Publications by authors named "Mercedes Bonfill"

39 Publications

Effect of gamma rays and colchicine on silymarin production in cell suspension cultures of Silybum marianum: A transcriptomic study of key genes involved in the biosynthetic pathway.

Gene 2021 Jul 5;790:145700. Epub 2021 May 5.

Department of Biology, Health, and Environment, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain.

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 600 Gy) 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 200 Gy as well as colchicine constitute a renewable resource with the potential to obtain large amounts of silymarin.
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http://dx.doi.org/10.1016/j.gene.2021.145700DOI Listing
July 2021

Improved biotechnological production of paclitaxel in Taxus media cell cultures by the combined action of coronatine and calix[8]arenes.

Plant Physiol Biochem 2021 Jun 27;163:68-75. Epub 2021 Mar 27.

Secció de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, Barcelona, Spain. Electronic address:

Paclitaxel (PTX), a widely used anticancer agent, is found in the inner bark of several Taxus species, although at such low levels that its extraction is ecologically unsustainable. Biotechnological platforms based on Taxus sp. cell cultures offer an eco-friendlier approach to PTX production, with yields that can be improved by elicitation. However, the also limited excretion of target compounds from the producer cells to the medium hampers their extraction and purification. In this context, we studied the effect of treating T. media cell cultures with the elicitor coronatine (COR) and calix[8]arenes (CAL), nanoparticles that can host lipophilic compounds within their macrocyclic scaffold. The highest taxane production (103.5 mg.L), achieved after treatment with COR (1 μM) and CAL (10 mg.L), was 15-fold greater than in the control, and PTX represented 82% of the total taxanes analyzed. Expression levels of the flux-limiting PTX biosynthetic genes, BAPT and DBTNBT, increased after the addition of COR, confirming its elicitor action, but not CAL. The CAL treatment significantly enhanced taxane excretion, especially when production levels were increased by COR; 98% of the total taxanes were found in the culture medium after COR + CAL treatment. By forming complexes with PTX, the nanoparticles facilitated its excretion to the medium, and by protecting cells from PTX toxicity, its intra-and extra-cellular degradation may have been avoided. The addition of COR and CAL to T. media cell cultures is therefore a bio-sustainable and economically viable system to improve the yield of this important anticancer compound.
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http://dx.doi.org/10.1016/j.plaphy.2021.03.047DOI Listing
June 2021

Production of Encecalin in Cell Cultures and Hairy Roots of (Hook.) A. Gray.

Molecules 2020 Jul 15;25(14). Epub 2020 Jul 15.

Área de Fisiología Vegetal, Departamento de Ingeniería y Ciencias Agrarias, Universidad de León, 24071 León, Spain.

Plant cell and organ cultures of , a medicinal plant whose roots are used by the Tarahumara Indians of Chihuahua, Mexico, to relieve several ailments, were established to identify and quantify some chromenes with biological activity, such as encecalin, and to evaluate their potential for biotechnological production. Gas chromatography-mass spectrometry (GC-MS) analysis corroborated the presence of quantifiable amounts of encecalin in cell cultures (callus and cell suspensions). In addition, hairy roots were obtained through three transformation protocols (prick, 45-s sonication and co-culture), using wild type A4. After three months, cocultivation achieved the highest percentage of transformation (66%), and a comparable production (FW) of encecalin (110 μg/g) than the sonication assay (120 μg/g), both giving far higher yields than the prick assay (19 μg/g). Stable integration of and genes in the transformed roots was confirmed by polymerase chain reaction (PCR). Hairy roots from cocultivation (six months-old) accumulated as much as 1086 μg/g (FW) of encecalin, over three times higher than the cell suspension cultures. The production of encecalin varied with growth kinetics, being higher at the stationary phase. This is the first report of encecalin production in hairy roots of , demonstrating the potential for a future biotechnological production of chromenes.
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http://dx.doi.org/10.3390/molecules25143231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397079PMC
July 2020

A Novel Hydroxylation Step in the Taxane Biosynthetic Pathway: A New Approach to Paclitaxel Production by Synthetic Biology.

Front Bioeng Biotechnol 2020 13;8:410. Epub 2020 May 13.

Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.

Engineered plant cell lines have the potential to achieve enhanced metabolite production rates, providing a high-yielding source of compounds of interest. Improving the production of taxanes, pharmacologically valuable secondary metabolites of spp., is hindered by an incomplete knowledge of the taxane biosynthetic pathway. Of the five unknown steps, three are thought to involve cytochrome P450-like hydroxylases. In the current work, after an in-depth characterization of four candidate enzymes proposed in a previous cDNA-AFLP assay, TB506 was selected as a candidate for the hydroxylation of the taxane side chain. A docking assay indicated TB506 is active after the attachment of the side chain based on its affinity to the ligand 3'-dehydroxydebenzoyltaxol. Finally, the involvement of TB506 in the last hydroxylation step of the paclitaxel biosynthetic pathway was confirmed by functional assays. The identification of this hydroxylase will contribute to the development of alternative sustainable paclitaxel production systems using synthetic biology techniques.
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http://dx.doi.org/10.3389/fbioe.2020.00410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247824PMC
May 2020

Transfecting Taxus � media Protoplasts to Study Transcription Factors BIS2 and TSAR2 as Activators of Taxane-Related Genes.

Plant Cell Physiol 2020 Mar;61(3):576-583

Departament de Ci�ncies Experimentals i de la Salut, Universitat Pompeu Fabra, Avda. Dr. Aiguader 80, E-08003 Barcelona, Spain.

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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http://dx.doi.org/10.1093/pcp/pcz225DOI Listing
March 2020

Biotechnological production of ruscogenins in plant cell and organ cultures of Ruscus aculeatus.

Plant Physiol Biochem 2019 Aug 29;141:133-141. Epub 2019 May 29.

Secció de Fisiologia i Biotecnologia Vegetal, Departament de Biologia, Sanitat i Medi Ambient, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, Joan XXIII 27-31, 08028, Barcelona, Spain. Electronic address:

Ruscus aculeatus is a threatened medicinal plant whose main bioactive components, the ruscogenins, have long been used in the treatment of hemorrhoids and varicose veins, but recently demonstrated activity against some types of cancer. Plant cell biofactories could constitute an alternative to the whole plant as a source of ruscogenins. In this pipeline, despite the in vitro recalcitrance of R. aculeatus, after many attempts we developed friable calli and derived plant cell suspensions, and their ruscogenin production was compared with that of organized in vitro plantlet and root-rhizome cultures. Root-rhizomes showed a higher capacity for biomass and ruscogenin production than the cell suspensions and the yields were greatly improved by elicitation with coronatine. Although ruscogenins accumulate in plants mainly in the root-rhizome, it was demonstrated that the aerial part could play an important role in their biosynthesis, as production was higher in the whole plant than in the root-rhizome cultures.
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http://dx.doi.org/10.1016/j.plaphy.2019.05.029DOI Listing
August 2019

Genomic methylation in plant cell cultures: A barrier to the development of commercial long-term biofactories.

Eng Life Sci 2019 Dec 17;19(12):872-879. Epub 2019 May 17.

Secció de Fisiologia Vegetal Facultat de Farmacia Universitat de Barcelona Barcelona Spain.

Plant cell biofactories offer great advantages for the production of plant compounds of interest, although certain limitations still need to be overcome before their maximum potential is reached. One obstacle is the gradual loss of secondary metabolite production during in vitro culture maintenance, which is an important impediment in the development of large-scale production systems. The relationship between in vitro maintenance and epigenetic changes has been demonstrated in several plant species; in particular, methylation levels have been found to increase in in vitro cultures over time. Higher DNA methylation levels have been correlated with a low yield of secondary metabolites in in vitro plant cell cultures. The longer the period of subculturing, the more methylated cytosines were found throughout the genome, and secondary metabolism decreased significantly. This review summarizes different studies on epigenetic changes during the maintenance of in vitro cell cultures and the insights they provide on the mechanisms involved. It concludes by looking at the perspectives for new approaches designed to avoid declines in metabolite production.
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http://dx.doi.org/10.1002/elsc.201900024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999079PMC
December 2019

Physiological and anatomical studies of two wheat cultivars irrigated with magnetic water under drought stress conditions.

Plant Physiol Biochem 2019 Feb 14;135:480-488. Epub 2018 Nov 14.

Plant Physiology Department, Faculty of Pharmacy, University of Barcelona, E-08028, Spain. Electronic address:

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 (November 20 to May 5 2014/2015 and 2015/2016). Pot experiments were carried out in a greenhouse in the experimental farm of the Faculty of Agriculture, Menoufia University, Shibin El-Kom, Egypt. A water deficit, particularly at 50% FC, significantly decreased growth and parameter values, above all in Sakha 93, and disrupted most physiological aspects, biochemical constituents and internal structural features of both wheat cultivars. Irrigation with magnetized water alleviated the negative consequences of drought stress on most physiological and biochemical parameters to a variable extent: the whole plant dry weight, total water content, total soluble sugar concentration in leaves, total free amino acids and proline increased by about 32, 12, 17, 27 and 73%, respectively, under 50% FC drought stress in Sids 9 compared to the control. As the levels of drought increased, the grain yield (g/plant) decreased considerably, from about 81% in Sakha 93 at 50% FC to 26% in Sids 9 at 75% FC. The use of magnetic water increased grain yield from 61% in Sakha 93 at 75% FC to about 268% in Sids 9 at 50% FC. Magnetic water also increased the thickness of the flag leaf midvein and lamina, as well as the metaxylem vessel diameter of Sakha 93 by 28.8, 11.7 and 20.0%, respectively, compared to the control. The application of magnetic water increased the growth and the other parameter values studied in both cultivars but above all in Sakha 93, whereas Sids 9 produced more grain yield under all levels of drought stress. As the growth and grain production increased in both cultivars when using magnetic water, this study recommends this type of irrigation for these wheat plants, which are widespread in Egypt.
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http://dx.doi.org/10.1016/j.plaphy.2018.11.012DOI Listing
February 2019

Advances in the Regulation of In Vitro Paclitaxel Production: Methylation of a Y-Patch Promoter Region Alters BAPT Gene Expression in Taxus Cell Cultures.

Plant Cell Physiol 2018 Nov;59(11):2255-2267

Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, E-08003 Barcelona, Spain.

Plant cell biofactories represent a promising solution to the increasing demand for plant-derived compounds, but there are still limiting factors that prevent optimal production, including the loss of yield during in vitro maintenance. Our results reveal a clear correlation between genomic methylation levels and a progressive decline in taxane production in Taxus spp. cell cultures. A comparative study of two cell lines, one 10 years old and low productive and the other new and high productive, revealed important differences in appearance, growth, taxane accumulation and expression levels of several taxane biosynthetic genes. Differences in taxane content and gene expression profile indicate an altered pathway regulation and that the BAPT gene, located in the center of the expression network of taxane biosynthetic genes, is active in a potentially flux-limiting step. The methylation patterns of the BAPT gene were studied in both cell lines by bisulfite sequencing, which revealed high rates of CHH methylated cytosines on the core promoter. Using a bioinformatics approach, this hotspot was identified as a Y-patch promoter element. The Y-patch may play a key role in the epigenetic regulation of the taxane biosynthetic pathway, which would open up novel genetic engineering strategies toward stable and high productivity.
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http://dx.doi.org/10.1093/pcp/pcy149DOI Listing
November 2018

Comparing aryltetralin lignan accumulation patterns in four biotechnological systems of Linum album.

J Plant Physiol 2018 Sep 15;228:197-207. Epub 2018 Jun 15.

Laboratory of Plant Physiology, Department of Biology, Health and Environment, University of Barcelona, 08028 Barcelona, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jplph.2018.06.006DOI Listing
September 2018

Effect of in vitro morphogenesis on the production of podophyllotoxin derivatives in callus cultures of Linum album.

J Plant Physiol 2018 Sep 18;228:47-58. Epub 2018 May 18.

Sección de Fisiologia Vegetal, Facultad de Farmacia, Universidad de Barcelona, E-08028 Barcelona, Spain. Electronic address:

The anticancer compound podophyllotoxin and other related lignans can be produced in Linum album in vitro cultures, although their biosynthesis varies according to the degree of differentiation of the plant material. In general, L. album cell cultures do not form the same lignans as roots or other culture systems. Our aim was to explore how the lignan-producing capacity of organogenic cell masses is affected by the conditions that promote their formation and growth. Thus, L. album biomass obtained from plantlets was cultured in darkness or light, with or without the addition of plant growth regulators, and the levels of podophyllotoxin, methoxypodophyllotoxin and other related lignans were determined in each of these conditions. The organogenic capacity of the cell biomass grown in the different conditions was studied directly and also with light and scanning electronic microscopy, leading to the observation of.several somatic embryos and well-formed shoots. The main lignan produced was methoxypodophyllotoxin, whose production was clearly linked to the organogenic capacity of the cell biomass, which to a lesser extent was also the case for podophyllotoxin.
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http://dx.doi.org/10.1016/j.jplph.2018.05.007DOI Listing
September 2018

Biotechnological Production of Pharmaceuticals and Biopharmaceuticals in Plant Cell and Organ Cultures.

Curr Med Chem 2018 ;25(30):3577-3596

Laboratori de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, Barcelona, Spain.

Background: Plant biofactories are biotechnological platforms based on plant cell and organ cultures used for the production of pharmaceuticals and biopharmaceuticals, although to date only a few of these systems have successfully been implemented at an industrial level. Metabolic engineering is possibly the most straightforward strategy to boost pharmaceutical production in plant biofactories, but social opposition to the use of GMOs means empirical approaches are still being used. Plant secondary metabolism involves thousands of different enzymes, some of which catalyze specific reactions, giving one product from a particular substrate, whereas others can yield multiple products from the same substrate. This trait opens plant cell biofactories to new applications, in which the natural metabolic machinery of plants can be harnessed for the bioconversion of phytochemicals or even the production of new bioactive compounds. Synthetic biological pipelines involving the bioconversion of natural substrates into products with a high market value may be established by the heterologous expression of target metabolic genes in model plants.

Objective: To summarize the state of the art of plant biofactories and their applications for the pipeline production of cosme-, pharma- and biopharmaceuticals.

Results: In order to demonstrate the great potential of plant biofactories for multiple applications in the biotechnological production of pharmaceuticals and biopharmaceuticals, this review broadly covers the following: plant biofactories based on cell and hairy root cultures; secondary metabolite production; biotransformation reactions; metabolic engineering tools applied in plant biofactories; and biopharmaceutical production.
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http://dx.doi.org/10.2174/0929867325666180309124317DOI Listing
October 2018

Specialized Plant Metabolism Characteristics and Impact on Target Molecule Biotechnological Production.

Mol Biotechnol 2018 Feb;60(2):169-183

Plant Physiology Laboratory, Center for Biotechnology and Department of Botany, UFRGS, Porto Alegre, RS, Brazil.

Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.
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http://dx.doi.org/10.1007/s12033-017-0056-1DOI Listing
February 2018

Isolation of the antibiotic methyl (R,E)-3-(1-hydroxy-4-oxocyclopent-2-en-1-yl)-acrylate EA-2801 from Trichoderma atroviridae.

J Antibiot (Tokyo) 2017 Nov 20;70(11):1053-1056. Epub 2017 Sep 20.

Centre National de la Recherche Scientifique, CNRS, Institut de Chimie des Substances Naturelles, ICSN, Gif-sur-Yvette, France.

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.
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http://dx.doi.org/10.1038/ja.2017.107DOI Listing
November 2017

Viability-reducing activity of Coryllus avellana L. extracts against human cancer cell lines.

Biomed Pharmacother 2017 May 1;89:565-572. Epub 2017 Mar 1.

Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Avda. Dr. Aiguader 80, E-08003, Barcelona, Spain. Electronic address:

The increasing rate of cancer incidence has encouraged the search for novel natural sources of anticancer compounds. The presence of small quantities of taxol and taxanes in Corylus avellana L. has impelled new potential applications for this plant in the field of biomedicine. In the present work, the cell viability-reducing activity of stems and leaves from three different hazel trees was studiedagainst three human-derived cancer cell lines (HeLa, HepG2 and MCF-7). Both leaf and stem extracts significantly reduced viability of the three cell lines either after maceration with methanol or using taxane extraction methods. Since maceration reduced cell viability to a greater extent than taxane extraction methods, we scaled up the maceration extraction process using a method for solid/liquid extraction (Zippertex technology). Methanol leaf extracts promoted a higher reduction in viability of all cell lines assayed than stem extracts. Fractionation of methanol leaf extracts using silica gel chormatography led to the purification and identification of two compounds by HPLC-MS and NMR: (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl) heptane 3-O-β-d-glucopyranoside and quercetin-3-O-rhamnoside. The isolated compounds decreased viability of HeLa and HepG2 cells to a greater extent than MCF-7 cells. Our results suggest a potential use of C. avellana extracts in the pharmacotherapy of cervical cancer and hepatocarcinoma and, to a lesser extent, breast cancer.
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http://dx.doi.org/10.1016/j.biopha.2017.02.046DOI Listing
May 2017

Plant Anti-cancer Agents and their Biotechnological Production in Plant Cell Biofactories.

Curr Med Chem 2016 ;23(39):4418-4441

Laboratori de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII sn, 08028 Barcelona, Spain.

Background: Bioactive plant secondary metabolites have complex chemical structures, which are specific to each plant species/family, and accumulate in tiny amounts. The growing market demand for many phytochemicals can lead to the over-harvesting of medicinal plants in their natural habitat, endangering species in the process.

Objective: An ongoing challenge for our society is therefore to develop a bio-sustainable production of phytochemicals, among other natural resources. Cancer is currently a major health problem, responsible for approximately 8.2 million deaths per year worldwide. We therefore focused this review on cancer therapeutic agents from plants and their biotechnological production.

Method And Results: An extensive review of the literature shows that although a wide range of phytochemicals have demonstrated anti-proliferative activity in vitro, only a few examples of plant-based drugs are included in the Anatomical Therapeutic Chemical (ATC) classification as antineoplastic agents. These include vinca alkaloids and their derivatives (L01CA), podophyllotoxin derivatives (L01CB), and paclitaxel and its derivatives (L01CD), as well as camptothecin derivatives (L01XX). These compounds all have in common a complex chemical structure, a scarce distribution in nature, and a high added value. After describing the chemical structures, natural sources and biological activities of these anticancer compounds, we focus on the state of the art in their biotechnological production in plant cell biofactories.

Conclusion: More in-depth studies are required on the biosynthesis of target plant metabolites and its regulation in order to increase their biotechnological production in plant cell factories and ultimately implement these biosustainable processes at an industrial level.
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http://dx.doi.org/10.2174/0929867323666161024145715DOI Listing
January 2017

Enhanced artemisinin yield by expression of rol genes in Artemisia annua.

Malar J 2015 Oct 29;14:424. Epub 2015 Oct 29.

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.

Background: Despite of many advances in the treatment of malaria, it is still the fifth most prevalent disease worldwide and is one of the major causes of death in the developing countries which accounted for 584,000 deaths in 2013, as estimated by World Health Organization. Artemisinin from Artemisia annua is still one of the most effective treatments for malaria. Increasing the artemisinin content of A. annua plants by genetic engineering would improve the availability of this much-needed drug.

Methods: In this regard, a high artemisinin-yielding hybrid of A. annua produced by the centre for novel agricultural products of the University of York, UK, was selected (artemisinin maximally 1.4 %). As rol genes are potential candidates of biochemical engineering, genetic transformation of A. annua with Agrobacterium tumefaciens GV3101 harbouring vectors with rol B and rol C genes was carried out with the objective of enhancement of artemisinin content. Transgenic lines produced were analysed by the LC-MS for quantitative analysis of artemisinin and analogues. These high artemisinin yielding transgenics were also analysed by real time quantitative PCR to find the molecular dynamics of artemisinin enhancement. Genes of artemisinin biosynthetic pathway were studied including amorphadiene synthase (ADS), cytochrome P450, (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1). Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis. Thus, real time qPCR analysis of the TAFR1 gene was carried out, and trichome density was determined.

Results: Transgenics of rol B gene showed two- to ninefold (the decimal adds nothing in the abstract, please simplify to two- to ninefold) increase in artemisinin, 4-12-fold increase in artesunate and 1.2-3-fold increase in dihydroartemisinin. Whereas in the case of rol C gene transformants, a fourfold increase in artemisinin, four to ninefold increase in artesunate and one- to twofold increase in dihydroartemisinin concentration was observed. Transformants with the rol B gene had higher expression of these genes than rol C transformants. TAFR1 was also found to be more expressed in rol gene transgenics than wild type A. annua, which was also in accordance with the trichome density of the respective plant.

Conclusion: Thus it was proved that rol B and rol C genes are effective in the enhancement of artemisinin content of A. annua, rol B gene being more active to play part in this enhancement than rol C gene.
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http://dx.doi.org/10.1186/s12936-015-0951-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625606PMC
October 2015

Genetic Transformation of Artemisia carvifolia Buch with rol Genes Enhances Artemisinin Accumulation.

PLoS One 2015 7;10(10):e0140266. Epub 2015 Oct 7.

Department of Biochemistry, Faculty of Biological sciences, Quaid-i-Azam University, Islamabad, Pakistan.

The potent antimalarial drug artemisinin has a high cost, since its only viable source to date is Artemisia annua (0.01-0.8% DW). There is therefore an urgent need to design new strategies to increase its production or to find alternative sources. In the current study, Artemisia carvifolia Buch was selected with the aim of detecting artemisinin and then enhancing the production of the target compound and its derivatives. These metabolites were determined by LC-MS in the shoots of A. carvifolia wild type plants at the following concentrations: artemisinin (8μg/g), artesunate (2.24μg/g), dihydroartemisinin (13.6μg/g) and artemether (12.8μg/g). Genetic transformation of A. carvifolia was carried out with Agrobacterium tumefaciens GV3101 harboring the rol B and rol C genes. Artemisinin content increased 3-7-fold in transgenics bearing the rol B gene, and 2.3-6-fold in those with the rol C gene. A similar pattern was observed for artemisinin analogues. The dynamics of artemisinin content in transgenics and wild type A.carvifolia was also correlated with the expression of genes involved in its biosynthesis. Real time qPCR analysis revealed the differential expression of genes involved in artemisinin biosynthesis, i.e. those encoding amorpha-4, 11 diene synthase (ADS), cytochrome P450 (CYP71AV1), and aldehyde dehydrogenase 1 (ALDH1), with a relatively higher transcript level found in transgenics than in the wild type plant. Also, the gene related to trichome development and sesquiterpenoid biosynthesis (TFAR1) showed an altered expression in the transgenics compared to wild type A.carvifolia, which was in accordance with the trichome density of the respective plants. The trichome index was significantly higher in the rol B and rol C gene-expressing transgenics with an increased production of artemisinin, thereby demonstrating that the rol genes are effective inducers of plant secondary metabolism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140266PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4596866PMC
June 2016

Changes in gene transcription and taxane production in elicited cell cultures of Taxus × media and Taxus globosa.

Phytochemistry 2015 Sep 16;117:174-184. Epub 2015 Jun 16.

Laboratori de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, Barcelona, Spain. Electronic address:

The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), supplied to the culture medium either separately or together, was studied. Two-stage Taxus globosa and Taxus media cell cultures were established and the elicitors were added at the beginning of the second stage. Growth, taxane production, and the expression of known taxol biosynthetic genes, including the recently characterized CoA ligase gene, were studied. Although CORO reduced the growth capacity of both cell lines, CD apparently counteracted this negative effect. Taxane production was significantly enhanced by the simultaneous addition of CD and CORO to the medium. The total taxane production in the T. media cell line was more than double that of T. globosa, but in the latter more than 90% of the taxanes produced were excreted to the medium. Individual taxane patterns also differed: at the height of production, the main taxanes in T. globosa cultures were cephalomannine and 10-deacetyltaxol, and in T. media, taxol and baccatin III. The low transcript levels of taxane biosynthetic genes found in T. globosa cells mirrored the lower taxane production in these cultures, while a high expression was strongly correlated with a high taxane production in T. media.
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http://dx.doi.org/10.1016/j.phytochem.2015.06.013DOI Listing
September 2015

Development of a hazel cell culture-based paclitaxel and baccatin III production process on a benchtop scale.

J Biotechnol 2015 Feb 3;195:93-102. Epub 2015 Jan 3.

Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Avda. Dr. Aiguader 80, E-08003 Barcelona, Spain. Electronic address:

The growing demand for the antitumorous agent paclitaxel and the difficulty in increasing its production by genetic engineering has prompted a search for new sources of taxanes. It has been reported that taxanes can be extracted from the angiosperm Corylus avellana L. Our aim was to improve taxane production by scaling up the process from mL-level to benchtop bioreactors, optimizing culture conditions and comparing the effect of two elicitors, 1 μM coronatine (Cor) and 100 μM methyl jasmonate (MeJA). Orbitally shaken flask cultures achieved a maximum fresh cell weight of 11.54 gDCW/L under control conditions, and MeJA- and Cor-treatment produced a statistically significant reduction in growth to 4.28 gDCW/L and 5.69 gDCW/L, while increasing the taxane content 3- and 27-fold, respectively. The enhancing effect of these elicitors on taxane production, despite affecting growth, was confirmed in orbitally shaken TubeSpin Bioreactors 50, where the highest taxane content (8583.3 μg/L) was obtained when 1μM Cor was used and elicitation took place at a packed cell volume of 50%. Two benchtop stirred bioreactors, BIOSTAT B plus and UniVessel SU, were compared, the latter providing a higher biomass of C. avellana cell suspension cultures. Transferring the established optimum culture conditions for taxane production to the UniVessel SU resulted in a total taxane content of 6246.1 μg/L, a 10-fold increase compared with shake flask experiments.
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http://dx.doi.org/10.1016/j.jbiotec.2014.12.023DOI Listing
February 2015

Xanthomicrol: a comprehensive review of its chemistry, distribution, biosynthesis and pharmacological activity.

Mini Rev Med Chem 2014 ;14(9):725-33

Laboratori de Fisiologia Vegetal. Facultat de Farmacia, Universitat de Barcelona. Av. Joan XXIII sn. 08028 Barcelona. Spain.

Highly methoxylated flavones, which have known potential as cancer chemopreventive agents, accumulate on the leaf surfaces of some plant species and their physiological role is to protect the plant against harmful UV radiation. Xanthomicrol is one of the methoxylated flavones currently attracting most attention from researchers worldwide because of its promising pharmacological activities, including anti-spasmodic, anti-platelet and anti-cancer effects, among others. This review covers the chemistry and biological origin, distribution and pharmacological activity of xanthomicrol. Knowledge of the botanical distribution of this compound will not only encourage the use of plant sources for pharmacological purposes, but will also serve as a reference in the search for this valuable flavonoid in another genus or family. New approaches to xanthomicrol production are also described, including biotechnological attempts to develop xanthomicrol-producing plant cell factories.
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http://dx.doi.org/10.2174/1389557514666140820122818DOI Listing
June 2015

Synergistic effect of cyclodextrins and methyl jasmonate on taxane production in Taxus x media cell cultures.

Plant Biotechnol J 2014 Oct 9;12(8):1075-84. Epub 2014 Jun 9.

Department of Plant Biology, Faculty of Biology, University of Murcia, Campus de Espinardo, Murcia, Spain.

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.
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http://dx.doi.org/10.1111/pbi.12214DOI Listing
October 2014

A rational approach to improving the biotechnological production of taxanes in plant cell cultures of Taxus spp.

Biotechnol Adv 2014 Nov 27;32(6):1157-67. Epub 2014 Mar 27.

Secció de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII sn., 08028 Barcelona, Spain. Electronic address:

Taxol is a complex diterpene alkaloid scarcely produced in nature and with a high anticancer activity. Biotechnological systems for taxol production based on cell cultures of Taxus spp. have been developed, but the growing commercial demand for taxol and its precursors requires the optimization of these procedures. In order to increase the biotechnological production of taxol and related taxanes in Taxus spp. cell cultures, it is necessary not only to take an empirical approach that strives to optimize in-put factors (cell line selection, culture conditions, elicitation, up-scaling, etc.) and out-put factors (growth, production, yields, etc.), but also to carry out molecular biological studies. The latter can provide valuable insight into how the enhancement of taxane biosynthesis and accumulation affects metabolic profiles and gene expression in Taxus spp. cell cultures. Several rational approaches have focused on studying the transcriptomic profiles of key genes in the taxol biosynthetic pathway in Taxus spp. cell cultures treated with elicitors such as methyl jasmonate, coronatine and cyclodextrins in relation with the taxane pattern, production and excretion to the culture medium. These studies have provided new insights into the taxol biosynthetic pathway and its regulation. Additionally, identifying genes with low levels of expression even in the presence of elicitors, together with metabolomics studies, has shed light on the limiting steps in taxol biosynthesis and could help define suitable metabolic targets for engineering with the main aim of obtaining highly productive Taxus cultured cells. In this review, we have summarized the latest endeavors to enhance the molecular understanding of the action mechanism of elicitors in Taxus spp. cell cultures. Developments in the ongoing search for new and more effective elicitation treatments and the application of metabolic engineering to design new transgenic cell lines of Taxus with an improved capacity for taxane production are described.
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http://dx.doi.org/10.1016/j.biotechadv.2014.03.002DOI Listing
November 2014

Bicyclic and tetracyclic diterpenes from a Trichoderma symbiont of Taxus baccata.

Phytochemistry 2014 Jan 21;97:55-61. Epub 2013 Nov 21.

Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles ICSN, Centre National de la Recherche Scientifique, CNRS, Avenue de la Terrasse 91198, Gif-sur-Yvette cedex, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.phytochem.2013.10.016DOI Listing
January 2014

Biosynthesis of panaxynol and panaxydol in Panax ginseng.

Molecules 2013 Jul 2;18(7):7686-98. Epub 2013 Jul 2.

Lehrstuhl für Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.

The natural formation of the bioactive C17-polyacetylenes (-)-(R)-panaxynol and panaxydol was analyzed by 13C-labeling experiments. For this purpose, plants of Panax ginseng were supplied with 13CO2 under field conditions or, alternatively, sterile root cultures of P. ginseng were supplemented with [U-13C6]glucose. The polyynes were isolated from the labeled roots or hairy root cultures, respectively, and analyzed by quantitative NMR spectroscopy. The same mixtures of eight doubly 13C-labeled isotopologues and one single labeled isotopologue were observed in the C17-polyacetylenes obtained from the two experiments. The polyketide-type labeling pattern is in line with the biosynthetic origin of the compounds via decarboxylation of fatty acids, probably of crepenynic acid. The 13C-study now provides experimental evidence for the biosynthesis of panaxynol and related polyacetylenes in P. ginseng under in planta conditions as well as in root cultures. The data also show that 13CO2 experiments under field conditions are useful to elucidate the biosynthetic pathways of metabolites, including those from roots.
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http://dx.doi.org/10.3390/molecules18077686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6270202PMC
July 2013

Coronatine, a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate.

J Plant Physiol 2013 Jan 23;170(2):211-9. Epub 2012 Oct 23.

Departament de Ciències Experimentals i de Salut, Universitat Pompeu Fabra, E-08003 Barcelona, Spain.

Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received much attention recently for its potential to act as a plant growth regulator and elicitor of plant secondary metabolism. To gain more insight into the mechanism by which elicitors can affect the biosynthesis of paclitaxel (Px) and related taxanes, the effect of coronatine (Cor) and methyl jasmonate (MeJA) on Taxus media cell cultures has been studied. For this study, a two-stage cell culture was established, in which cells were first cultured for 14 days in a medium optimised for growth, after which the cells were transferred to medium optimised for secondary metabolite production. The two elicitors were added to the medium at the beginning of the second stage. Total taxane production in the cell suspension was significantly enhanced by both elicitors, increasing from a maximum level of 8.14mg/L in control conditions to 21.48mg/L (day 12) with MeJA and 77.46mg/L (day 16) with Cor. Expression analysis indicated that the txs, t13oh, t2oh, t7oh, dbat, pam, bata and dbtnbt genes were variably induced by the presence of the elicitors. Genes encoding enzymes involved in the formation of the polihydroxylated hypothetical intermediate (TXS, T13OH, T2OH, T7OH) and the phenylalanoil CoA chain (PAM) were stronger induced than those encoding enzymes catalysing the last steps of the Px biosynthetic pathway (DBAT, BAPT and DBTNBT). Notably, although taxane accumulation differed qualitatively and quantitatively following MeJA- or Cor-elicitation, gene expression induction patterns were similar, inferring that both elicitors may involve distinct but yet uncharacterised regulatory mechanisms.
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http://dx.doi.org/10.1016/j.jplph.2012.09.004DOI Listing
January 2013

Living between two worlds: two-phase culture systems for producing plant secondary metabolites.

Crit Rev Biotechnol 2013 Mar 29;33(1):1-22. Epub 2012 Feb 29.

Department of Plant Biology, Institute of Biology, State University of Campinas, Campinas, Brazil.

The two-phase culture system is an important in vitro strategy to increase the production of secondary metabolites (SMs) by providing an enhanced release of these compounds from plant cells. Whereas the first phase supports cell growth, the second phase provides an additional site or acts as a metabolic sink for the accumulation of SMs and also reduces feedback inhibition. This review is focused on several aspects of the two-phase culture system and aims to show the diverse possibilities of employing this technique for the in vitro production of SMs from plant cells. Depending on the material used in the secondary phase, two-phase culture systems can be broadly categorised as liquid-liquid or liquid-solid. The choice of material for the second phase depends on the type of compound to be recovered and the compatibility with the other phase. Different factors affecting the efficiency of two-phase culture systems include the choice of material for the secondary phase, its concentration, volume, and time of addition. Factors such as cell elicitation, immobilization, and permeabilization, have been suggested as important strategies to make the two-phase culture system practically reliable on a commercial scale. Since there are many possibilities for designing a two-phase system, more detailed studies are needed to broaden the range of secondary phases compatible with the various plant species producing SMs with potential applications, mainly in the food and pharmacology industries.
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http://dx.doi.org/10.3109/07388551.2012.659173DOI Listing
March 2013

Isolation and characterization of Stemphylium sedicola SBU-16 as a new endophytic taxol-producing fungus from Taxus baccata grown in Iran.

FEMS Microbiol Lett 2012 Mar 10;328(2):122-9. Epub 2012 Jan 10.

Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Tehran, Iran.

In this study, a total of 25 endophytic fungi were successfully isolated from the inner bark of Taxus baccata grown in Iran by the aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of the Taxus taxadiene synthase (ts) gene, which encodes the enzyme catalyzing the first committed step of taxol biosynthesis. Four of 25 isolated endophytic fungi isolates showed PCR positive for the ts gene. Subsequently, taxol and 10-deacetylbaccatin III (10-DAB III) were extracted from culture filtrates and mycelia of the PCR positive isolates and analyzed by high-performance liquid chromatography and mass spectrometry. The analysis showed that one isolate (SBU-16) produced taxol (6.9 ± 0.2 μg L(-1) ) and its intermediate compound, 10-DAB III (2.2 ± 0.1 μg L(-1) ). The isolate SBU-16 was identified as Stemphylium sedicola SBU-16, according to its morphological characteristics as well as the internal transcribed spacer nuclear rDNA gene sequence analysis. Interestingly, this is the first report of the genus Stemphylium as a taxol-producing taxon.
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http://dx.doi.org/10.1111/j.1574-6968.2011.02488.xDOI Listing
March 2012

Metabolic responses of Taxus media transformed cell cultures to the addition of methyl jasmonate.

Biotechnol Prog 2010 Jul-Aug;26(4):1145-53

Laboratorio de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, Joan XXIII s/n, Barcelona, Spain.

Dedifferentiated Taxus media cell cultures presenting the same genetic characteristics as the parent culture were established from transformed roots. Two transformed cell lines were studied: Rol C, carrying the T-DNA of A. rhizogenes 9,402 and TXS, carrying both the T-DNA of A. rhizogenes and the txs transgene of T. baccata under the control of the 35S CaMV promoter. In the second part of a previously optimized two-stage system, the transformed cell lines were cultured in a production medium supplemented with the elicitor methyl jasmonate. Taxane production in the transformed cultures was compared with an untransformed T. media cell line cultured in the same conditions. The highest taxane production was observed in the TXS cell line when cultured in the optimized production medium with methyl jasmonate, being 265% greater than in the untransformed control and 170% greater than in the Rol C cell line. However, txs expression and the activity of the enzyme taxadiene synthase in the TXS cells were lower than in the line carrying only the rol genes (Rol C). It is also noteworthy that the taxane production as well as the txs gene expression and TXS activity in all the cell lines, both transformed and untransformed, were clearly dependent on the elicitor action.
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http://dx.doi.org/10.1002/btpr.424DOI Listing
December 2010

Conversion of alpha-amyrin into centellosides by plant cell cultures of Centella asiatica.

Biotechnol Lett 2010 Feb 17;32(2):315-9. Epub 2009 Oct 17.

Laboratori de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, 08028 Barcelona, Spain.

Plant cell cultures of Centella asiatica produce small quantities of centellosides: madecassosid > asiaticosid > madecassic acid > asiatic acid. To obtain a more efficient production system of these bioactive triterpenoid compounds, we developed a process where the substrate, alpha-amyrin, was converted into centellosides by cell suspensions of C. asiatica. When alpha-amyrin in acetone was added at 0.01 mg/ml(-1) to the culture medium, together with the permeabilizing agent DMSO, after 7 days nearly 50% had penetrated the plant cells, of which almost 84% was transformed into centellosides. The system therefore efficiently converts alpha-amyrin into centellosides, thus opening a new possibility for the production of these compounds.
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http://dx.doi.org/10.1007/s10529-009-0143-xDOI Listing
February 2010