Rafael Zárate

Dichloromethane as a Solvent for Lipid Extraction and Assessment of Lipid Classes and Fatty Acids from Samples of Different Natures

The usefulness of the solvent mixture dichloromethane/methanol for lipid extraction and the determination of lipid classes and fatty acids in samples of different natures was conducted. Two different extraction methods were compared, one containing chloroform/methanol, another containing dichloromethane/methanol. Total lipid extraction showed some minor differences but no variation in the lipid classes. Regarding the fatty acid profile, in Echium virescens seeds, 17 major fatty acids could be identified and quantified, and all were equally extracted when either solvent system was employed. In Echium acanthocarpum hairy roots, 17 major fatty acids were quantified, showing some statistical differences for one cell line in favor of chloroform. The data obtained from the liquid nutrient medium were also comparable. The cod roe sample showed 31 major fatty acids, showing no statistical differences between the two solvent systems. Contrarily, the CH 2Cl 2 method was able to extract 31 main fatty acids found in European seabass dorsal muscle more efficiently than the CHCl 3 method. The results indicate that, for lipid extraction and fatty acid assessment, dichloromethane/methanol can readily replace the commonly employed chloroform/methanol, thus avoiding the major health, security, and regulatory problems associated with the use of chloroform.

Strategies for the genetic modification of the medicinal plant Catharanthus roseus (L.) G. Don

This review describes the different plant transformation techniques, including guided infection with Agrobacterium tumefaciens and A. rhizogenes, particle bombardment and protoplast fusion, that have been attempted to create transgenic Catharanthus roseus (L.) G. Don cell cultures, hairy roots and whole plants. The review also focuses on the different approaches used to manipulate and improve secondary metabolite yields in various culture systems, with special attention to the most relevant results achieved. Finally, under future perspectives, the authors propose several approaches which would likely be implemented with this species, to try to boost the accumulation of the anti-tumour agents, vinblastine and vincristine. Some comments on how the future of the genetic manipulation of medicinal plants may proceed aiming at achieving higher secondary metabolite yields are also given.

Significance of long chain polyunsaturated fatty acids in human health

In the last decades, the development of new technologies applied to lipidomics has revitalized the analysis of lipid profile alterations and the understanding of the underlying molecular mechanisms of lipid metabolism, together with their involvement in the occurrence of human disease. Of particular interest is the study of omega-3 and omega-6 long chain polyunsaturated fatty acids (LC-PUFAs), notably EPA (eicosapentaenoic acid, 20:5n-3), DHA (docosahexaenoic acid, 22:6n-3), and ARA (arachidonic acid, 20:4n-6), and their transformation into bioactive lipid mediators. In this sense, new families of PUFA-derived lipid mediators, including resolvins derived from EPA and DHA, and protectins and maresins derived from DHA, are being increasingly investigated because of their active role in the “return to homeostasis” process and resolution of inflammation. Recent findings reviewed in the present study highlight that the omega-6 fatty acid ARA appears increased, and omega-3 EPA and DHA decreased in most cancer tissues compared to normal ones, and that increments in omega-3 LC-PUFAs consumption and an omega-6/omega-3 ratio of 2–4:1, are associated with a reduced risk of breast, prostate, colon and renal cancers. Along with their lipid-lowering properties, omega-3 LC-PUFAs also exert cardioprotective functions, such as reducing platelet aggregation and inflammation, and controlling the presence of DHA in our body, especially in our liver and brain, which is crucial for optimal brain functionality. Considering that DHA is the principal omega-3 FA in cortical gray matter, the importance of DHA intake and its derived lipid mediators have been recently reported in patients with major depressive and bipolar disorders, Alzheimer disease, Parkinson’s disease, and amyotrophic lateral sclerosis. The present study reviews the relationships between major diseases occurring today in the Western world and LC-PUFAs. More specifically this review focuses on the dietary omega-3 LC-PUFAs and the omega-6/omega-3 balance, in a wide range of inflammation disorders, including autoimmune diseases. This review suggests that the current recommendations of consumption and/or supplementation of omega-3 FAs are specific to particular groups of age and physiological status, and still need more fine tuning for overall human health and well being.

Production of pulchelin E in hairy roots, callus and suspension cultures of Rudbeckia hirta L.

Agrobacterium rhizogenes transformed hairy roots, callus and suspension cultures of Rudbeckia hirta produced pulchelin E, a compound of the sesquiterpene lactone group, which is not present in the intact plant. During the research the level of the investigated lactone was optimised yielding 8.3 mg g−1 in the callus, 9.1 mg g−1 in the suspension cultures and 14.7 mg g−1 in the transformed roots (per dry weight basis). The content of pulchelin E in the cultures was calculated by thin-layer chromatography method with densitometric detection. Pulchelin E production in R. hirta tissues occurred only under constant light and was correlated with the growth cycle. In suspension cultures pulchelin E was partly released into the medium; this was not observed in transformed root cultures. The exceptional influence of 2,3,5-triiodobenzoic acid (TIBA) on pulchelin E production was stated.

Effects of Elicitors on Tropane Alkaloids and Gene Expression in Atropa baetica Transgenic Hairy Roots

Elicitation of transgenic Atropa baetica overexpressing the h6h gene with salicylic acid (SA), acetylsalicylic acid (ASA),or methyl jasmonate (MeJ) was conducted to boost tropane alkaloid yields. Scopolamine (1) amounts increased after treatment with ASA and MeJ, but not with SA. The highest enhancement of 1 was achieved with MeJ followed by ASA dissolved in EtOH. Transcriptomic analyses showed a direct relationship between content of 1 and gene expressions;the engineered h6h gene and other biosynthetic genes were stimulated. ASA dissolved in EtOH showed a high h6h gene expression, increasing 25-fold and 5-fold compared to controls; tr-I also displayed a 5-fold increase. The controls to which EtOH was added showed a 5-fold increase in h6h gene expression and 125-fold for pmt, demonstrating that EtOH also functioned as an enhancer of 1. MeJ was the best elicitor, displaying a 25-fold increase in h6h expression level, not affecting the expression of the other three genes analyzed, and it appears to possibly stimulate the phenylpropanoids branch of the tropane alkaloid pathway.

Tropane Alkaloid Distribution in Atropa baetica Plants

Fifteen different tropane alkaloids and derivatives were identified by GC-MS in various plant organs of Atropa baetica. The main root and leaves displayed the largest number of tropane alkaloids, most of which were also present in lateral roots. In contrast, only five of these alkaloids appeared in stem tissue. Quantitative analysis by HPLC showed the presence of the two major tropane alkaloids, atropine and scopolamine, in all the samples studied. Atropine was more abundant, with the highest concentration in the main root (ca. 10.0 mg/g dry wt) followed by leaves (ca. 3.0 mg/g dry wt); scopolamine was present in highest concentration in the main root (0.6 mg/g dry wt) followed by leaves (0.4 mg/g dry wt). The lowest concentrations of these compounds were detected in stem tissue, followed by the lateral roots. The main root constitutes the major tropane alkaloid storage site; moreover, the distribution of these compounds does not appear to be organ dependent. These latter two characteristics are in contrast to closely related Atropa genera.

Terpenoid indole alkaloid profile changes in Catharanthus pusillus during development

The terpenoid indole alkaloid content of Catharanthus pusillus was investigated during development from young to old plants. Different plant organs were assessed showing that the new leaves were the main repository site with vindoline ( approximately 4.8 mg/g DW) and catharanthine ( approximately 2.2 mg/g DW) being the major metabolites with the highest yields at the second and third sampling time (51 and 70 days, respectively). The other samples analysed, from old, oldest and yellow leaves followed in accumulation levels. The roots and stems were the least accumulative organs, although for the case of tubotaiwine the root was the most important organ. It appeared that the alkaloid content changed coinciding with the different developmental stages of the plants, particularly at flowering and fruiting stages. Moreover, this species seems to constitute a precious source of the monomerics, vindoline and catharanthine, intermediates in the synthesis of the two important antitumor dimerics vincristine and vinblastine, which did not accumulate in this species.

Cloning, characterization and analysis of expression profiles of a cDNA encoding a hyoscyamine 6β-hydroxylase (H6H) from Atropa baetica Willk

A new cDNA encoding hyoscyamine 6beta-hydroxylase (H6H, EC 1.14.11.11), a bifunctional enzyme catalyzing the last two steps in the biosynthesis of scopolamine, was isolated from Atropa baetica roots (GenBank accession no. EF442802). The full cDNA sequence showed an ORF of 1035bp, coding for a protein with 344 amino acid residues. Sequence analyses at the nucleotide level showed that this ORF shares high identity with other H6H from different plant species, such as Anisodus tanguticus and Hyoscyamus niger with 90% identity, and an almost total identity with A. belladonna (98%). Tissue expression analyses showed that the gene transcript was tissue dependent, appearing exclusively in roots, thus being the only biosynthetic site for the production of scopolamine. Furthermore, Southern hybridization experiments revealed that this gene was not part of a multigene family as appears in low copy number. Phylogenetic tree analysis indicated that A. baetica H6H had a very close relationship with A. belladonna and to a lesser extent with H. niger.

The lupane-type triterpene 30-oxo-calenduladiol is a CCR5 antagonist with anti-HIV-1 and anti-chemotactic activities

The existence of drug-resistant human immunodeficiency virus (HIV) viruses in patients receiving antiretroviral treatment urgently requires the characterization and development of new antiretroviral drugs designed to inhibit resistant viruses and to complement the existing antiretroviral strategies against AIDS. We assayed several natural or semi-synthetic lupane-type pentacyclic triterpenes in their ability to inhibit HIV-1 infection in permissive cells. We observed that the 30-oxo-calenduladiol triterpene, compound 1, specifically impaired R5-tropic HIV-1 envelope-mediated viral infection and cell fusion in permissive cells, without affecting X4-tropic virus. This lupane derivative competed for the binding of a specific anti-CCR5 monoclonal antibody or the natural CCL5 chemokine to the CCR5 viral coreceptor with high affinity. 30-Oxo-calenduladiol seems not to interact with the CD4 antigen, the main HIV receptor, or the CXCR4 viral coreceptor. Our results suggest that compound 1 is a specific CCR5 antagonist, because it binds to the CCR5 receptor without triggering cell signaling or receptor internalization, and inhibits RANTES (regulated on activation normal T cell expressed and secreted)-mediated CCR5 internalization, intracellular calcium mobilization, and cell chemotaxis. Furthermore, compound 1 appeared not to interact with β-chemokine receptors CCR1, CCR2b, CCR3, or CCR4. Thereby, the 30-oxo-calenduladiol-associated anti-HIV-1 activity against R5-tropic virus appears to rely on the selective occupancy of the CCR5 receptor to inhibit CCR5-mediated HIV-1 infection. Therefore, it is plausible that the chemical structure of 30-oxo-calenduladiol or other related dihydroxylated lupane-type triterpenes could represent a good model to develop more potent anti-HIV-1 molecules to inhibit viral infection by interfering with early fusion and entry steps in the HIV life cycle.

Induction and development of adventitious shoots of Atropa baetica as a means of propagation

In vitro propagation of Atropa baetica was established employing axillary buds. Single buds were cultured through a multiple shoot induction phase, rooting phase, and then followed by acclimatization in soil. For multiple shoot induction, Murashige and Skoog (MS) medium with 3% sucrose, supplemented with either 0.75 or 1.25 mg l-1 of BAP provided the best results with an average of 5.6 shoots per explant after 31 days of culture. Similar results were obtained with higher BAP concentrations (1.75–2.0 mg l-1); however, these media had a negative effect on the subsequent root induction due to residual BAP effect. Medium containing only 0.25 mg l-1 of BAP induced a significantly lower number of shoots. Root induction occurred spontaneously after transferring the shoots onto MS medium lacking any plant growth regulator. Moreover, root induction also occurred on media supplemented with 0.125 and 0.25 mg l-1 of NAA. On these two rooting media, this response was more prominent and with a higher number of roots per explant. Nevertheless, after 28 days on root induction medium, the number of rooted plantlets was similar on the three media. Acclimatization of plantlets in soil was very successful (95.52%). However, all plantlets which died during acclimatization were rooted on medium containing 0.25 mg l-1 NAA suggesting a negative carry over effect of this medium upon plantlet survival, irrespective of the initial BAP treatment used. On the other hand, karyological studies showed no variation in the number of chromosome (2n=72) in root tips of the plantlets produced.