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Quantitative genotoxicity assays for analysis of medicinal plants: A systematic

review

Article  in  Journal of ethnopharmacology · December 2015

DOI: 10.1016/j.jep.2015.10.026

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 Journal of Ethnopharmacology 178 (2016) 289–296

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Review

Quantitative genotoxicity assays for analysis of medicinal plants: A

systematic review
Graziela Sponchiado a, Mônica Lucia Adam b, Caroline Dadalt Silva c, Bruna Silva Soley c,
Cristina de Mello-Sampayo d, Daniela Almeida Cabrini c, Cassyano Januário Correr a,
Michel Fleith Otuki c,e,n
a
Departamento de Ciências Farmacêuticas, Universidade Federal do Paraná, Curitiba, PR, Brazil
b
Universidade Federal de Pernambuco- UFPE/CAV, Brazil
c
Departamento de Farmacologia, Universidade Federal do Paraná, Centro Politécnico, Curitiba, Brazil
d
Pharmacological Sci Department, Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
e
Departamento de Ciências Farmacêuticas, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Medicinal plants are known to contain numerous biologically active com-
Received 15 April 2015 pounds, and although they have proven pharmacological properties, they can cause harm, including DNA damage.
Received in revised form Aim of the study: Review the literature to evaluate the genotoxicity risk of medicinal plants, explore the geno-
17 October 2015
toxicity assays most used and compare these to the current legal requirements.
Accepted 17 October 2015
Available online 8 December 2015
Material and methods: A quantitative systematic review of the literature, using the keywords “medicinal plants”,
“genotoxicity” and “mutagenicity”, was undertakenQ to identify the types of assays most used to assess geno-
Keywords: toxicity, and to evaluate the genotoxicity potential of medicinal plant extracts.
Medicinal plants Results: The database searches retrieved 2289 records, 458 of which met the inclusion criteria. Evaluation of the
Systematic review selected articles showed a total of 24 different assays used for an assessment of medicinal plant extract gen-
Herbal extracts
otoxicity. More than a quarter of those studies (28.4%) reported positive results for genotoxicity.
Genotoxicity assays
Conclusions: This review demonstrates that a range of genotoxicity assay methods are used to evaluate the
genotoxicity potential of medicinal plant extracts. The most used methods are those recommended by reg-
ulatory agencies. However, based on the current findings, in order to conduct a thorough study concerning the
possible genotoxic effects of a medicinal plant, we indicate that it is important always to include bacterial and
mammalian tests, with at least one in vivo assay. Also, these tests should be capable of detecting outcomes that
include mutation induction, clastogenic and aneugenic effects, and structural chromosome abnormalities. In
addition, the considerable rate of positive results detected in this analysis further supports the relevance of
assessing the genotoxicity potential of medicinal plants.
& 2016 Published by Elsevier Ireland Ltd.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

Abbreviations: A, Acetone extract; AAE, Water–acetone extract; AC, Alliun cepa; ACE, Acetate extract; AE, Aqueous extract; AEE, Hydroalcoholic extract; AI, Apoptosis-inducing; Ames,
Bacterial reverse mutation assay; AN, Aspergillus nidulans; ANVISA, Agência Nacional de Vigilância Sanitária; C, Crude extract; CA, Chromosomal Aberration; CDSCO, Central Drugs
Standard Control Organization; CE, Chloroform extract; CHE, Cyclohexane extract; CHO, Chinese hamster ovary cells; DCME, Dichloromethane extract; DMSOE, Dimethylsulfoxide
extract; EAE, Ethyl acetate extract; EC, European Commission; EE, Ethanol extract; EFTA, European Free Trade Area; EMEA or EMA, European Medicines Agency; EO, Essential oil; ERA,
E. coli WP2 Reversion Assay; EURL ECVAM, EU Reference Laboratory for Alternatives to Animal Testing; FDA, Food and Drug Administration; FM, Forward mutagenesis; GE, Gly-
coalkaloid extract; HE, Hexane extract; HGPRT, HGPRT or HPRT gene mutation test; HME, Hydromethanolic; HMPC, Committee for herbal medicinal products; ICH, Harmonization of
Technical Requirements for Registration of Pharmaceuticals for Human Use; ICPM, Induction of Cytoplasmic Petite Mutations; L, Latex; ME, Methanolic extract; MHW, Japanese
Ministry of Health and Welfare; MI, Mitotic Index; MN, Micronuclei; NC, Not Clear; OECD, Organization for Economic Co-operation and Development; PDB, Plasmid DNA breakage; PE,
Phenolic extract; PEE, Petroleum ether extract; RI, Replication Indice; RXL, Ring-X-loss test; S, Seiva; SC, Saccharomyces cerevisiae; SCE, Sister Chromatid Exchange; SCGE, Single-
Stranded DNA Breaks-Comet; SHA, Sperm Head Anomaly; SLRL, Sex-linked recessive lethal; SMART, Somatic Mutations and Recombination Test; SOS, SOS chromotest/inductest; T,
Tincture; UC, Umu – C; UDS, Unscheduled DNA Synthesis; VIT, VITOTOX; WHO, World Health Organization
n
Corresponding author at: Departamento de Farmacologia, Universidade Federal do Paraná, Centro Politécnico, Curitiba, Brazil.
E-mail address: michelotuki@yahoo.com.br (M.F. Otuki).

http://dx.doi.org/10.1016/j.jep.2015.10.026
0378-8741/& 2016 Published by Elsevier Ireland Ltd.
290 G. Sponchiado et al. / Journal of Ethnopharmacology 178 (2016) 289–296

3. Results and discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
Appendix A. Supplementary material. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294

1. Introduction interaction may lead to chromosomal aberrations and/or changes

in DNA structure that may affect the fidelity of the message and
In the last decade, the use of medicinal plants has increased lead to irreversible changes in the cell (Varanda et al., 2002). The
substantially, either as agents employed in traditional medicine consequences of such DNA impairment could be the establishment
(Harvey, 2000) and/or as source material for the production of of and/or predisposition to diseases, increased morbidity/mortal-
dietary supplements, both in Western and Asian cultures. Fur- ity, changes in heritable characteristics and impaired reproductive
thermore, according to the World Health Organization (WHO) 70– capacity (Lázaro et al., 2010).
80% of the world population depends exclusively on herbs for their Thus, the aim of this study was to undertake a quantitative
primary health care (Chan, 2003; Muhammad et al., 2011). Despite systematic review of the literature in order to identify the assays
the extensive development of methods for synthesis of pharma- used to evaluate the genotoxicity of medicinal plants, explore the
ceuticals, medicinal plants still represent important sources of most used genotoxicity assays and compare them to the current
new molecular identities, mainly due to the fact that plants can legal requirements. This type of analysis is of valuable interest
synthesize and produce constituents that are difficult to obtain via since medicinal plants are used widely by the world's population
chemical synthesis. Compounds obtained from natural sources and are a potential source of therapeutic drugs. Therefore, safety
also can serve as prototypes for the synthesis of new drugs with assessments are crucial to validate the continuous use of medicinal
similar biological and therapeutic activities or be slightly modified plants and phytotherapy.
in order to make them more effective or less toxic (Harvey, 2000;
Munari et al., 2010; Regner et al., 2011; Robbers et al., 1996; Turolla
and Nascimento, 2006). 2. Methods
Although centuries of traditional usage apparently support an
often mistaken belief about the safety of medicinal plants, con- A systematic review was performed within articles published
trary to allopathic drugs, the toxicity of traditional herbal medi- from 2000 to February (4) 2015 (in the databases Medline (by
cines has not been evaluated completely in most cases and med- Pubmed), Science Direct, Web of Science (by ISI Web of Knowl-
icinal plants can be extremely harmful to human health. Studies edge), Scopus and Lilacs) using the keywords “medicinal plants”,
have revealed that some plants frequently used in folk medicine “genotoxicity” and “mutagenicity” and their MeSH terms. The
are potentially genotoxic (Ananthi et al., 2010; Marques et al., search strategy for Medline (by Pubmed) was ("mutagenicity
2003; Melo-Reis et al., 2011; Regner et al., 2011; Shin et al., 2011). tests"[MeSH Terms] OR ("mutagenicity"[All Fields] AND "tests"[All
The species identified as genotoxic include Cochlospermum re- Fields]) OR "mutagenicity tests"[All Fields] OR ("genotoxicity"[All
gium Pilg (Schrank) (Andrade et al., 2008; Castro et al., 2004), Fields] AND "testing"[All Fields]) OR "genotoxicity testing"[All Fields])
Ocotea duckei VATT (Marques et al., 2003), and Copaifera langsdorfii AND ("plants, medicinal"[MeSH Terms] OR ("plants"[All Fields] AND
Desfon (Chen-Chen and Sena, 2002). "medicinal"[All Fields]) OR "medicinal plants"[All Fields] OR ("medi-
Therefore, it is extremely important to assess genotoxicity cinal"[All Fields] AND "plants"[All Fields])); for Science Direct and
during the preclinical evaluation of herbal extracts/substances in Scopus was ("mutagenicity tests" OR ("mutagenicity" AND "tests")
order to verify their mutagenic potential for both safety and eco- OR ("genotoxicity" AND "testing") OR "genotoxicity testing") AND
nomic concerns since medicinal plants are widely used in folk ("plants, medicinal" OR ("plants" AND "medicinal") OR "medicinal
medicine and as a resource for the development of new drugs (Di plants" OR ("medicinal" AND "plants")); for Web of Science (by ISI
Stasi et al., 2002; Melo-Reis et al., 2011; Silva et al., 1995). On the Web of Knowledge) was (TS ¼("mutagenicity tests" OR ("muta-
other hand, these tests can also identify those plant extracts that genicity" AND "tests") OR ("genotoxicity" AND "testing") OR "geno-
have an antagonistic effect to genotoxicity, such as an anti-geno- toxicity testing") AND TS ¼("plants, medicinal" OR ("plants" AND
toxic, anti-mutagenic and/or anti-carcinogenic effect (Felício et al., "medicinal") OR "medicinal plants" OR ("medicinal" AND "plants")))
2011; Grover and Bala, 1993; Idaomar et al., 2002; Romero-Jimé- OR (TI¼ ("mutagenicity tests" OR ("mutagenicity" AND "tests") OR
nez et al., 2005; Villasenor et al., 1996). ("genotoxicity" AND "testing") OR "genotoxicity testing") AND TI ¼
Over the last 30 years, different methodologies, strategies and ("plants, medicinal" OR ("plants" AND "medicinal") OR "medicinal
approaches have been developed to assess chemicals that could plants" OR ("medicinal" AND "plants"))); and for Lilacs was ("mu-
demonstrate genotoxic and/or carcinogenic effects. The establish- tagenicity tests" OR ("mutagenicity" AND "tests") OR ("genotoxicity"
ment of these protocols has been based on studies demonstrating AND "testing") OR "genotoxicity testing") AND ("plants, medicinal"
the correlation between carcinogenicity and mutagenicity, and the OR ("plants" AND "m("mutagenicity tests" OR ("mutagenicity" AND
correlation of both parameters with genotoxicity (Dearfield et al., "tests") OR ("genotoxicity" AND "testing") OR "genotoxicity testing")
1991; Waters et al., 1999). Since then, established procedures have AND ("plants, medicinal" OR ("plants" AND “medicinal") OR "medic-
been used for evaluation of the genotoxic risk associated with the inal plants" OR ("medicinal" AND "plants")) AND db:("LILACS").
usage of drugs, food additives, pesticides, industrial and environ- In order to be considered in this analysis, articles had to meet
mental chemicals and also natural products, including medicinal the following two inclusion criteria: the genotoxicity assays must
plants and their oils (Chen-Chen and Sena, 2002; Costa et al., have been performed with crude extract, but not with isolated
2005; Vilar et al., 2008; Waters et al., 1999). compounds, formulations, mixtures of herbs, or fractions isolated
Genotoxicity assays are normally used to identify extracts/ from herbal extracts; and the articles must have been published in
substances that have the ability to interact with nucleic acids at English, Spanish or Portuguese. In order to ensure the consistency
low concentrations. When a toxic agent interacts with DNA, this and reproducibility of the process, we followed the
 G. Sponchiado et al. / Journal of Ethnopharmacology 178 (2016) 289–296 291

recommendations of the Cochrane Collaboration and PRISMA Guidelines on genotoxicity testing of defined pharmaceutical or
statement (Higgins and Green, 2011; Liberati et al., 2009) for chemical active ingredients intended for human use were estab-
systematic reviews. lished in the 1990s by the Organisation for Economic Co-operation
Two independent reviewers evaluated the articles by their ti- and Development (OECD, 1997a, 1997b, 1997c, 2014a, 2014b,
tles and abstracts, selecting them according to the inclusion/ex- 2014c, 2014d, 2014e) and by the International Conference on
clusion criteria set out above. Subsequently, the articles selected in Harmonization of Technical Requirements for Registration of
this initial screening were carefully evaluated and excluded or Pharmaceuticals for Human Use (ICH). Since then, these guidelines
permanently maintained in the review. In a detailed evaluation, have been adopted by the research-based pharmaceutical industry
data regarding the genotoxicity assays described in the articles associations and by the regulatory authorities of the three ICH
were extracted and descriptively analyzed. regions (European Union, Japan and USA) (BRASIL, 2010; CDSCO,
The assays and biomarkers were quantified according to the 2010; EMEA, 2008a; FDA, 1992, 1997; ICH-S2(R1), 2011; MHW,
number of times they were used and the frequency was calculated 1989). Efforts have been made to harmonize these requirements at
by comparing it to the total number of employed assays. The fre- the global level for ICH observers [Canada, the European Free
quency of positive genotoxic results was calculated as the sum of Trade Area (EFTA) and WHO] and non-ICH countries. Re-
plants with positive genotoxic results compared to the total commendations for implementation of these guidelines have been
number of articles selected. Lastly, the frequency of techniques developed by non-ICH countries, for example the Brazilian agency
that used human biological material was compared to the total ANVISA (ANVISA, Guideline on Nonclinical Safety Studies required
number of tests and biomarkers counted in this review. for medical products development or Phytomedicines Registration
– Genotoxicity assays), and by the regulatory agencies in other
countries including India and African countries (BRASIL, 2010;
3. Results and discussions
CDSCO, 2010; EMEA, 2008a; FDA, 1992, 1997; ICH-S2(R1), 2011;
MHW, 1989).
The database search retrieved 2289 records and 458 of those
The European Medicines Agency (EMA formerly EMEA) adop-
articles met the previously established inclusion criteria. The
ted the ICH guidelines on genotoxicity testing (ICH-S2) as EMA
evaluation of these selected articles included identification of the:
guidelines (CPMP/ICH/141/95, CPMP/ICH/174/95 both amended by
(i) medicinal plant, (ii) type of extract, (iii) biomarker, (iv) geno-
EMA/CHMP/ICH/126642/2008), which defines a battery approach
toxicity assay employed and (v) positive or negative genotoxic
for genotoxicity analysis including tests in mammalian cells in
result (Table 1). The evaluation of all of the selected articles re-
vitro and mandatory tests in mammalian models in vivo followed
vealed that a total of 24 different genotoxicity assay methods were
by an initial bacterial reverse mutation test, with and without
used and in most cases more than one technique was used to
metabolic activation (EMEA, 2008b; ICH-S2(R1), 2011). A brief
assess the genotoxicity of the herbal extracts.
summary of the two options for the standard battery of geno-
The assays most often employed by the authors for evaluating
toxicity testing present in ICH-S2 (ICH-S2(R1), 2011) is shown in
the genotoxicity of medicinal plant extracts included the Chro-
mosomal Aberration (CA), the Assessment of Mitotic Index (MI), Fig. 2. The committee for herbal medicinal products (HMPC), re-
the Allium cepa Assay (AC), the Salmonella/microsome or bacterial sponsible for preparing the EMA's opinion on herbal medicines
reverse mutation assay (Ames), the E. coli WP2 reversion assay according to regulation (EC) No.726/2004, has adapted the step-
(ERA), the VITOTOX Assay (VIT), the Umu-C Assay (UC), the SOS wise approach of genotoxicity testing in order to address both
Induc test (SOS), the Assessment of Somatic Segregation in As- scientific aspects of genotoxicity testing and the special needs of
pergillus nidulans (AN), the Gene Mutation in Saccharomyces cere- herbal medicinal products (EMEA, 2008b). In this approach, ad-
visiae (SC), the Somatic Mutations and Recombination Test ditional testing in mammalian cells in vitro and in vivo are only
(SMART), the Assessment of Recessive Mutation Lethal X-linked required in cases of equivocal or positive results in the bacterial
(SLRL) in Drosophilla melanogaster, The Ring-X-Loss test (RXL), the reverse mutation test (OECD, 1997a) and when those results can-
Sperm Head Anomaly (SHA), the Micronuclei assay (MN), The not be explained or clearly attributed to specific constituents with
Single-Stranded DNA Breaks (Comet- SCGE), the Apoptose-Indu- a well-established safety profile (for example quercetin) (EMEA,
cing (AI), the Locus of Hypoxanthine Guanine Phosphoribosyl-
Transferase (HGPRT) assay or HPRT gene mutation test, the Cell
Replication Indice (RI), the Unscheduled DNA Synthesis (UDS), the
Sister Chromatid Exchange (SCE), the Plasmid DNA Breakage
(PDB), the Forward Mutagenesis (FM) and the Induction of Cyto-
plasmic Petite Mutations (ICPM). Table 2 provides a brief de-
scription of the principles of each technique used for assaying
genotoxicity included in this review. As shown in Table 2, although
all reported assay methods share the same purpose, each one has
its own particular attributes and specificity in assessing DNA da-
mage. Most of the assays reported in our systematic review cor-
roborate the findings of Ouedraogo et al. (2012), who reported
current and "omics" methods for assessing the toxicity (geno-
toxicity, teratogenicity and nephrotoxicity) of herbal medicines
and mushrooms.
Of those assays described in Table 2, the micronucleus assay
(172 times), Ames test (171 times), comet assay (109 times) and
the chromosomal aberration assay (77 times) were the most fre-
quently used to evaluate the genotoxicity of herbal extracts (Fig. 1).
This observed frequency might reflect the implementation of Fig. 1. Frequency of most commonly used genotoxicity tests in the evaluation of
specific legal guidelines or requirements on the development of medicinal plant extract, as calculated from 458 article retrieved in the database
new drugs and medicinal products. search according to the specified inclusion criteria.
292 G. Sponchiado et al. / Journal of Ethnopharmacology 178 (2016) 289–296
2008b).
In the U.S., genotoxicity information is required for herbal
medicines, just as it is for active chemical ingredients, when re-
questing marketing authorization (Chen et al., 2008; FDA, 1992). In
the European Union, in addition to the full application for mar-
keting authorization as in the U.S., an abbreviated dossier can be
filed for many traditional herbal medicinal products. The main
requirement for a non-clinical assessment of well-established or
traditional herbal medicinal products is a documented history of
medicinal use with bibliographic data (EMEA, 2006). However,
particular attention should be paid to effects that are difficult or
even impossible to detect clinically. A complete lack of genotoxi-
city studies may present a safety concern (EMEA, 2008a). In ad-
dition, when data on genotoxicity are inadequate or absent, it will
not be possible for HMPC to endorse the herbal substance or
herbal preparation for inclusion in the Community list (EMEA,
2006, 2008a).
Nonetheless, one must keep in mind that herbal products are
marketed under different regulatory systems in diverse jurisdic-
tions with differing regulatory requirements depending on the
particular category, viz. herbal medicinal products, dietary sup-
plements/botanicals, and cosmetics (EMA, 1998; FDA, 1992, 1997;
ICH-S2(R1), 2011; Miroddi et al., 2013; Quintus and Schweim,
2012; Turolla and Nascimento, 2006).
In general, one of the primary aims is to know whether a
particular plant is capable of damaging genetic material, regard-
less of the organism. Thus the methodologies that make use of
microorganisms are the most used. Among these, we highlight the
Ames Test. The advantage of using tests with microorganisms is
mainly the speed with which results may be obtained due to the
short life cycle of the bacteria. Furthermore, the cost is low and
these tests do not involve a large burden on the laboratory.
However, these tests restrict the assessment of genetic damage
only to mutation induction. In this case, clastogenic and aneugenic
effects and structural chromosome abnormalities are not detected.
Thus, additional tests are needed in order to detect all possible
forms of genotoxicity (Benigni et al., 2010; Kirkland et al., 2011;
Lee et al., 2014; Mortelmans and Zeiger, 2000; Zhang et al., 2012).
The fact that the micronucleus assay (in vivo and/or in vitro) has
Fig. 2. A brief summary of the two options for the standard battery of genotoxicity
testing described in ICH-S2, assumed and adapted by the regulatory agencies in
other countries (e.g. Brazil, Africa and India). Option 1 – Bacterial assay and chro-
mosomal damage in vitro and in vivo. Option 2 – Bacterial assay and chromosomal
damage in vivo.
been adopted by regulatory medicine and advisory bodies as a
follow up test if the Ames and chromosome aberration (CA) assays
do not provide conclusive results or have positive results, re-
spectively, could explain why it has the highest frequency of use
(BRASIL, 2010; EC, 2009; ICH-S2(R1), 2011). The micronucleus as-
say (in vivo and in vitro) is highly reliable, rapid, and capable of
determining a vast spectrum of DNA damage at the chromosomal
level, especially for the assessment of mutagenic hazard (Hov-
hannisyan, 2010; OECD, 1997b). A recent publication by the EU
Reference Laboratory for Alternatives to Animal Testing (EURL
ECVAM), highlights the importance of utilizing an in vivo mam-
malian assay when positive Ames results are obtained. In their
database search, substances with positive Ames results did not
always have a positive in vivo mammalian cell test result. This
suggests that an Ames-positive chemical is not guaranteed to be
carcinogenic or genotoxic in vivo (Benigni et al., 2012; Kirkland
et al., 2014). However, in this review we found that positive Ames
test results (25% of 64% that only use Ames) were not always
followed up by another assay (in vivo or not) to elucidate the
genotoxicity potential of the herbal extract.
The comet assay (SCGE) in vivo or in vitro is the third most
frequently used assay in genotoxicity studies of herbal extracts
observed in this review, actually it is a second option of techniques
not specified by regulatory agencies. Despite not being a com-
monly required test in the guidelines' battery of genotoxicity tests
for the development of new medicinal drugs, this assay, together
with the micronucleus (MN) assay, has been recognized as an al-
ternative in vivo genotoxicity assay (ICH, 2012). The advantages of
the (SCGE) include its speediness, high sensitivity (Hovhannisyan,
2010; Kang et al., 2013), relative simplicity and low cost (Belpaeme
et al., 1998). It allows for quantification when needed and the re-
sults are viewed from a single cell obtained from in vitro or in vivo
samples (Hovhannisyan, 2010; Kang et al., 2013; Singh et al., 1988).
The assay is relevant to an increasing number of applications and
include, among others, evaluation of the genotoxic effects of sev-
eral agents on somatic and germ cells (Hartmann and Speit, 1997),
evaluation of DNA repair (Laffon et al., 2002), clinical applications
(Kassie et al., 2000; Sardas et al., 1998), and human (Frenzilli et al.,
2000; Kan et al., 2002) and environmental biomonitoring (Kassie
et al., 2001; Monarca et al., 2001).
Since medicinal plants are to be used in animals (with a
growing rate of usage) and mainly by humans, the application of
genotoxicity tests that use biomonitors and biomarkers that are
phylogenetically closer to these species are desired. The phyloge-
netic proximity allows greater accuracy in the observed response,
as close species respond to genomic damage in a similar way
(Adam et al., 2013; Willmer et al., 2009; Withers, 1992). This is due
to the fact that DNA repair mechanisms are evolutionarily con-
served. Thus, methodologies that make use of mammalian tissues
(especially blood) are recommended (Fenech et al., 2011). In this
review it was found that SCGE, MN, CA and SCE are among the
assays most used in humans with a rate of 53.33%, 32.00%, 25.33%
and 17.33%, respectively. However, methods requiring cell culture
are used only in specific cases, for example, to detect specific da-
mage on a given chromosome. The lower rate of use of these cell
culture-requiring techniques is also due to their high cost. Con-
versely, the MN and SCGE tests provide a safe, low-cost demon-
stration of a proven genotoxic action at the level of both macro-
and micro-lesions (Heddle et al., 1991; Speit and Hartmann, 1999).
Both techniques can detect mutagenic, clastogenic and aneugenic
effects, and are quantitative, not qualitative, methodologies. Thus,
non-specific damage is detected (Heddle et al., 1991).
A variety of biomarkers also can be used as biomonitors (Al-
bertini et al., 2000; Borras and Nadal, 2004; van der Oost et al.,
2003) to detect genotoxic effects in several organisms (fish, in-
sects, plants and mammals), tissues, and cells following exposure
 G. Sponchiado et al. / Journal of Ethnopharmacology 178 (2016) 289–296
to a feasibly toxic agent. When extrapolating the results to hu-
mans, the use of vertebrate organisms is recommended. Many
authors have considered the possibility of using fruit flies and
plants. However, vertebrates are phylogenetically closer to hu-
mans, and therefore have similar responses to genotoxic effects as
mentioned above (Adam et al., 2013; Willmer et al., 2009; Withers,
1992).
The analysis of the articles selected in this review revealed a
variety of cell types and species (experimental models/bio-
markers) used to assess the genotoxic effect of tested agents
(Fig. 3). These experimental models were divided into eight
groups: microorganisms, cell culture, insects, plants, crustaceans,
molluscs, fish and mammals. In each group a predominant cell
type/species was observed; for microorganisms the most common
was Salmonella thypimurium (76%), for cell culture it was Chinese
hamster ovary cells (CHO) (30%), for insects it was Drosophila
melanogaster (100%), for plants it was the Allium cepa (96%), for
crustaceans it was Brine shrimp (100%), for molluscs it was Helix
aspersa (100%), for fish it was Channa punctatus (100%) and for
mammals it was Swiss mice (52%) (Fig. 3). These results were not a
surprise since the most used genotoxicity tests use these cells and
species as experimental models, as mentioned in Table 2.
The Salmonella mammalian microsome mutagenicity assay has
been central to the field of genetic toxicology since the 1970s
(Maron and Ames, 1983). Some authors consider this assay a
model for the development of 21st century in vitro toxicology as-
says in terms of implementation of standard procedures, ability to
test various agents, transferability across laboratories, validation
testing, and structure–activity analysis (Claxton et al., 2010).
Genotoxicity tests should be able to assess the potential for
DNA damage. This damage can manifest as a mutation, numerical
changes or chromosomal recombination. Herbal extracts with
positive genotoxicity test results may be indicative of potential
carcinogenicity and/or mutagenicity risk to humans (BRASIL, 2010;
EMEA, 2008a; Ribeiro et al., 2003).
Of the 458 studies evaluated in this review, and published since
2000, 28.4% reported genotoxic activity for the tested medicinal
plant extracts (Table 1). Before an extract or another product from
a medicinal plant is used as a therapeutic agent, it is extremely
important to assess its genotoxic potential.
The incidence of false positives or false negatives is likely due
to methodological errors, considering that all techniques are
carefully optimized so that any observed genotoxic effect is due to
the test substance (Fowler et al., 2012). A false positive or false
negative result is mainly due to error and/or methodological
Fig. 3. Frequency of the most used biomarkers for assessment of the genotoxicity
of medicinal plant extracts, divided into eight groups: microorganisms, cell culture,
insects, plants, crustaceans, molluscs, fish and mammals; and for each group a
higher frequency of a particular cell type/species observed is shown.
293
artifacts, therefore it is recommended to test in duplicate and/or
triplicate to ensure the accuracy and reproducibility of the results
(FDA, 2012a; OECD, 1998; WHO, 2001).
Whereas both point mutations and chromosomal changes
(losses, inversions, duplications, breaks, translocations, amplifica-
tions, etc.) can activate genes related to oncogenesis, and since
genotoxic agents generally are not target-specific, any of the
methodologies may indicate the carcinogenic risk from exposure
to the tested substances (Bonassi et al., 2007; Mitelman, 2000;
Mitelman et al., 2007).
Ideally, for obtaining safety data regarding the use of medicines
in case of repeated administrations, such as medicinal plant ex-
tracts, more than one methodology and target organism will be
required (BRASIL, 2010; EMEA, 2008a; FDA, 2012b). However, it
should be considered that each individual responds in a particular
way according to his or her genetic individuality.
In actuality, a comparison of the sensitivity, false positive or
false negative rates between studies on the techniques used to
evaluate the genotoxicity of plants is almost impractical with the
data collected in this review. The studies are performed with dif-
ferently prepared extracts, using distinct solvents and sometimes,
even with different parts of the plant, as well as differences in
concentrations/doses. Thus, the use of different strategies to obtain
plant extracts results in the isolation of different chemical com-
ponents, possibly leading to different results in efficacy, as well as
in toxicity. Additionally, it is possible that the in vivo bio-
transformation of plant compounds could result in new com-
pounds (metabolites) or combinations of compounds that convey
toxic effects whereas the original extract did not, supporting the
need to include in vivo tests.
It is very difficult to draw conclusions from this critical analysis
of the sensitivity of employed techniques and the results derived
therein. This was because studies rarely use the same type of plant
product, concentrations, doses, or plant parts. This can be ob-
served in Table 1, and for example we highlight the cases of Aloe
vera (L.) Burm f. (Mehrabian et al., 2012; Sehgal et al., 2013; Vizoso
Parra et al., 2000a) and Anacardium occidentale L. (Cavalcante et al.,
2005; da Silva et al., 2013; Konan et al., 2007). In both plants,
studies were performed with different kinds of extracts (aqueous,
crude, hydroalcoholic, ethanolic, chloroform and methanolic) and
only one type of extract from each plant presented genotoxic ac-
tivity. Moreover, the evaluations of the reports on other plants
show a similar pattern, such as Brassica oleaceae L. var. acephala D.
C. (Gonçalves et al., 2012; Heres-Pulido et al., 2010), Moringa
oleifera* (Al-Anizi et al., 2014; Arora and Onsare, 2014; Rolim et al.,
2011), Liquidambar orientalis Mill. (Karadeniz et al., 2013; Saraç
and Şen, 2014), Ocimum basilicum L. (Beric et al., 2008; García
López et al., 2000), Persea americana* (Kulkarni et al., 2010; Pa-
dilla-Camberos et al., 2013).
The comparison of genotoxic studies of Moring oleifera shows
the importance of using a large range of concentrations or doses
and more than one technique to confirm genotoxicity (Al-Anizi
et al., 2014; Arora and Onsare, 2014; Rolim et al., 2011). Further-
more, Verma et al. (2012) study demonstrated that Aloe vera
(L) chloroform extract (CE) extract was positive in Mitotic Index
(MI) and Chromosome Aberration (CA) test results, but Sehgal
et al. (2013) and Vizoso Parra et al. (2000b) studies with crude
extract (C) and hydroalcoholic extract (AEE) extracts showed no
genotoxicity at the same or even higher doses. This is an example
of the different effects that can be observed between distinct
products from the same plant. The investigations of Liquidambar
orientalis Mill. had similar findings (Karadeniz et al., 2013; Saraç
and Şen, 2014).
Many examples are included in Table 1 that highlight the im-
portance of using both in vivo and in vitro techniques in order to
confirm the genotoxic effect of a medicinal plant. The studies of
294 G. Sponchiado et al. / Journal of Ethnopharmacology 178 (2016) 289–296
Cavalcante et al. (2005) and da Silva et al. (2007) demonstrated
that Anacardium occidentale L. has no genotoxic action in SMART
and Ames tests. However, an in vivo MN study, conducted by Ko-
nan et al. (2007), was positive, emphasizing the importance of an
in vivo analysis. Similar observations have been made with Aloe
vera (L.) Burm f. (Mehrabian et al., 2012; Sehgal et al., 2013; Vizoso
Parra et al., 2000a). Actually, in all of these situations the tests
using microorganisms presented negative results, whereas the
in vivo studies were positive. Are the microorganism results in
vitro a false negative result? These apparent conflicts possibly
could be explained by differences in cell specificity and sensitivity,
or the formation of metabolites with genotoxicity by in vivo me-
tabolism. There may be a different explanation for each case.
Again, this stresses the importance of running a wider range of
genotoxic tests.
In some situations the opposite results are obtained, ques-
tioning the possibility of false positive genotoxicity findings in
microorganisms. The plant Artemisia absinthium L. was positive in
the Ames test (Samonella typhimurium) but the effect was not
confirmed in MN testing in mice (Piloto Ferrer et al., 2000; Ta-
herkhani, 2014). However, once again, the plant extracts in the two
studies were prepared differently.
Yet, examples of the reproducibility of results were found when
evaluating the same extract by the same techniques and at the
same concentrations, as observed for Chenopodium multifidum L.*
(Gadano et al., 2000, 2006), Cynara Scolymus L. (Jacociunas et al.,
2012, 2013, 2014; Zan et al., 2013), Gentiana asclepiadea L. (Hu-
decová et al., 2012; Mihailović et al., 2013) and Cryptolepis san-
guinolenta* (Ansah and Gooderham, 2002; Ansah et al., 2005).
Hence further studies about the genotoxicity of medicinal plants
are needed, and an improved critical analysis is fundamental to
understand the genotoxic potential of plants and their extracts.
4. Conclusions
Evaluation of the genotoxic potential of plants intended for use
as medicinal products is essential to ensure that they are safe for
use. This systematic review has revealed that a wide range of
methods are used to evaluate the genotoxicity of crude plant ex-
tracts and indicates that the most commonly used methods are
those recommended by regulatory agencies. Among the published
studies, a high percentage have revealed positive genotoxic results.
In addition, we have noted the importance of always including
genotoxic assays in bacterial and mammalian species, with at least
one in vivo test, and using the same route as indicated for med-
icinal use. This recommended range of tests is capable of detecting
mutation induction, clastogenic and aneugenic effects, and struc-
tural chromosomal abnormalities, therefore covering all the pos-
sibilities of DNA damage and providing better safety outcomes for
the community. The aggregated results demonstrate another ex-
tremely important outcome of genotoxic tests: they can dis-
criminate the magnitude of damage to DNA of exposed in-
dividuals. Thus, the effects observed in the tests are also the most
likely effects to be observed in the target population. These ob-
servations highlight the fact that medicinal plants cannot be as-
sumed to be safe simply because they are natural. Hence, appro-
priate safety studies are needed as part of the development pro-
cess for plant extracts to be used as medicinal products, as well as
to define the safety of ancient ones.
Acknowledgements
G.S. is PhD student in pharmaceutical sciences and thanks
CAPES Grantee – Proc. n° 9878/11-4, B.S.S. is PhD and C. D. S. is
MSc student in pharmacology and thanks CAPES fellowship sup-
port. This study was supported by grants from Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) (Grant
nos. 307559/2012-2 and 480293/2012-0) and Fundação Araucária
(PR, Brazil) (Grant no. 279/2014).
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.jep.2015.10.026.
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