In silico and experimental analysis of DNA markers for Citrus aurantium identification in herbal medicines
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abstract
Citrus aurantium L. belongs to the Rutaceae family and it is popularly known as bitter orange, Seville
orange, sour orange, bigarade orange, or marmalade orange. In traditional Chinese medicine, the fruits
are known as "zhi shi", being used as a stimulant of gastrointestinal function. In addition to the fruit itself,
the leaves are used in herbal infusions as aids in digestion, the flowers are used for their sedative
properties in cases of flu and the peel of the fruit is used as regulator of digestion. The fruit is traditionally
used as an antiemetic, antitussive, diaphoretic, digestive and expectorant. Adrenergic amines
(octopamine, synephrine, tyramine, methyltyramine and hordenine) are considered to be the most
important active compounds of C. aurantium. Synephrine, tyramine and hordenine act in fat metabolism,
activating lipolysis through adrenergic action and stimulation of beta-3 receptor [1]. Synephrine is a
chiral amine present in nature as (R)-(-)-p-synephrine, which is chemically similar to other
sympathomimetic amines, such as ephedrine (from Ephedra sinica). Therefore, bitter orange has been
used in weight-loss formulations with increased popularity [2]. Since plants in herbal preparations can
be substituted by others due to adulteration or to misidentification/accidental swap, to ensure
consumer’s safety and to verify labelling compliance, the development of adequate analytical methods
is mandatory. So far, DNA-based methods have been reported as highly appropriate tools for plant
authentication [3].
Therefore, the aim of this study was to develop a methodology for the specific identification of C.
aurantium using DNA-based methods. For this purpose, voucher leaves of C. aurantium, as well as
other 23 Citrus species (including C. sinensis – sweet orange) were gently provided by Germplasm
Banks. Commercial samples of herbal infusions having bitter orange as ingredient (n=6) were acquired
in supermarkets. DNA was extracted from all samples using the commercial Nucleospin Plant II kit. An
in silico study using 263 sequences of C. aurantium, available at NCBI database, was performed to
eliminate high homology with other Citrus spp. Eight genomic sequences (NCBI accession no.
HM172529, KM201256, KM201257, HM172529, HM172533, M172528, JN681151.1 and JN681151.1)
were selected and used to design new primers. Qualitative PCR results showed the identification of
Citrus genus targeting a resistance-like protein RGAs4-8-like encoding gene (NCBI accession no.
HM172533) and RGAs4-1-like encoding gene for the identification of C. aurantium (NCBI accession no.
M172528). Real-time PCR coupled to high resolution melting analysis using EvaGreen dye was also
performed with C. aurantium specific primers. The developed methodologies were further applied to the
analysis of the 6 commercial infusions labelled as containing bitter orange. PCR results showed
incompliances for 5 of these samples due to the absence of bitter orange DNA. This result can possibly
be related to a misidentification/swap of bitter orange (C. aurantium) with sweet orange (C. sinensis).
Future work will comprise the development of a methodology for the specific identification of C. sinensis
to confirm this hypothesis.
This work was supported by the project UID/QUI/50006/2013 – POCI/01/0145/FEDER/007265 with financial support from FCT/MEC through national funds and co-financed by FEDER.
