Percorrer por autor "Batista, Andreia"
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- Botanical authentication of globe artichoke-containing foods: Differentiation of Cynara scolymus by a novel HRM approachPublication . Grazina, Liliana; Batista, Andreia; Amaral, Joana S.; Costa, Joana; Mafra, IsabelCynara scolymus L., known as globe artichoke, is a medicinal plant widely used in plant food supplements (PFS) and herbal infusions due to its beneficial health properties. The high demand for artichoke-containing products can lead to adulteration practices. In this work, a real-time polymerase chain reaction (PCR) system coupled to high-resolution melting (HRM) analysis was proposed to differentiate C. scolymus from other Cynara species. Hence, a Cynara-specific real-time PCR assay was successfully developed with high analytical performance, achieving a sensitivity of 0.4 pg of globe artichoke DNA. HRM analysis enabled the discrimination of C. scolymus, with a high level of confidence (>98%), corroborating sequencing data. Application results to artichokecontaining PFS and mixed herbal infusions allowed confirming the presence of C. scolymus in 38% of the samples, suggesting the substitution/mislabelling of globe artichoke in 2 samples and the need for further efforts to increase DNA amplifiability of PFS.
- Cynara Scolymus clustering in plant food supplements by high resolution melting analysisPublication . Batista, Andreia; Costa, Joana; Fernandes, Telmo J.R.; Amaral, Joana S.; Ramos, Fernando; Oliveira, Beatriz; Mafra, IsabelPlant 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.
- Detection of botanical adulterations in plant food supplements by molecular biology techniquesPublication . Amaral, Joana S.; Costa, Joana; Fernandes, Telmo J.R.; Batista, Andreia; Oliveira, Beatriz; Mafra, IsabelIn 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.
- DNA extraction from plant food supplements: Influence of different pharmaceutical excipientsPublication . Costa, Joana; Amaral, Joana S.; Fernandes, Telmo J.R.; Batista, Andreia; Oliveira, Beatriz; Mafra, IsabelThe 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.
- 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, IsabelArtichoke (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.
- High resolution melting analysis to discriminate artichoke (Cynara scolymus) in plant food supplementsPublication . Batista, Andreia; Costa, Joana; Fernandes, Telmo J.R.; Amaral, Joana S.; Oliveira, Beatriz; Mafra, IsabelArtichoke (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.