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DNA extraction from plant food supplements: Influence of different pharmaceutical excipients

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

The consumption of plant food supplements (PFS) has been growing globally, with an increase of misleading labeling and fraudulent practices also being reported. Recently, the use of molecular biology techniques has been proposed to detect botanical adulterations, one of the possible frauds in PFS. However, difficulties in recovering DNA from some PFS samples have been described. Aiming at using DNA-based methods for the unequivocal identification of plant species in PFS, adequate DNA isolation is required. However, PFS often contain pharmaceutical excipients known to have adsorbent properties that might interfere with DNA extraction. Thus, the aim of this work was to assess the effect of different excipients (talc, silica, iron oxide and titanium dioxide) on the recovery/amplification of DNA. For that purpose, known amounts of template maize DNA were spiked either to PFS or to model mixtures of excipients and quantified by real-time PCR. The tested excipients evidenced clear adsorption phenomena that justify the hampering effect on DNA extraction from PFS. The use of either 10% talc or 0.5% dyes completely adsorbed DNA, resulting in negative PCR amplifications. For the first time, pharmaceutical excipients were shown to affect DNA extraction explaining the inability of recovering DNA from some PFS samples in previous studies.

2015Journal article

Open access

High resolution melting analysis to discriminate artichoke (Cynara scolymus) in plant food supplements

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 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.

2015Conference object

Open access

Novel Strategies for Genetically Modified Organism Detection

Publication . Plácido, Alexandra; Amaral, Joana S.; Costa, Joana; Fernandes, Telmo J.R.; Oliveira, Beatriz; Delerue-Matos, Cristina; Mafra, Isabel

All rights reserved. Interest in DNA biosensors for genetically modified organism (GMO) testing is been growing due to their possibility for automation and microfabrication based on simple and portable detection systems. Biosensors can provide rapid, low-cost, sensitive, real-time, and high-throughput measurements suitable for in-field analysis. The efficiency of GMO diagnostics could be improved by simultaneously analyzing several targets, which is presently being exploited using the microarray platforms. As promising alternatives to conventional polymerase chain reaction, isothermal amplification strategies, such as loop-mediated isothermal amplification (LAMP), are low-cost and suitable in-field, enabling visual and electrochemical detection. This chapter intends to provide an overview on the advances in novel biosensing technologies, namely optical, piezoelectric, and electrochemical biosensors, as well as microarrays and LAMP applied to GMO testing.

2015Book part

Restricted 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

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