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In vitro cultures of undifferentiated plant cells of Tessaria absinthioides, a native herb popularly recognized and used for its health benefits, were studied as potential food supplements. These tissues were incubated under two light conditions, and the biomass obtained was freeze-dried and oven-dried. To evaluate their nutritional value, their physicochemical and functional properties were determined. Although in some cases there were significant differences in the results according to the drying methodology applied, all these tissues presented a high proportion of proteins (23.6-28.3%), a low percentage of fats (< 2%) constituted mainly by phytosterols, and a significant amount of crude fibers (6.9-9.0%) and ashes (> 10%). In addition, the freeze-dried calli resulted in a product with better functional properties. On the other hand, their phytochemical profiles and antioxidant capacity were studied and compared with tissues from wild specimens and with green tea and chamomile as reference extracts.

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Although many countries banned the insecticide endosulfan, it is still an environmental pollutant. Plants metabolize the two diastereomers of the formulations known as technical grade endosulfan (TGE) by two phase I pathways: hydrolysis leading to less toxic derivatives and oxidation giving endosulfan sulfate which is as toxic as endosulfan itself. We assessed the removal, bioaccumulation and phase I metabolization of TGE from water matrices using hairy root clones (HRs) of three edible species, Brassica napus, Raphanus sativus and Capsicum annuum. B. napus and C. annuum HRs removed 86% of TGE from the bioreaction media in 2 and 96 h, respectively, whereas R. sativus HRs removed 91% of TGE within 6 h of biotreatment. In the experiments with B. napus, only endosulfan sulfate was detected in both biomass and medium, whereas R. sativus and C. annuum accumulated endosulfan sulfate and endosulfan alcohol. Besides, endosulfan lactone was detected in C. annuum reaction medium. Acute ichthyotoxicity assays toward Poecilia reticulata showed that media contaminated with TGE lethal levels did not produce mortality after the phytotreatments. This research highlights the feasibility of using HRs to evaluate plant enzymatic abilities toward xenobiotics and their potential for the design of ex situ decontamination processes.

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Plants in the Amaryllidaceae family synthesize a diversity of bioactive alkaloids. Some of these plant species are not abundant and have a low natural multiplication rate. The aims of this work were the alkaloids analysis of a Habranthus cardenasianus bulbs extract, the evaluation of its inhibitory activity against cholinesterases, and to test several propagation strategies for biomass production. Eleven compounds were characterized by GC-MS in the alkaloid extract, which showed a relatively high proportion of tazettine. The known alkaloids tazettine, haemanthamine, and the epimer mixture haemanthidine/6-epi-haemanthidine were isolated and identified by spectroscopic methods. Inhibitory cholinesterases activity was not detected. Three forms of propagation were performed: bulb propagation from seed, cut-induced bulb division, and micropropagated bulbs. Finally, different imbibition and post-collection times were evaluated in seed germination assays. The best propagation method was cut-induced bulb division with longitudinal cuts into quarters (T1) while the best conditions for seed germination were 0-day of post-collection and two days of imbibition. The alkaloids analyses of the H. cardenasianus bulbs showed that they are a source of anti-tumoral alkaloids, especially pretazettine (tazettine) and T1 is a sustainable strategy for its propagation and domestication to produce bioactive alkaloids.

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While many redox enzymes are nowadays available for synthetic applications, the toolbox of ene-reductases is still limited. Consequently, the screening for these enzymes from diverse sources in the search of new biocatalyst suitable for green chemistry approaches is needed. Among 13 plant tissue cultures, Medicago sativa and Tessaria absinthioides calli, as well as Capsicum annuum hairy roots, were selected due to their ability to hydrogenate the CC double bond of the model substrate 2-cyclohexene-1-one. The three axenic plant cultures showed more preference toward highly activated molecules such as nitrostyrene and maleimide rather than the classical substrates of the well-known Old Yellow Enzymes, resembling the skills of the NAD(P)H-dependent flavin-independent enzymes. When the three biocatalytic systems were applied in the reduction of chalcones, T. absinthioides showed high chemoselectivity toward the CC double bond whereas the other two demonstrated abilities to biohydrogenate the CC double bounds and the carbonyl groups in a sequential fashion.

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Endosulfan is a Persistent Organic Pollutant insecticide still used in many countries. It is commercially available as mixtures of two diastereomers, α- and ß-endosulfan, known as technical grade endosulfan (TGE). A laboratory model based on the use of axenic plant cell cultures to study the removal and metabolization of both isomers from contaminated water matrixes was established. No differences were recorded in the removal of the two individual isomers with the two tested endemic plants, Grindelia pulchella and Tessaria absinthioides. Undifferentiated cultures of both plant species were very efficient to lower endosulfan concentration in spiked solutions. Metabolic fate of TGE was evaluated by analyzing the time course of endosulfan metabolites accumulation in both plant biomass and bioremediation media. While in G. pulchella we only detected endosulfan sulfate, in T. absinthioides the non-toxic endosulfan alcohol was the main metabolite at 48h, giving the possibility of designing phytoremediation approaches.

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A chemoenzymatic strategy for the synthesis of enantiomerically pure novel alkaloids (1S,3R)-1-benzyl-2,3-dimethyl-1,2,3,4-tetrahydroisoquinolines is presented. The key steps are the biocatalytic stereoselective reductive amination of substituted 1-phenylpropan-2-one derivatives to yield chiral amines employing microbial ω-transaminases, and the diastereoselective reduction of a Bischler-Napieralski imine intermediate by catalytic hydrogenation in the presence of palladium on charcoal, leading exclusively to the desired cis-isomer.

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A screening based on undifferentiated plant cells allowed identifying Gardenia jasminoides as the best biocatalyst to perform the kinetic resolution of 1-phenylethanol. This species was further tested for its ability to oxidize stereoselectively the (S)-isomers from racemic mixtures of secondary alcohols leaving their antipodes unaffected in Tris-HCl buffer. Those substrates which afforded the best results in the kinetic resolution were subjected to a chemo-enzymatic sequence of deracemization. G. jasminoides immobilized cells in calcium alginate were used for the oxidation of the (S)-enantiomers and, in a second step, NaBH(4) was added to the same vessel for the reduction of the corresponding ketone. The sequential repetition of these two steps allowed obtaining the R-alcohols in 82-90% yield in high optical purity (71-96% ee). Despite the viability of the cells is affected by the chemical reagent, their enzymes remain active due to the protective environment of the calcium alginate beads.

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Coumarin metabolism by several Aspergillus strains was studied. Aspergillus ochraceus and Aspergillus niger carried out the reduction of the C3-C4 double bond to yield dihydrocoumarin in 24h. Meanwhile, the first strain did not transform dihydrocoumarin after 7d, A. niger demonstrated to have two divergent catabolic pathways: (a) the lactone moiety opening and further reduction of the carboxylic acid furnishing the primary alcohol 2-(3-hydroxypropyl)phenol and, (b) the hydroxylation of the aromatic ring of dihydrocoumarin at a specific position to give 6-hydroxy-3,4-dihydrochromen-2-one. Aspergillus flavus did not perform double bond reductions, and only produced oxygenated metabolites, mainly 5-hydroxycoumarin. Enzyme-specific inhibitors and a coumarin analogous were useful to confirm the A. niger catabolic route.

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5,6-Epoxycholestan-3beta-ol derivatives were hydrolyzed in a diastereoconvergent manner by growing and resting cells of several strains of Aspergillus niger, particularly A. niger ATCC 11394. These strains displayed opposite regioselectivity toward each isomer in an alpha and beta epoxide mixture, thus, the nucleophilic attack took place at the less substituted and the most substituted carbon atom on each diasteromer, respectively. These biocatalysts opened trisubstituted oxiranes but were unable to hydrolyze the disubstituted oxiranes in the tested sterol derivatives. These findings suggest that A. niger strains possess another hydrolytic ability different from the commercial A. niger epoxide hydrolase (EH) that did not accept this kind of steroidal oxiranes as substrates.

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Grindelia pulchella callus and cell suspension cultures were established from seedling leaves. When several phytoregulator supplementations were assayed in solid Murashige and Skoog medium containing 3 percent (w/v) of sucrose (MS medium), combinations of indole-3-butyric acid (IBA) and N6-benzylaminopurine (BA) resulted the most appropriate conditions to generate fast growing friable calli with detectable levels of grindelic acid. Moreover, the same basal media supplemented with 20.0 µM IBA/4.4 µM BA was found to be optimal for cell growth in submerged cultures (µmax = 0.26 days-1) while the addition of 20.0 µM IBA/18.0 µM BA resulted in a relative higher metabolite production (4.55 mg/gDW) when the inocula was 5 percent (v/v). Furthermore, three different stress factors and combinations of them were used to elicit cell suspensions. These experiments demonstrated that the combination of CuSO4 and dimethylsulfoxide (DMSO) increase the grindelic acid production to 2.63 mg/gDW in the elicited essay versus 0.756 mg/gDW in the control, at expense of cell growth. In contrast, the addition of jasmonic acid (JA) alone and combined with DMSO neither affected cell growth nor grindelic acid accumulation.

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