Papers by Mercedes Bonfill
Biomedical Chromatography, Jan 2, 2002
A reversed-phase high-performance liquid chromatographic assay for the simultaneous quantitative ... more A reversed-phase high-performance liquid chromatographic assay for the simultaneous quantitative determination of seven ginsenosides, Rb(1), Rb(2), Rc, Rd, Rg(1), Re and Rf in pharmaceutical preparations is described. Chromatographic separation was achieved in less than 20 min using a 250 x 4 mm Lichrospher, 5 microm, 100 A diol column with detection at 203 nm. The method was validated over the range of 2.5-20 ng/microL using a 20 microL sample volume. The average accuracy at five concentrations was 90-100%, and the within-day and between-day precision ranged from 1 to 7% expressed as coefficient of variation. The detection limit and the quantitation limit of the method were 20 and 50 ng injected for each ginsenoside, respectively.
Phytochemistry, 2014
Trichoderma atroviridae UB-LMA is an endophytic fungus isolated from Taxus baccata trees. Liquid-... more Trichoderma atroviridae UB-LMA is an endophytic fungus isolated from Taxus baccata trees. Liquid-state fermentation coupled to in situ solid phase extraction (SPE) was applied, and four compounds were discovered. Compounds 2-4 belong to the harziane tetracyclic diterpene family. Bicylic compound 1 may represent the biosynthetic precursor of this scarce family of compounds.
The Journal of Antibiotics, Sep 20, 2017
The endophytic Trichoderma atroviridae UB-LMA was isolated as a symbiont of Taxus baccata and ana... more The endophytic Trichoderma atroviridae UB-LMA was isolated as a symbiont of Taxus baccata and analyzed for its antimicrobial activity. By applying an original approach consisting of solid-state cultivation coupled with solid-phase extraction, a new methyl (R,E)-3-(1-hydroxy-4-oxocyclopent-2-en-1-yl)-acrylate derivative named EA-2801 (1) was isolated together with the previously reported isonitrin A and dermadin methyl ester. The chemical structure of 1 was determined by NMR and MS. Compound 1 showed antimicrobial activity against a panel of Gram-positive and-negative bacteria.
Antioxidants, Apr 5, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Proceedings of the National Academy of Sciences of the United States of America, Apr 14, 2004
Frontiers in Plant Science
Salinity is an abiotic stress that reduces the seed germination and productivity of wheat. The ob... more Salinity is an abiotic stress that reduces the seed germination and productivity of wheat. The objective of this study was to assess the impact of irrigation with magnetically treated seawater on the germination, growth, certain physiological and anatomical parameters, and production attributes of wheat (Triticum aestivum L.) cv. Sakha 93 plants. Experiments were conducted in the Experimental Farm of the Faculty of Agriculture, Menoufia University, Egypt, during two consecutive winter seasons. Pot experiments involved ten treatments with non-magnetized and magnetized water with various degrees of salinity. Plant samples were taken 95 days after sowing. Irrigation with magnetically treated seawater was found to have beneficial effects on plant growth, water relations, biochemical characteristics, and yield components compared with untreated plants. The germination of wheat seeds increased 13% when treated with magnetic seawater. On the yield scale, the spike length was increased by 4...
Plant Physiology and Biochemistry, 2018
The aim of this study was to assess some physiological parameters and anatomical changes in two w... more The aim of this study was to assess some physiological parameters and anatomical changes in two wheat plant cultivars (Triticum aestivum L. cvs. Sakha 93 and Sids 9) in response to irrigation with magnetized water under two levels of drought stress (field capacity (FC) of 75% and 50%) and the control (FC 100%) in two consecutive winter growing seasons (
Frontiers in Plant Science, Sep 2, 2022
Hairy roots are made after the integration of a small set of genes from Agrobacterium rhizogenes ... more Hairy roots are made after the integration of a small set of genes from Agrobacterium rhizogenes in the plant genome. Little is known about how this small set is linked to their hormone profile, which determines development, morphology, and levels of secondary metabolite production. We used C. asiatica hairy root line cultures to determine the putative links between the rol and aux gene expressions with morphological traits, a hormone profile, and centelloside production. The results obtained after 14 and 28 days of culture were processed via multivariate analysis and machine-learning processes such as random forest, supported vector machines, linear discriminant analysis, and neural networks. This allowed us to obtain models capable of discriminating highly productive root lines from their levels of genetic expression (rol and aux genes) or from their hormone profile. In total, 12 hormones were evaluated, resulting in 10 being satisfactorily detected. Within this set of hormones, abscisic acid (ABA) and cytokinin isopentenyl adenosine (IPA) were found to be critical in defining the morphological traits and centelloside content. The results showed that IPA brings more benefits to the biotechnological platform. Additionally, we determined the degree of influence of each of the evaluated genes on the individual hormone profile, finding that aux1 has a significant influence on the IPA profile, while the rol genes are closely linked to the ABA profile. Finally, we effectively verified the gene influence on these two specific hormones through feeding experiments that aimed to reverse the effect on root morphology and centelloside content.
Frontiers in Plant Science, Jan 26, 2023
Paclitaxel (PTX) and its derivatives are diterpene alkaloids widely used as chemotherapeutic agen... more Paclitaxel (PTX) and its derivatives are diterpene alkaloids widely used as chemotherapeutic agents in the treatment of various types of cancer. Due to the scarcity of PTX in nature, its production in cell cultures and plant organs is a major challenge for plant biotechnology. Although significant advances have been made in this field through the development of metabolic engineering and synthetic biology techniques, production levels remain insufficient to meet the current market demand for these powerful anticancer drugs. A key stumbling block is the difficulty of genetically transforming the gymnosperm Taxus spp. This review focuses on the progress made in improving taxane production through genetic engineering techniques. These include the overexpression of limiting genes in the taxane biosynthetic pathway and transcription factors involved in its regulation in Taxus spp. cell cultures and transformed roots, as well as the development and optimization of transformation techniques. Attempts to produce taxanes in heterologous organisms such as bacteria and yeasts are also described. Although promising results have been reported, the transfer of the entire PTX metabolic route has not been possible to date, and taxane biosynthesis is still restricted to Taxus cells and some endophytic fungi. The development of a synthetic organism other than Taxus cells capable of biotechnologically producing PTX will probably have to wait until the complete elucidation of its metabolic pathway.
Gene, Jul 1, 2021
The aim of this study was to investigate secondary metabolite production in Silybum marianum L. c... more The aim of this study was to investigate secondary metabolite production in Silybum marianum L. cell suspension cultures obtained from seeds treated with gamma rays (200 and 600Gy) and 0.05% colchicine. The effects of these treatments on callus induction, growth, viability, and silymarin production were studied, along with the changes in the transcriptome and DNA sequence of chalcone synthase (CHS) genes. The effect of gamma radiation (200 and 600 Gy) on silymarin production in S. marianum dry seeds was also studied using HPLC-UV. All three treatments induced high callus biomass production from leaf segments. The viability of the cell suspension cultures was over 90%. The flavonolignan content measured in the extracellular culture medium of the S. marianum cell suspension was highest after treatment with 600 Gy, followed by 0.05% colchicine, and finally, 200 Gy, after a growth period of 12 days. In general, an increased expression of CHS1, CHS2, and CHS3 genes, accompanied by an increase of silymarin content, was observed in response to all the studied treatments, although the effect was greatest on CHS2 expression. Bioinformatics analysis confirmed that the three CHS2 clones exhibited the highest genetic variation, both in relation to each other and to the CHS1 and CHS3 clones. Based on the results, S. marianum plants obtained from seeds previously exposed to 600 and 200Gy as well as colchicine constitute a renewable resource with the potential to obtain large amounts of silymarin.
Biomedical Chromatography, Feb 1, 2002
RSC Advances, 2015
Since the recent discovery of taxol and other taxanes in Corylus avellana, this plant species has... more Since the recent discovery of taxol and other taxanes in Corylus avellana, this plant species has attracted interest as a potential new source of these compounds. However, its low taxane content in comparison with Taxus spp. has restricted research to analytical identification or global quantitation. A feasible and sensitive method based on liquid chromatography-tandem mass spectrometry using a triple quadrupole analyzer was developed for the analysis of taxol and four other taxanes in a Corylus avellana cell suspension medium. Taxanes were extracted from the cell culture medium with dichloromethane and analyzed using electrospray ionization and quantified by multiple-reaction monitoring mode. Methanol and matrix-matching calibration curves using docetaxel as the internal standard were analyzed. Linearity was confirmed over the whole calibration range (0.3-2.1 mg mL À1). The inter-and intra-day precision of taxanes ranged from 80% to 120% and the recovery rates were higher than 80%. Limits of detection were between 0.24-38 ng mL À1 and the limits of quantification were between 0.8-125 ng mL À1. The low detection and quantitation values obtained allowed us to detect small quantities of the released taxanes (120 ng mL À1 of B, 151 ng mL À1 of CF and 105 ng mL À1 of T), which correspond to about 0.5 ng mL À1 of each taxane, in the 20 mL Corylus avellana cell suspension culture medium extracted, even at the beginning of the culture. These results were confirmed by high resolution mass spectrometry.
Frontiers in Plant Science
Hairy roots are made after the integration of a small set of genes from Agrobacterium rhizogenes ... more Hairy roots are made after the integration of a small set of genes from Agrobacterium rhizogenes in the plant genome. Little is known about how this small set is linked to their hormone profile, which determines development, morphology, and levels of secondary metabolite production. We used C. asiatica hairy root line cultures to determine the putative links between the rol and aux gene expressions with morphological traits, a hormone profile, and centelloside production. The results obtained after 14 and 28 days of culture were processed via multivariate analysis and machine-learning processes such as random forest, supported vector machines, linear discriminant analysis, and neural networks. This allowed us to obtain models capable of discriminating highly productive root lines from their levels of genetic expression (rol and aux genes) or from their hormone profile. In total, 12 hormones were evaluated, resulting in 10 being satisfactorily detected. Within this set of hormones, a...
Journal of Plant Physiology, Sep 1, 2018
Linum album is a herbaceous plant with medical interest due to its content of podophyllotoxin (PT... more Linum album is a herbaceous plant with medical interest due to its content of podophyllotoxin (PTOX), an aryltetralin lignan with cytotoxic activity. Previous studies in our laboratory showed that cell suspension cultures of L. album produced more PTOX than methoxypodophyllotoxin (6-MPTOX), both lignans being formed from the same precursor after divergence close to the end of the biosynthetic pathway. In contrast, the hairy roots produced more 6-MPTOX than PTOX. Taking into account this variability, we were interested to know if the lignan profile of an in vitro PTOX-producing L. album plant changes according to the biotechnological system employed and, if so, if this is due to cell dedifferentiation and/or transformation events. With this aim, we established four biotechnological systems: (1) Wild type cell suspensions, (2) transformed cell suspensions, (3) adventitious roots and (4) hairy roots. We determined the production of four aryltetralin lignans: PTOX, 6-MPTOX, deoxypodophyllotoxin (dPTOX) and β-peltatin. The results show that in vitro plantlets, WT cells and transformed cells predominantly produced PTOX, production being 11-fold higher in the plantlets. Otherwise, the adventitious and hairy roots predominantly produced 6-MPTOX, the adventitious roots being the most productive, with MPTOX levels 1.58-fold higher than in transformed roots. We can infer from these results that in the studied plants, cell differentiation promoted the formation of 6-MPTOX over PTOX, while transformation did not influence the lignan pattern.
Plant and Cell Physiology, Mar 1, 1995
Frontiers in Plant Science, Jul 8, 2022
Environmental conditions are key factors in the modulation of the epigenetic mechanisms regulatin... more Environmental conditions are key factors in the modulation of the epigenetic mechanisms regulating gene expression in plants. Specifically, the maintenance of cell cultures in optimal in vitro conditions alters methylation patterns and, consequently, their genetic transcription and metabolism. Paclitaxel production in Taxus x media cell cultures is reduced during its maintenance in in vitro conditions, compromising the biotechnological production of this valuable anticancer agent. To understand how DNA methylation influences taxane production, the promoters of three genes (GGPPS, TXS, and DBTNBT) involved in taxane biosynthesis have been studied, comparing the methylation patterns between a new line and one of ~14 years old. Our work revealed that while the central promoter of the GGPPS gene is protected from cytosine methylation accumulation, TXS and DBTNBT promoters accumulate methylation at different levels. The DBTNBT promoter of the old line is the most affected, showing a 200 bp regulatory region where all the cytosines were methylated. This evidence the existence of specific epigenetic regulatory mechanisms affecting the last steps of the pathway, such as the DBTNBT promoter. Interestingly, the GGPPS promoter, a regulatory sequence of a non-specific taxane biosynthetic gene, was not affected by this mechanism. In addition, the relationship between the detected methylation points and the predicted transcription factor binding sites (TFBS) showed that the action of TFs would be compromised in the old line, giving a further explanation for the production reduction in in vitro cell cultures. This knowledge could help in designing novel strategies to enhance the biotechnological production of taxanes over time.
New Biotechnology, Sep 1, 2009
Plant Biotechnology Journal, Jun 9, 2014
Methyl jasmonate and cyclodextrins are proven effective inducers of secondary metabolism in plant... more Methyl jasmonate and cyclodextrins are proven effective inducers of secondary metabolism in plant cell cultures. Cyclodextrins, which are cyclic oligosaccharides, can form inclusion complexes with nonhydrophilic secondary products, thus increasing their excretion from the producer cells to the culture medium. In the present work, using a selected Taxus x media cell line cultured in a two-stage system, the relationship between taxane production and the transcript profiles of several genes involved in taxol metabolism was studied to gain more insight into the mechanism by which these two elicitors regulate the biosynthesis and excretion of taxol and related taxanes. Gene expression was not clearly enhanced by the presence of cyclodextrins in the culture medium and variably induced by methyl jasmonate, but when the culture was supplemented with both elicitors, a synergistic effect on transcript accumulation was observed. The BAPT and DBTNBT genes, which encode the last two transferases involved in the taxol pathway, appeared to control limiting biosynthetic steps. In the cell cultures treated with both elicitors, the produced taxanes were found mainly in the culture medium, which limited retroinhibition processes and taxane toxicity for the producer cells. The expression level of a putative ABC gene was found to have increased, suggesting it played a role in the taxane excretion. Taxol biosynthesis was clearly increased by the joint action of methyl jasmonate and cyclodextrins, reaching production levels 55 times higher than in nonelicited cultures.
Plant and Cell Physiology, Dec 16, 2019
Taxane diterpenes are secondary metabolites with an important pharmacological role in the treatme... more Taxane diterpenes are secondary metabolites with an important pharmacological role in the treatment of cancer. Taxus spp. biofactories have been used for taxane production, but the lack of knowledge about the taxane biosynthetic pathway and its molecular regulation hinders their optimal function. The difficulties in introducing foreign genes in Taxus spp. genomes hinder the study of the molecular mechanisms involved in taxane production, and a new approach is required to overcome them. In this study, a reliable, simple and fast method to obtain Taxus  media protoplasts was developed, allowing their manipulation in downstream assays for the study of physiological changes in Taxus spp. cells. Using this method, Taxus protoplasts were transiently transfected for the first time, corroborating their suitability for transfection assays and the study of specific physiological responses. The two assayed transcription factors (BIS2 and TSAR2) had a positive effect on the expression of several taxane-related genes, suggesting their potential use for the improvement of taxane yields. Furthermore, the results indicate that the developed method is suitable for obtaining T.  media protoplasts for transfection with the aim of unraveling regulatory mechanisms in taxane production.
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Papers by Mercedes Bonfill