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Projeto de investigação
novel methods and approaches for detecting the illegal addition of Pharmaceutical drugs and bOtanIcal adulteRatiOn in planT food supplements
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Mass spectrometry‐based approaches to assess the botanical authenticity of dietary supplements
Publication . Grazina, Liliana; Mafra, Isabel; Monaci, Linda; Amaral, Joana S.
Dietary supplements are legally considered foods despite frequently including
medicinal plants as ingredients. Currently, the consumption of herbal dietary
supplements, also known as plant food supplements (PFS), is increasing worldwide
and some raw botanicals, highly demanded due to their popularity,
extensive use, and/orwell-established pharmacological effects, have been attaining
high prices in the international markets. Therefore, botanical adulteration
for profit increase can occur along the whole PFS industry chain, from raw
botanicals to plant extracts, until final PFS. Besides the substitution of highvalue
species, unintentional mislabeling can happen in morphologically similar
species. Both cases represent a health risk for consumers, prompting the development
of numerous works to access botanical adulterations in PFS. Among
different approaches proposed for this purpose, mass spectrometry (MS)-based
techniques have often been reported as the most promising, particularly when
hyphenated with chromatographic techniques. Thus, this review aims at describing
an overview of the developments in this field, focusing on the applications
of MS-based techniques to targeted and untargeted analysis to detect botanical
adulterations in plant materials, extracts, and PFS.
Molecularly imprinted polymer-based sensor for the determination of 2,4-dinitrophenol in food supplements
Publication . Torre, Ricarda; Rebelo, Patrícia; Seguro, Isabel; Pacheco, João G.; Amaral, Joana S.; Nouws, Henri P.A.; Delerue-Matos, Cristina
The rising demand for weight-loss solutions, driven by obesity concerns and societal pressure, has led to an increasing growth in both the consumption and market of food supplements. However, several studies have identified food supplements as common targets of economically motivated adulteration by the addition of pharmaceutical drugs to enhance the product's effects. Despite its toxicity and associated health risks, 2,4-dinitrophenol (2,4-DNP) is prone to be illegally added to weight-loss products due to its thermogenic effect, emphasizing the need for reliable detection methods. In this study, we report the first electrochemical sensor integrating a molecularly imprinted polymer (MIP) onto a paper-based platform for the selective detection of 2,4DNP. This innovative approach combines the high selectivity of MIPs with the cost-effectiveness, portability, and disposability of paper-based devices, paving the way for practical, on-site monitoring of 2,4-DNP in food supplements. The sensor features a paper-based transducer with a gold and carbon ink working electrode that was modified with the MIP, which was synthesized through precipitation polymerization. The sensor's performance was thoroughly evaluated, demonstrating a linear range of 50 - 1000 mu mol L-1 and a limit of detection of 16.3 mu mol L-1. The sensor was successfully applied to the analysis of various commercially available weight-loss food supplements (e.g., slimming activators, fat burners, thermoshape) in capsule form, yielding satisfactory recoveries ranging from 85 to 109%. These results highlight the potential of this MIP-based sensor for the rapid, sensitive, and selective determination of 2,4-DNP in food supplements, contributing to consumer safety and regulatory compliance.
Towards Botanical Authentication of Ginkgo Food Supplements: A Holistic Approach Based on Phytochemical and Genomic Markers
Publication . Grazina, Liliana; Paíga, Paula; Amaral, Joana S.; Costa, Joana; Moreira, Manuela M.; Delerue-Matos, Cristina; Mafra, Isabel
Ginkgo biloba is one of the most consumed medicinal plants and broadly included as an ingredient in plant food supplements (PFS) and herbal infusions, being potential targets for economically motivated adulteration. This work aimed at comparing the use of DNA and phytochemical markers to authenticate the botanical origin of ginkgo-leaf extracts and PFS. Quantitative real-time PCR was used to detect ginkgo DNA, while ultra-high performance liquid chromatography with tandem mass spectrometry detection (UHPLC-MS/MS) determined its main phytochemicals (terpene lactones and flavonol aglycones). DNA was detected in all ginkgo leaf extracts, mainly water, while the highest levels of phytochemicals were obtained using ethanol or acetone as solvents. The results suggested that 4 out of a total of 19 PFS samples were adulterated, with two samples evidencing the addition of quercetin from sources other than ginkgo. The other two samples showed low amounts of ginkgo phytochemicals, which was corroborated by low DNA content, suggesting the use of reduced amounts of G. biloba leaf material.
Development of a DNA-based methodology for the identification of Pfaffia glomerata in herbal products
Publication . Richter, Camila Palacio; Amaral, Joana S.; Maniglia, Thiago Cintra; Rodrigues, Vânia
Pfaffia glomerata (Brazilian ginseng) is a medicinal plant widely recognized for its adaptogenic, anti-inflammatory, and immunomodulatory properties. Due to its growing commercial value, as well as to its common name that includes the word “ginseng”, and the morphological similarity to other ginsengs, it is increasingly prone to adulteration. This study aimed to develop and validate a species-specific real-time PCR method for the identification and quantification of P. glomerata in commercial herbal products. Primers targeting the ITS1 region were designed and evaluated both in silico and experimentally. Specificity was assessed against 51 medicinal plant samples, including other species known for their adaptogenic properties.
Sensitivity assays confirmed an absolute detection limit of 0.001 ng of P. glomerata DNA.
Quantitative analysis using binary mixtures with Withania somnifera and the ∆Cq normalization method reliably detected P. glomerata at levels as low as 0.1% (w/w) and allowed the accurate estimate of the percentage of P. glomerata material in the range of 50% to 0.1% (m/m). The method was applied to 25 commercial products labeled as P. glomerata, Pfaffia paniculata, Pfaffia, Brazilian ginseng, or other possibly adulterant species of Brazilian ginseng, seven samples showed inconsistencies with the label and were considered as adulterated. P. glomerata DNA was also found to be present in nine other samples, but only at trace amounts (
Comparison of DNA barcoding and metabarcoding approaches for the authentication of herbal infusions in the Portuguese market
Publication . Pinto, M. Alice; Santos, Joana; Quaresma, Andreia; Honrado, Mónica; Rufino, José; Amaral, Joana S.
The authentication of commercial herbal products such as herbal teas and infusions is essential to ensure consumer safety and product integrity. In this study, 100 commercial herbal infusions, including 94 single-species products and six polyherbal formulations, were analysed using DNA barcoding (matK and rbcL) and ITS2 metabarcoding. Moreover, the performance of two custom reference databases (NCBI and BOLD) was compared in ITS2 metabarcoding. DNA extraction was successful for 94 samples, while six single-species products failed to amplify any of the tested barcodes. Among the 88 remaining single-species samples, ITS2 showed the highest amplification success (100 %), outperforming the barcodes rbcL (94 %) and matK (84 %). Sanger sequencing confirmed the labelled species in 69.3 % of cases with rbcL and 48.9 % with matK. While 63 samples would be considered authentic solely based on barcoding (i.e., if either rbcL or matK matched the label), ITS2 metabarcoding revealed that many of these contained additional undeclared species, indicating that barcoding alone overestimated product authenticity. Of the 85 samples successfully analysed by ITS2 metabarcoding, only 27 (32 %) fully matched their label, while 58 (68 %) contained either additional undeclared species or complete substitutions. Several products contained undeclared species in significant proportions, indicating potential economic adulteration. Analysis of mock-mixtures demonstrated that ITS2 metabarcoding can approximate ingredient proportions, though recovery bias may occur. Overall, the results revealed (i) the importance of curated and comprehensive databases, with a higher number of species being identified by NCBI database, (ii) the superior sensitivity of ITS2 metabarcoding, and (iii) the widespread mislabelling in commercial herbal products.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
Concurso de Projetos IC&DT em Todos os Domínios Científicos
Número da atribuição
PTDC/SAU-PUB/3803/2021