High resolution melting analysis to discriminate artichoke (Cynara scolymus) in plant food supplements
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abstract
Artichoke (Cynara scolymus L.) is a medicinal plant mainly used for its antioxidant,
diuretic, choleretic and hepatoprotective properties, being frequently included in weightloss
plant food supplements (PFS) (Lattanzio et al, 2009). PFS are legally considered as
foods under EU Directive 2002/46/EC, which means that PFS are not submitted to any
safety assessment prior to their commercialisation. This can lead to adulteration issues.
such as accidental swap ofplants or deliberate substitution ofhigh value plant material by
other species of lower cost. In arder to ensure consumer's safety, the development of
analytical methods for plant species identifícation in complex matrices hás become cmcial.
Só far DNA-based methods have been reported as the most adequate tools for plant
authentication (Kazi et al, 2013). Thus, the main goal of the present study was to
discriminate C. scolymus from other Cynara spp. in PFS by real-time polymerase chain
reaction (PCR) coupled to high resolution melting (HRM) analysis. For this purpose,
differeat Cynara species (C. scolymus, C. cardunculus, C. humilis and C. syriaca} were
obtained from Portuguese, Spanish and French germplasm banks. A total of eight PFS
samples containing artichoke were acquired at local herbal stores. DNA fi-om plant
material and PFS was exbracted using the commercial NucleoSpin Plant II kit/The
specificity and sensitivity of the designed primers targeting the C. scolymus (GenBank
EU744973. 1) were assayed by qualitative and real-time PCR. Prior to the specific
amplification of C. scolymus, DNA extracts were positively tested targeting an universal
eukaryotic sequence (18S rRNA gene). The application of the specific PCR assay was
successfül for the detection ofthe genus Cynara in some ofthe PFS samples. The results of
real-time PCR coupled to HRM analysis showed that different Cynara ~spp. were included
in three distinct clusters with a levei of confidence above 99.4%, thus discriminating
artichoke from other Cynara species. The proposed HRM analysis allowed confirming the
unequivocal presence of C. scolymus in the tested PFS with high levei of confídence
(>98. 8%). To our knowledge, this is the first successfül attempt for the rapid
discrimination ofC. scolymus in PFS.
This work was supported by European Union (FEDER fünds through COMPETE) and National Fundds (FCT Fundação para a ciência e Tecnologia) through projecïs EXPL/DTP-SAP/1438/2013 and UID/QUI/50006/2013. T.J.R. Fernandes and J. Costa are grateful to FCT grants (SFRH/BD/93711/2013 and SFRH/BPD/102404/2014, respectively) financed by POPH-QREN (subsidised by FSE and MCTES).
