Cynara Scolymus clustering in plant food supplements by high resolution melting analysis
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abstract
Plant food supplements (PFS) have been regarded with special interest since they are
concentrated sources of nutrients or other substances with a nutritional or physiological
effect, contributing to a better homeostatic balance. Weight-loss PFS, one of the most
consumed PFS, may include Cynara scolymus (artichoke) as an ingredient due to its
antioxidant, diuretic, choleretic and hepatoprotective properties [1]. Considered as foods
under the EU Directive 2002/46/EC, the PFS are not usually subjected to any safety
assessment prior to their commercialisation. This can lead to adulteration issues, such as
accidental swap of plants or deliberate substitution of higher cost botanicals by other
similar, but cheaper species. Thus, to ensure consumer’s safety, the development of
analytical methods for the correct identification of different plant species in PFS has
become essential. Until now, DNA-based methods have been reported as highly appropriate
tools for plant authentication [2]. The aim of this study was to discriminate C. scolymus
from other Cynara spp. using real-time polymerase chain reaction (PCR) coupled to high
resolution melting (HRM) analysis.
For this purpose, different Cynara species (C. scolymus, C. syriaca, C. cardunculus and C.
humilis) were obtained from Portuguese and French Germplasm Banks. A total of 8 PFS
tablets for weight-loss containing artichoke were acquired at local herbal stores. DNA from
plant material and PFS was extracted using the commercial NucleoSpin Plant II kit. Former
to DNA extraction, PFS were pre-treated with a phosphate buffer 1M (pH8, 15% ethanol)
to enhance the purity and quality of the extracts. The specificity and sensitivity of the
designed primers targeting C. scolymus were assayed by qualitative PCR and real-time
PCR with HRM analysis. The application of the specific PCR assay was successful in the
detection of Cynara spp. in some of the PFS samples. The results of real-time PCR with
HRM analysis showed that different Cynara spp. were included in three distinct clusters
with a level 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 level of confidence (>98.8%). To our knowledge, this
is the first successful attempt for the rapid discrimination of C. scolymus in PFS.
This work was supported by European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through project EXPL/DTPSAP/1438/2013 and PEst-C/EQB/LA0006/2013
