Micropropagation for the production of high quality phytochemicals

Plants area source of many valuable secondary metabolites that find a broad field of applications, ranging from the agrochemical to the pharmaceutical industries. Establishing a suitable source for extraction of phytochemicals is, however, not always straightforward. In many instances the production by chemical synthesis is not economically viable due to their complex structures and conservation issues may arise when they are harvested from natural sources. In vitro culture techniques offer an attractive alternative to these issues. Natural grown plants can be replaced by in vitro produced biomass with the advantage that several strategies can be implemented to increase production yields, such as genotype selection, altering growth conditions and use of elicitors, so that the higher investment costs are justified. Also, because plant tissue cultures can be generated on a continuous year-round basis without seasonal constraints, they can guarantee reliable and predictable production levels, which is of great importance for efficient process down-stream. Plant tissue culture techniques offer the possibility of establishing cultures from leaves, stems, roots and meristems, meaning that metabolites produced in specific plant organs can also be prospected. The successful production of a large number of phytochemicals from micropropagated biomass has been reported, and it seems that only in a few cases cultures fail to accumulate compounds of interest. The advantages and the range of possibilities offered by plant tissue culture techniques suggest that these might become a valuable and indispensable tool for the production of phytochemicals. In this work, the example of the prospection of plumbagin from micropropagated D. intermedia plants is described. Plumbagin is a naphthoquinone with potential pharmaceutical applications and results obtained by several hyphenated analytical techniques confirm that an end product with high purity and recoveries can be obtained from in vitro cultured plants. © ISHS 2013

In vitro plantlet production of the endangered Pinguicula vulgaris

This study describes the development of a micropropagation protocol for Pinguicula vulgaris using cultures initiated from in vitro produced seedlings. P. vulgaris is a carnivorous plant with a northern, disjunctly circumpolar distribution and specific habitat requirements, and is hence becoming increasingly rare. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher proliferation rates in 1/4MS, but was not affected by the addition of 0.1 mg/L 6-benzyladenine (BA) or zeatin (Zea). The best medium for propagating P. vulgaris was plant growth regulator (PGR) free A1/4MS. An average of 7.62 new shoots per initial explant could be obtained after 8 weeks of culture, of which over 79% produced roots during proliferation. Moreover, rooting percentages of 100% were obtained for the initial explants in all the tested media, including media without PGRs. The plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development

Aluminium speciation and internal detoxification mechanisms in plants: where do we stand?

Aluminium (Al) typically has a deleterious impact on plant growth and development but some plants can accumulate otherwise toxic concentrations of this metal by deploying internal detoxification mechanisms (tolerance mechanisms). Although there are scattered reports concerning the regulation of Al uptake, transport and storage in metabolically innocuous forms, the overall picture remains incomplete and there are no accounts of a general overview of Al tolerance mechanisms. This review aims to compile the current knowledge on the physiological basis of Al tolerance and speculate on possible underlying molecular mechanisms regarding the uptake of Al, speciation in internal organs, transport to upper organs and storage in non-vital structures. Given the difficulties in detecting and studying the speciation of Al, special attention will be given to the approaches used so far and new developments that allow greater sensitivity and accuracy in uncovering the fate of Al in planta. © 2013 The Royal Society of Chemistry

Phytochemical studies and biological activity of carnivorous plants from the Mediterranean region

Tese de doutoramento, Ciências Biotecnológicas (Biotecnologia Vegetal), Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2010In this thesis several studies were conducted with four carnivorous plant species which occur on Portuguese territory: Pinguicula lusitanica, Pinguicula vulgaris, Drosera intermedia and Drosera rotundifolia. Most habitats of these plants are threatened and natural populations are scarce, therefore micropropagation protocols were developed to produce biomass for the subsequent studies. Efficient micropropagation protocols were developed for P. lusitanica and D. intermedia enabling large scale biomass production, while protocols for the other two species have still to be optimized (in Chapter 2). The in vitro established cultures represent active germplasm collections of Portuguese natural populations and contribute therefore for their conservation. In Chapter 3 extracts prepared from micropropagated plant material were analyzed using state of the art HPLC-ESI-MS and HPLC-SPE-NMR equipment which enabled the identification of the major secondary metabolites produced by P. lusitanica and D. intermedia, directly from essentially crude extracts. The metabolites identified in P. lusitanica belong to the iridoid glucosides and caffeoyl phenylethanoid glycosides and D. intermedia was shown to produce mainly flavonoid glucosides, ellagic acid derivatives and the naphthoquinone plumbagin. The evaluation of the biological activities of these extracts, compiled in Chapter 4, showed that the methanol extract of P. lusitanica has considerable antioxidant activity and that the n-hexane extract of D. intermedia has high antimicrobial potential. In Chapter 5 a method for the extraction of plumbagin from micropropagated D. intermedia plants was optimized and its potential as an alternative for bioprospection evaluated. It was shown that the commercial exploitation of plumbagin from D. intermedia cultures might be viable and that UAE with n-hexane followed by an SPE purification step is an efficient procedure for obtaining large quantities of high purity plumbagin. It is hoped that this study represents an enrichment of the knowledge on these plants and contributes to their conservation and valorisation

Approach to the multisystem inflammatory syndrome in children associated with covid-19

A maioria dos casos de COVID-19 em idade pediátrica caracterizam-se por doença ligeira ou assintomática. Porém, desde maio de 2020, que têm sido reportados casos de hospitalizações em cuidados intensivos, devido a uma síndrome inflamatória multissistêmica em crianças associada à COVID-19 (MIS-C). Apesar de ser uma entidade recente sobre a qual existem muitas incertezas, sabe-se que um acesso atempado a cuidados intensivos e à terapêutica adequada melhora o prognóstico. Assim sendo, este trabalho pretende apresentar o estado da arte quanto à abordagem de um caso suspeito de MIS-C.info:eu-repo/semantics/publishedVersio

Antioxidant activity and verbascoside content in extracts from two uninvestigated endemic Plantago spp.

This work describes for the first time the antioxidant activity of extracts from wild plants and in vitro cultures of two previously uninvestigated Plantago spp. endemic to Portugal, Plantago algarbiensis Samp. and Plantago almogravensis Franco. The Well-known and widely distributed species Plantago lagopus L. was also studied for comparison. Although the extracts from the three species exhibited radical scavenging and iron-reducing properties, the extracts from the two endemic species showed greater activity irrespectively of plant material source. Both P. algarbiensis extracts showed similar antioxidant activity whereas, in the case of P. almogravensis higher activity was observed for extracts from wild plants compared to extracts from in vitro cultures. Liquid chromatography-diode array detection analyses indicate verbascoside as a major constituent of all extracts and the highest content was obtained from in vitro cultures algarbiensis. In addition, a high correlation-between-the anti-oxidant-activity measured with three. methods and the total phenolic content was found, but no correlation was observed between antioxidant activity and verbascoside content. The results suggest that P. algarbiensis and P. almogravensis are sources of health-beneficial phytochemicals and indicate that in vitro cultures represent a promising alternative for the biosynthesis of verbascoside. (C) 2014 Elsevier B.V. All rights reserved

In vitro propagation of Drosera intermedia in a single step

A simple and efficient protocol for the micropropagation of Drosera intermedia, using cultures initiated from in vitro produced seedlings, is described. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher multiplication rates for ¼ MS (the lowest concentration), but was not affected by the addition of 0.1 mg dm-3 kinetin. In all cases a multiplication percentage above 90 % was recorded. High rooting percentages (up to 100 %) were obtained in multiplication phase on ¼ MS medium without growth regulators. In average 15.8 plantlets per initial shoot was produced after 8 weeks of culture. All plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development.T. Grevenstuk and S. Gonçalves acknowledge a grant from the Portuguese Science and Technology Foundation(FCT, Grant SFRH/BD/31777/2006 and SFRH/BPD/31534/2006, respectively)

An efficient in vitro propagation protocol for Pinguicula lusitanica, a rare insectivorous plant

In this study, an efficient protocol was developed for in vitro propagation of Pinguicula lusitanica L., a rare insectivorous plant with pharmacological value and limited reproductive capacity. The effects of two concentrations (0.1 and 0.5 mg l-1) of a range of plant growth regulators, including cytokinins (BA, KIN, and ZEA) and auxins (IAA, IBA, and NAA), and three concentrations of MS medium macronutrients (total, 1/2 and 1/4MS) on proliferation and rooting, were investigated. P. lusitanica shoots showed abundant proliferation and rooting capacity, both of which were significantly influenced by MS medium strength. The use of 1/2MS supplemented with 0.5 mg l-1 BA or KIN ensure a 29-fold rate of proliferation. Best rooting frequency and higher root number and length were attained in 1/4MS medium containing 0.2 mg l-1 IAA. Sixty percent of the plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development. These plantlets can be used to replenish declining populations in the wild and/or for the extraction of bioactive compounds, reducing pressure on wild stocks.S. Gonçalves and T. Grevenstuk acknowledge a grant from Portuguese Science and Technology Foundation (FCT, Grant SFRH/BPD/31534/2006, Grant SFRH/BD/31777/2006, respectively)

Plumbagin recovery in field specimens of Drosophyllum lusitanicum (L.) Link

The naphthoquinone plumbagin has a broad spectrum of biological activities. The aim of this study was to investigate the efficiency of two extraction methods (Soxhlet and ultrasound-assisted extraction) and three solvents (methanol, chloroform and hexane) to recover plumbagin from fresh and dried tissues of field specimens of Drosophyllum lusitanicum (L.) Link. The highest extraction yields were obtained with methanol as solvent and using fresh plant material. The obtained extracts were analysed by gas chromatography with mass spectrometric detection and plumbagin was the major compound present. Plumbagin was quantified in the extracts using the external standard methodology. The results obtained showed that the best recoveries of plumbagin were attained using fresh plant material and there were no significant differences between Soxhlet and ultrasound-assisted extraction. Moreover, hexane proved to be the more appropriate solvent for the extraction of plumbagin, providing high recoveries and the most concentrated extracts, yielding 2.42 mg of plumbagin per gram of plant material with the highest degree of purity. This method is a simple and efficient one to extract large amounts of plumbagin from D. lusitanicum field specimens.Tomás Grevenstuk acknowledges a grant from Fundação para a Ciência e a Tecnologia (SFRH/BD/31777/2006

Aluminium stress disrupts metabolic performance of Plantago almogravensis plantlets transiently.

Little is known about how tolerant plants cope with internalized aluminium (Al). Tolerant plants are known to deploy efficient detoxification mechanisms, however it is not known to what extent the primary and secondary metabolism is affected by Al. The aim of this work was to study the metabolic repercussions of Al stress in the tolerant plant Plantago almogravensis. P. almogravensis is well adapted to acid soils where high concentrations of free Al are found and has been classified as a hyperaccumulator. In vitro reared plantlets were used for this purpose in order to control Al exposure rigorously. The metabolome of P. almogravensis plantlets as well as its metabolic response to the supply of sucrose was characterized. The supply of sucrose leads to an accumulation of amino acids and secondary metabolites and consumption of carbohydrates that result from increased metabolic activity. In Al-treated plantlets the synthesis of amino acids and secondary metabolites is transiently impaired, suggesting that P. almogravensis is able to recover from the Al treatment within the duration of the trials. In the presence of Al the consumption of carbohydrate resources is accelerated. The content of some metabolic stress markers also demonstrates that P. almogravensis is highly adapted to Al stress