytoestrogens can be regarded as endocrine disruptors and could potentially be
harmful to human health. However, many health benefits have been associated with their use,
including the alleviation of menopausal symptoms, especially the reduction of the frequency
of hot flushes, and a lowered risk of developing atherosclerosis, ischaemic heart disease,
osteoporosis and breast cancer (Chen et al., 2015; Patisaul and Jefferson, 2010; Schmidt et al.,

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2016). As previously mentioned, we are exposed to phytoestrogens on a daily basis because
they are widely distributed in plants that are components of our diets. In spite of their weak
affinity for oestrogen receptors (ER), they therefore cannot be neglected as EDCs, and their
consumption could have profound physiological effects (Patisaul and Jefferson, 2010). In the
next subsection, we consider the mechanism of action of phytoestrogens and reveal that their
interaction with oestrogen receptors is in some way similar to the SERM/ER interaction.

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Fugure 6. Structures of three isoflavones which are characteristic for soy and certain other
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legumes, such as red clover, alfalfa and peanuts.
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Although the ER signalling is characterized as the principal mechanism of the

phytoestrogen action, phytoestrogens do not exert their effects solely through ERs. Instead,
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they have the potential to affect a wide array of intracellular signalling mechanisms that are
important for regulating cellular growth, proliferation and protection against harmful
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environmental influences (Oseni et al., 2008; Ruiz-Larrea et al., 1997). Several

phytoestrogens are powerful antioxidants and antiinflammatory agents, including isoflavones
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and resveratrol (Ruiz-Larrea et al., 1997). Daidzein and genistein are the two best-
characterized isoflavones. Human exposure to these compounds occurs primarily through the
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consumption of soy-based food and beverage products. Some isoflavones, most notably
genistein, inhibit pathways that are important for cell growth and proliferation, thereby
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affecting multiple organ systems (Oseni et al., 2008; Piontek et al., 1993). Genistein, for
instance, inhibits the activity of protein tyrosine kinases, which are very important regulatory
proteins (Piontek et al., 1993).
In vitro assays have shown that although most phytoestrogens, including the
isoflavones, bind both ERα and ERβ and activate ER-dependent gene transcription through
both receptor subtypes, they generally have a higher relative binding affinity for ERβ than for
ERα (Kuiper et al., 1997; Kuiper et al., 1998). Once bound, isoflavones do not act like typical
oestrogen agonists; instead, they act like SERMS, such as tamoxifen, which acts as an ER
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agonist in the uterus and bone but as an antagonist in the breast, where it has proven
anticancer activity (Oseni et al., 2008). This differential activity by phytoestrogens and
SERMS results, in part, from the profile of co-activator and corepressor proteins in the cell
being affected (Kushner et al., 2000; Oseni et al., 2008). Once bound to ERs, phytoestrogens
can initiate transcription either classically, through interactions with oestrogen response
elements (ERE), or by binding to early immediate genes, such as Jun and Fos (Kushner et al.,

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2000).
Phytoestrogens, particularly the isoflavones, fit the definition of an endocrine

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disruptor. In animal models, they produce effects such as premature reproductive senescence,
compromised fertility, disruption of the lactation and the timing of puberty, the inhibition of

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the sexual behaviour etc. (Jefferson et al., 2007; Patisaul et al., 2001; Patisaul and Jefferson,
2010; Setchell et al., 1987). The exposure to high levels of phytoestrogens could therefore
pose a risk to human reproductive health, especially when taken by women diagnosed with

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endometriosis or those experiencing irregular menstrual cycles. In addition, phytoestrogens
AN
should be consumed in moderate amounts by women diagnosed with estrogene-sensitive
cancers and during the critical periods, such as puberty and adolescence, pregnancy or
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lactation. Phytoestrogens have been namely shown to be able to perturb normal menstrual
cycles in humans, for instance, their intake in high amounts can prolong the menstrual
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bleeding (Strom et al., 2001) or cause dysmenorrhea, worsen the clinical course of
endometriosis, induce postmenopausal bleedings or elicit endometrial prolifeartion with
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polyps (Chandrareddy et al., 2008). Nevertheless, there is a relative lack of studies among
humans regarding effects of the phytoestrogen-induced hormonal disbalance on fecundity and
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fertility. Some studies have even demostrated that the phytoestrogen supplementation in
women can be even associated with improved reproductive outcomes, such as higher
ovulation and implantation rates (Kohama et al., 2005; Unfer et al., 2004), shorter time to
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pregnancy (Mumford et al., 2014), higher pregnancy rates among couples with unexplained
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infertility (Shahin et al., 2008). Studies in men mainly reported no harmful effects of
phytoestrogens on their reproductive capacity (Beaton et al., 2010; Mitchell et al., 2001),
except for two reports which associated their intake with lower sperm concentration
(Chavarro et al., 2008) and with idiopathic male infertility (Xia et al., 2013).
Amog researchers, there is still no definite arrangement whether the phytoestrogens
increase or reduce the risk of developing breast cancer (Enderlin et al., 2009). While the
majority of epidemiological studies and meta-analyses concluded that the high intake of soy
isoflavones is associated with reduced breast cancer risk in pre- and postmenopausal women

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(Boucher et al., 2013; Lee et al. 1991; Schmidt et al., 2016; Trock et al., 2006; Verheus et al.,
2007; Wu et al., 2008), some studies state that the opposite might be true (Maskarinec et al.,
2004; Padilla-Banks et al., 2006). Recently, it has been demonstrated that the use of soy
isoflavones is safe in women with the history of breast cancer and is not contraindicated in
women on hormone therapy with tamoxifen and anastrozole (Kang et al., 2010; Schmidt et
al., 2016; Wu et al., 2007). Studies even indicate that the isoflavone intake improves the

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prognosis of breast cancer patients, prolonging their life and decreasing the cancer recurrence
rates (Marini et al., 2008; Shu et al., 2009).

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4.3 “Oestrogenic” Plant Species

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Mainly through our diet, we are exposed to a continuous and complex mixture of plant

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substances that, as outlined above, probably have a substantial impact on our endocrine
system. “Oestrogenic” plant compounds are widespread in food, including herbs and
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seasonings (e.g., garlic and parsley), grains (wheat, rye, and oat), vegetables (e.g., soybeans
and beans), fruits (cherries and apples), and drinks (coffee). In Table 6, we present the plants
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that are known to contain very high levels of phytoestrogens (Geller and Studee, 2006;
Poluzzi et al., 2014; Thompson et al., 2006); the list is not exhaustive.
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Table 6. Plant species with potentially endocrine-disrupting potential as a result of their high
level of phytoestrogens.
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Plant Species Major Phytoestrogenic Constituents Chemical Group

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Glycine max * Genistein, daidzein, glycitein, coumestrol Isoflavones, coumestans

Arachis hypogea Genistein, resveratrol Isoflavones, stilbenes

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Cicer arietinum Biochanin A Isoflavones

Psoralea corylifolia Isobavachalcone, bavachin, corylin Isoflavones

Bituminaria bituminosa Bitucarpin A and B Isoflavonoids (pterocarpans)

Medicago sativa * Spinasterol, coumestrol, coumestan, Phytosterols, coumestans,

formononetin
isoflavones

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Trifolium pratense * Coumestrol, trifoliol, formononetin, Coumestans,
biochanin A
isoflavones

Trifolium repens Coumestrol, trifoliol Coumestans

Linum usitatissimum Secoisolariciresinol, matairesinol Lignans

Sesamum indicum Sesamolin, sesamine, pinoresinol, Lignans

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lariciresinol, hydroxymatairesinol

Secale cereale Syringaresinol, lariciresinol Lignans

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Triticum spp. Hydroxymatairesinol, Syringaresinol, Lignans
Secoisolariciresinol

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Avena sativa Pinoresinol, lariciresinol, Lignans
hydroxymatairesinol

Brassica spp. Pinoresinol, lariciresinol, coumestrol Lignans, coumestans

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Humulus lupulus 8-Prenylnaringenin (hopein), xanthohumol, Prenylflavonoids (flavanones)
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isoxanthohumol

Glycyrrhiza spp. (some Liquiritigenin, glabrene, glabridine Prenylflavonoids (flavanones),

species) * isoflavones
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Vitis spp. Resveratrol Stilbenes

Morus spp. Resveratrol Stilbenes

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Fallopia japonica Resveratrol Stilbenes

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* Found in medicinal products used to treat perimenopausal ailments.

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4.4 Pseudohyperaldosteronism and Glycyrrhiza spp.

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Pseudohyperaldosteronism is a condition that clinically mimics hyperaldosteronism,

including the suppression of plasma renin activity and aldosterone levels. It can be a
consequence of an overconsumption of licorice, carbenoxolone or grapefruit juice, but it can
also be caused by genetic (Liddle syndrome) and endocrinal (congenital adrenal hyperplasia)
defects. The excessive use of licorice is by far its most significant dietary cause (Obolentseva
et al., 1999; Palermo et al., 2003).
The fabacean genus Glycyrrhiza consists of approximately 20 species. Two of them,
G. glabra and G. uralensis, are generally recognized as a source of licorice because of the

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distinctive sweet taste of their roots and stolons (Society of Japanese Pharmacopoeia, 2012).
This taste can be attributed to the triterpenoid substance glycyrrhizin, which is a mixture of
potassium, calcium and magnesium salts of glycyrrhizic acid (Obolentseva et al., 1999). The
level of glycyrrhizin that is present in the roots of different Glycyrrhiza species varies from 2
to 25 %, depending on growth conditions (Obolentseva et al., 1999).
Licorice is popular not only as a food and drink sweetener. It has also been used by

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tobacco companies as a flavouring agent and in herbal medicines, especially in China and
Japan. There, it has been used for decades to treat gastric ulcers (Obolentseva et al., 1999).

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However, this particular use of licorice has become obsolete as a result of the emergence of
newer, more powerful and safer prescription drugs. Nevertheless, licorice continues to be

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included in many health products and could even gain new medical interests as a result of
recent research findings which show that licorice has antiviral, antiinflamatory,
antithrombotic and hepatoprotective activities (Coon and Ernst, 2004; Marianecci et al., 2012;

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Mendes-Silva et al., 2003; Tsuruoka et al., 2009).
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The traditional belief that licorice is a healthy, natural substance without side effects
drives its broad consumption, which can in some cases be dangerous. The active compound in
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licorice extracts, that is the glycyrrhizic acid, and its metabolites that are produced in the
intestine and in the liver, i.e., glycyrrhetinic acid and 3-monoglucuronyl-glycyrrhetinic acid
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have mineralocorticoid-like activities and can cause pseudohyperaldosteronism (Joshino et al.,

2014; Makino, 2014; Stewart et al., 1987). This results from its direct binding to
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mineralocorticoid receptors in the kidneys (minor reason) and the inhibition of two enzymes
that are involved in the corticosteroid metabolism (major reason) (Calo et al., 2004; Joshino et
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al., 2014; Whorwood et al., 1993). Licorice extract (especially 3-monoglucuronyl-

glycyrrhetinic acid) specifically inhibits 11-ß-hydroxysteroid dehydrogenase (11-ß-HSD) type
2, which metabolises cortisol to cortisone. Subsequently, the activity of cortisol, which binds
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as avidly to the mineralocorticoid receptor as aldosterone, is increased (Funder et al., 1988).

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Furthermore, glycyrrhetinic acid also inhibits the hepatic metabolism of aldosterone by

suppressing 5-ß reductase activity (Latif et al., 1990; Makino, 2014).
Licorice overconsumption is most often characterised by muscle weakness, pain and
numbness, oedema of the face and lower extremities, and variously severe hypertension that is
accompanied by a reduction in plasma renin and aldosterone levels. This differentiates its
symptoms from those of primary or secondary hyperaldosteronism (Joshino et al., 2014; Lin
et al., 2003; Makino, 2014; Stewart et al., 1987). In addition to low serum levels of potassium
(due to its increased secretion in the kidneys), metabolic alkalosis (resulting from an increase

27
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in renal proton secretion) and a raised cortisol to cortisone ratio in the peripheral venous
plasma (resulting from the inhibition of 11-ß-HSD) are also found in most cases (Joshino et
al., 2014; Stewart et al., 1987). In cases with rhabdomyolysis (due to severe and long lasting
hypokalemia), creatine phosphokinase may be also elevated, and mioglobinuria with acute
kidney failure may ensue (Joshino et al., 2014; Mumoli and Cei, 2008; Murphy et al., 2009;
Omar et al., 2012; Stewart et al., 1987).

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The main difficulty in dosing licorice lies in in the fact that it is available in various
food and medicinal items that contain highly variable levels of licorice. In 1991, the European

RI
Union proposed a provisional dose of 100 mg/day as the upper limit for the ingestion of
glycyrrhizin (approximately the amount found in 60–70 g of licorice) (Murphy et al., 2009;

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Omar et al., 2012). Based on data obtained in studies of human volunteers, this upper limit
was later confirmed by the Scientific Committee on Food, which also stated that human
toxicity studies were too limited to define an exact safe average daily intake for glycyrrhizic

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acid (SCF, 2003b).
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5. Conclusions
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D

While there has long been widespread awareness among people regarding the acute
toxicity of herbs and their preparations, their potential for inducing chronic toxicity has
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received the attention it deserves only in the last few decades. A number of compounds with
the potential to induce chronic toxicity have been found in plants that are used in various
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botanical preparations. Among these, alkenylbenzenes and pyrrolizidine alkaloids present a

special concern for human health because of their genotoxic, carcinogenic and teratogenic
potential. The basic intention of this review was therefore to gather and systematically present
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what is currently known about these aspects of herbal toxicity and to describe how they could
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have a significant impact on public health.

Issues related to the adverse reactions that have been associated with different herbal
products, the use of which is growing exponentially, have become increasingly exposed in
recent times. Safety assessments of herbal medicines have become an important issue for
consumers, regulatory authorities, and healthcare professionals, but analyses of the adverse
events that are related to these products is much more complex than analyses of conventional
pharmaceuticals (Ekor, 2014). This is especially true in the case of chronic toxicity, where
causality can be very difficult to establish.

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Botanicals are self-prescribed and widely available, which make them difficult to
control. Furthermore, folklore and folk practices, and not scientific studies, are often used as a
means of obtaining information on the indications, efficacy and safety of medicinal plants and
herbal medicines (Lather et al., 2011). Many gatherers, manufacturers and consumers of
herbal medicines lack the appropriate level of botanical and toxicological knowledge. They
may be prone to misidentifying plant species, and they may be unfamiliar with the scientific

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binomial classification of plants. This lack of knowledge can lead to mistakes resulting from
confusion that is created by the numerous common names that are in use among people (Ekor,

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2014; Farah et al., 2000). In addition, the variability of secondary metabolites with
medicinally valuable or toxic effects in herbal products, which are not under strict control, is

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very high, as it depends on a myriad of factors, e.g., the plant phenotype, the soil and climate
characteristics, the harvest time, the preparation procedure etc. All of these constraints also
limit the efficiency of monitoring the safety of herbal medicines, which essentially requires

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the close collaboration of different experts in the field. The same basic standards should
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therefore apply to the recognition and reporting of herbal toxicity that apply to prescription
medications. The systematically collected data about the frequency of toxic reactions and the
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understanding of their etiopathogenesis and clinical manifestations are needed to enable

improvements in the safety of herbal medicine use.
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European Medicinal and Edible Plants Associated with Subacute and
Chronic Toxicity

Part I: Plants with Carcinogenic, Teratogenic and Endocrine-Disrupting Effects

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HIGHLIGHTS

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• Many edible plant species contain alkenylbenzenes, which have genotoxic and
carcinogenic properties.

•
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Many plants from Asteraceae and Boraginaceae contain carcinogenic pyrrolizidine
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alkaloids.

• Humans are mainly exposed to bracken fern carcinogen ptaquiloside through plant
material and milk consumption.
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• Although phytoestrogens have many beneficial properties, they could also pose a risk
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to human reproductive health.

• Licorice overconsumption leads to pseudohyperaldosteronism characterized by muscle

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weakness, oedema and hypertension.

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