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Detection of botanical adulterations in plant food supplements by molecular biology techniques

Publication . Amaral, Joana S.; Costa, Joana; Fernandes, Telmo J.R.; Batista, Andreia; Oliveira, Beatriz; Mafra, Isabel

In the last years, botanicals have become increasingly available in the EU market in the form of plant food supplements (PFS), which are legally considered as foods under Directive 2002/46/EC and consequently not submitted to safety assessment prior to commercialisation. A concern related with PFS regards its botanical composition since unintentional swap of plants has been reported and also because adulterations by the substitution of higher cost botanicals for closely related, but cheaper species, can occur. Thus, there is a need for reliable methodologies to authenticate botanicals in commercialised PFS. Recently, molecular biology techniques have been suggested for this purpose. However, difficulties in recovering DNA from some PFS samples have been described (1). Thus, as part of a study for the botanical authentication of PFS, this work aimed at assessing the interference of pharmaceutical excipients on the recovery/amplification of DNA. Different PFS (tablets and capsules) were submitted to DNA extraction and amplified by real-time polymerase chain reaction (PCR) targeting universal eukaryotic and plant genes using species-specific primers for Hypericum DNA barcode loci. However, some samples gave consistently negative PCR amplifications irrespective of the target gene or DNA extraction method used, raising the question of whether some excipients could interfere with DNA extraction from PFS. To address this question, model mixtures of pharmaceutical excipients and water as control, were spiked with known amounts of template maize DNA. Each mixture was then submitted to DNA extraction and maize DNA quantified by real-time PCR. The use of either 10% talc or 0.5 % dyes (iron oxide or titanium dioxide) completely adsorbed DNA, resulting in negative PCR amplifications. The use of 1% talc or 10% silica, both frequently used as diluents in PFS, allowed recovering very low amounts of maize DNA (7.1 % and 2.5%, respectively). The results showed a clear adsorption phenomena that justify the hampering effect on DNA extraction from PFS explaining the inability of recovering DNA from some samples reported in previous works. Thus, a strategy to release plant DNA from excipients, allowing its extraction and further analysis was also assayed. Hypericum species were not detected in four PFS, although being described on the label.

2015Conference object

Open access

Exploiting DNA markers for the authentication of Hypericum food supplements

Publication . Amaral, Joana S.; Costa, Joana; Fernandes, Telmo J.R.; Oliveira, Beatriz; Mafra, Isabel

During the last years. the consumption of plant food supplements CPFS) containing medicinal plants has been growing in popularity. Consequently, there has been an increasing demand for plant material that can result in a higher number of frauds (substitution of a higher cost medicinal plant for a closely related. but cheaper species) and possibility of unintentional swap/misidentiflcation of plants. In botll cases. the PFS integrity. efficacy and safety are compromised. Therefore. methodologies for the unequivocal identification of plant species in PFS are required. In this work. DNA-markers were used to specifically identify Hypericum perforatum (used in for its antidepressive properties) and H. androsaemum (used as cholagogue and hepatic protector) in several PFS samples (tablets. capsules. tintures and ampoules). Different DNA extraction protocols. including in-house methods and commercial kits were tested. Tile extracts were amplified by real-time PCR targeting reference genes (universal eukaryotic and plant rubisco genes) and using species-specific primers targeting a DNA barcode loci CmatK gene). Best results were achieved for capsules an tablets using Nucleospin Plant 11 extraction method. whi le for liquid samples using an in-11ouse method based on DNA precipitation with ethanol and centrifugation. Although labeled. three samples tested negative for H. perfuratum. For some samples. negative amplification was obtained regard less of the targeted gene and DNA extraction method. pointing to some matnx interference. possibly due to DNA adsorption phenomena to pharmaceutica l excipients.

2015Conference object

Open access

Development and validation of an HPLC-DAD-FL method for the determination of food supplements adulteration with undeclared phosphodiesterase type-5 inhibitor drugs

Publication . Rocha, Tiago; Santos, J.V. Araújo; Amaral, Joana S.; Oliveira, Beatriz

The consumption of food supplements has been increasing in developed countries. However, regulations and guidelines for this type of products reveal several gaps, and do not guarantee an efficient quality control, allowing for the possibility of intentional adulteration. Supplements used for improvement of male sexual performance are among the most popular food supplements. One of the major concerns in these products is the possible adulteration with synthetic drugs used for the treatment of erectile dysfunction, namely phosphodiesterase type-5 (PDE-5) inhibitor drugs, such as sildenafil, vardenafil and tadalafil. The side effects of these compounds and possible interactions with other drugs are well documented, thus its illegal addition to food supplements could pose a serious risk for consumers with known health constraints [1]. In the last years, the presence of this type of drugs have been detected by FDA in the US and reported in food supplements commercialized in Asia and the EU. Recently, Portuguese legal authorities reported the apprehension of some food supplements due to the presence of illegal PDE-5 inhibitor drugs. In this work, an high performance liquid chromatography (HPLC) based method was developed and validated for the detection of four PDE-5 inhibitors, namely sildenafil, vardenafil, tadalafil and yohimbine, in three sexual performance enhancement supplements. The analyses were performed by HPLC-DAD-FL in a JASCO chromatograph following the conditions of a previously published method [2]. A YMC-Triart C18 analytical column (3 μm, 250 × 4.6 mm) was used, together with (A) 50 mM Ammonium acetate; (B) acetonitrile/ methanol (50:50) as eluents. A simple liquid-liquid extraction with sonication using acetonitrile/methanol (50:50) was used in all samples. To validate the proposed methodology, the limits of detection (LOD) and quantification (LOQ), linearity range, intra- and inter-day precision and accuracy of the method were determined, showing high reproducibility scores and adequate recoveries for the tested compounds. One of the analyzed supplements showed the illegal addition of sildenafil.

2015Conference object

Open access

Assessment of plant food supplements adulteration with psychopharmaceutical drugs

Publication . Rodrigues, Manuela J.E.; Paíga, Paula; Santos, Lúcia H.M.L.M.; Amaral, Joana S.; Oliveira, Beatriz; Correia, Manuela; Delerue-Matos, Cristina

The purpose of this study was to compare three different extraction methods (two based on ethanol extraction and one on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERs) method) to assess the possible addition of psychopharmaceutical drugs (fluoxetine, sertraline, citalopram, venlafaxine, paroxetine, trazodone, and diazepam) as adulterants in St. John's wort (Hypericum perforatum) based plant food supplements (PFS). Methodology: Analysis was performed in a Nexera Ultra-High Performance Liquid Chromatograph (UHPLC) coupled to a triple-quadrupole mass spectrometer (LCMS-8030 Shimadzu) with an electrospray ionization source (ESI), operating in positive ion mode, using a Kinetex C18 fused core column (150 × 2.10 mm i.d.; 1.7 m) (Phenomenex). Multiple reaction monitoring mode (MRM) was selected and pharmaceuticals were quantified by internal standard calibration method. Calibration curves were constructed in the range 10 – 1000 g/L. The three different extraction methods were compared based on the analysis of spiked samples.

2015Conference object

Open access

High resolution melting analysis as a new tool to authenticate plant food supplements: the case of artichoke (Cynara Scolymus)

Publication . Batista, Andreia; Costa, Joana; Fernandes, Telmo J.R.; Amaral, Joana S.; Oliveira, Beatriz; Mafra, Isabel

Artichoke (Cynara scolymus L.) is a medicinal plant mainly used for its antioxidant, diuretic, choleretic and hepatoprotective properties, being frequently included in herbal infusions and plant food supplements (PFS) marketed for weight‐loss (Lattanzio et al, 2009). Both types of products can be adulteration targets, either by the deliberate substitution of other lower‐cost plant species, or by the accidental swap of plants owing to misidentification. Therefore, to ensure consumer’s safety, analytical methods for plant species identification in complex matrices are crucial. For this purpose, DNA‐based methods have been reported as the most adequate tools for plant authentication. Genetic composition of each plant is unique and independent from the part of the plant used (Kazi et al., 2013). Moreover DNA molecules are very stable, not affected by the plant’s age, physical conditions or environmental factors, in opposition to chemical markers. In this work, a molecular approach based on real‐time PCR coupled to high resolution melting (HRM) analysis to discriminate C. scolymus from other Cynara species was developed and applied to the analysis of herbal mixtures and PFS labelled as containing artichoke as ingredient. For this purpose, different Cynara voucher species (C. scolymus, C. cardunculus, C. humilis and C. syriaca) were obtained from germplasm banks, while samples of herbal infusions (6) and PFS (8) were acquired at local herbal and dietetic stores. DNA from plant material and PFS was extracted using the commercial NucleoSpin Plant II kit. For Cynara spp. differentiation, new primers were designed on a microsatellite region of C. cardunculus (GenBank EU744973.1) for the development of qualitative polymerase chain reaction (PCR) and real‐time PCR assays. Prior to the specific PCR assays, DNA extracts were positively tested targeting a universal eukaryotic sequence (18S rRNA gene). The qualitative PCR results were specific for Cynara genus. Further development of real‐time PCR coupled to HRM analysis showed that the tested Cynara spp. were grouped in three distinct clusters with a level of confidence above 99.4%, thus enabling the discrimination of C. scolymus from the others. The analysis of commercial samples showed that, with the exception of one PFS sample, all samples were positive for the presence of the universal eukaryotic gene. All herbal infusions and three PFS were positive for the presence of Cynara spp. based on the qualitative PCR assay. The application of the proposed method of HRM analysis confirmed the unequivocal presence of C. scolymus with high level of confidence (>98.8%) in the tested samples. To our knowledge, this is the first successful attempt for the rapid discrimination of C. scolymus in PFS.

2016Conference object

Open access