The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

Chemical constituents from red algae Bostrychia radicans (Rhodomelaceae): new amides and phenolic compounds

This study describes the isolation and structural determination of two amides, isolated for the first time: N,4-dihydroxy-N-(2'-hydroxyethyl)-benzamide (0.019%) and N,4-dihydroxy-N-(2'-hydroxyethyl)-benzeneacetamide (0.023%). These amides, produced by the red macroalgae Bostrychia radicans, had their structures assigned by NMR spectral data and MS analyses. In addition, this chemical study led to the isolation of cholesterol, heptadecane, squalene, trans-phytol, neophytadiene, tetradecanoic and hexadecanoic acids, methyl hexadecanoate and methyl 9-octadecenoate, 4-(methoxymethyl)-phenol, 4-hydroxybenzaldehyde, methyl 4-hydroxybenzeneacetate, methyl 2-hydroxy-3-(4-hydroxyphenyl)-propanoate, hydroquinone, methyl 4-hydroxymandelate, methyl 4-hydroxybenzoate, 4-hydroxybenzeneacetic acid and (4-hydroxyphenyl)-oxo-acetaldehyde. This is the first report concerning these compounds in B. radicans, contributing by illustrating the chemical diversity within the Rhodomelaceae family.

SEASONAL CHEMICAL COMPOSITIONS OF THE ESSENTIAL OILS OF TWOEugenia SPECIES AND THEIR ACARICIDAL PROPERTIES

The leaf essential oils of Eugenia lutescens Cambess andEugenia langsdorffii O. Berg, collected in the rainy (RS) and dry seasons (DS), were extracted by hydrodistillation and then characterized by a gas chromatography-flame ionization detector and a gas chromatography-mass spectrometer. The potential acaricidal activity and oviposition deterrence of these oils were evaluated against Tetranychus urticae . The oil yields were higher in the RS for E. lutescens, while those forE. langsdorffii were higher in the DS. α-Pinene and β-pinene were determined to be the major constituents of the oils fromE. lutescens, while bicyclogermacrene, spathulenol, and β-caryophyllene predominated in E. langsdorffii . Seasonal variations in the oils were primarily related to chemical diversity, and E. lutescens was more affected than was E. langsdorffii . The E. langsdorffii oil collected in the DS was most toxic to the spider mite, while the oils of E. lutescens and E. langsdorffii collected in the RS drastically reduced its egg quantities. This study successfully determined the periods of greater oil production and acaricidal activity.

Sobre o uso de métodos quimiométricos em química combinatória

Combinatorial chemistry has emerged as a tool to circumvent a major problem of pharmaceutical industries to discover new lead compounds. A rapid and massive evaluation of a myriad of newly synthesised compounds can be carried out. Combinatorial synthesis leads to high throughput screening en masse towards another myriad of biological targets. The design of a set of compounds based upon combinatorial chemistry may be envisaged by using of QSPR-SIMCA and QSAR-SIMCA as tools for classification purposes. This work deals with the definition and establishment of a spanned substituent space (SSS) that reduces the analogue numbers with no exclusion of global content. The chemical diversity may be set properly within a specified pharmacological field. This allows a better use of its potentiality without loosing information.

Os produtos naturais e a química medicinal moderna

Natural products have been utilized by humans since ancient times and the relief and cure of their diseases was the first purpose for using natural products in medicine. The history of the oriental and occidental civilizations is very rich in examples of the utilization of natural products in medicine and health care. Chinese traditional medicine is one of the most important examples of how natural products can be efficient in the treatment of diseases, and it points to the importance of scientific research on natural products, concerning the discovery of new active chemical entities. The complexity, chemical diversity and biological properties of natural products always fascinated people, and during the last 200 years, this led to the discovery of important new drugs. In the last 30 years, the development of new bioassay techniques, biotechnology methods, bio-guided phytochemical studies, automated high throughput screening and high performance analytical methods, have introduced new concepts and possibilities of rational drug design and drug discovery. In this context, natural products have played an important and decisive role in the development of modern medicinal chemistry.

Alcalóides indólicos isolados de espécies do gênero Aspidosperma (Apocynaceae)

Species from genus Aspidosperma (Apocynaceae) are popularly employed to treat various diseases. This genus is characterized by the occurrence of indole alkaloids. Taking into account the various biological activities attributed to these alkaloids, the description of the chemical diversity in genus Aspidosperma is important. A review of simple carbolinic alkaloids isolated from species of various genera was published in 1979. In 1987, it was published another one dealing with the relationships between the chemical structures of the indole alkaloids and the evolution of Aspidosperma species. This work updates the information about the indole alkaloids isolated from Aspidosperma species.

Variabilidade sazonal e biossíntese de terpenóides presentes no óleo essencial de Lippia alba (Mill.) N. E. Brown (Verbenaceae)

The essential oil of the leaves of Lippia alba chemotype linalool-1,8-cineol was extracted by hidrodistillation at different seasons and analyzed by GC/MS. Qualitative and quantitative variations in regard to the period of harvesting have been performed and the results were correlated with meteorological data. The essential oil yield varied from 0.33 to 0.67%. The chemical diversity of the constituents increased throughout the year, being 1,8-cineol and linalool the major components. Possible biosynthetic routes of mono and sesquiterpenoids present in the essential oil are discussed.

Tropical biodiversity: has it been a potential source of secondary metabolites useful for medicinal chemistry?

The use of natural products has definitely been the most successful strategy in the discovery of novel medicines. Secondary metabolites from terrestrial and marine organisms have found considerable use in the treatment of numerous diseases and have been considered lead molecules both in their natural form and as templates for medicinal chemistry. This paper seeks to show the great value of secondary metabolites and emphasize the rich chemical diversity of Brazilian biodiversity. This natural chemical library remains understudied, but can be a useful source of new secondary metabolites with potential application as templates for drug discovery.

Explorando produtos naturais microbianos nas fronteiras da Química e da Biologia

The chemistry of natural products has been remarkably growing in the past few decades in Brazil. Aspects related to the isolation and identification of new natural products, as well as their biological activities, have been achieved in different laboratories working on this subject in the country. More recently, the introduction of new molecular biology tools has strongly influenced the research on natural products, mainly those produced by microorganisms, creating new possibilities to assess the chemical diversity of secondary metabolites. This paper describes some ideas on how the research on natural products can have a considerable input from molecular biology in the generation of chemical diversity. We also explore the role of microbial natural products in mediating interspecific interactions and their relevance to ecological studies. Examples of the generation of chemical diversity are highlighted by using genome mining, mutasynthesis, combinatorial biosynthesis, metagenomics, and synthetic biology, while some aspects of microbial ecology are also discussed. The idea to bring up this topic is linked to the remarkable development of molecular biology techniques to generate useful chemicals from different organisms. Here, we focus mainly on microorganisms, even though similar approaches have also been applied to the study of plants and other organisms. Investigations in the frontier of chemistry and biology require interactions between different areas, characterizing the interdisciplinarity of this research field. The necessity of a real integration of chemistry and biology is pivotal to finding correct answers to a number of biological phenomena. The use of molecular biology tools to generate chemical diversity and control biosynthetic pathways is largely explored in the production of important biologically active compounds. Finally, we briefly comment on the Brazilian organization of research in this area, the necessity of new strategies for the graduation programs, and the establishment of networks as a way of organization to overcome some of the problems faced in the area of natural products.

Metabolômica, uma abordagem otimizada para exploração da biodiversidade brasileira: estado da arte, perspectivas e desafios

Brazilian biodiversity is a colossal source of secondary metabolites with remarkable structural features, which are valuable in further biodiscovery studies. In order to fully understand the relations and interactions of a living system with its surroundings, efforts in natural product chemistry are directed toward the challenge of detecting and identifying all the molecular components present in complex samples. It is plausible that this endeavor was born out of recent technological sophistication in secondary metabolite identification with sensitive spectroscopic instruments (MS and NMR) and higher resolving power of chromatographic systems, which allow a decrease in the amount of required sample and time to acquire data. Nevertheless, the escalation of data acquired in these analyses must be sorted with statistical and multi-way tools in order to select key information. Chromatography is also of paramount importance, more so when selected compounds need to be isolated for further investigation. However, in the course of pursuing a "greener" environment, new policies, with an aim to decrease the use of energy and solvents, are being developed and incorporated into analytical methods. Metabolomics could be an effective tool to answer questions on how living organisms in our huge biodiversity work and interact with their surroundings while also being strategic to the development of high value bio-derived products, such as phytotherapeutics and nutraceuticals. The incorporation of proper phytotherapeutics in the so-called Brazilian Unified Health System is considered an important factor for the urgent improvement and expansion of the Brazilian national health system. Furthermore, this approach could have a positive impact on the international interest toward scientific research developed in Brazil as well as the development of high value bio-derived products, which appear as an interesting economic opportunity in national and global markets. Thus, this study attempts to highlight the recent advances in analytical tools used in detection of secondary metabolites, which can be useful as bioproducts. It also emphasizes the potential avenues to be explored in Brazilian biodiversity, known for its rich chemical diversity.

Analysis of genetic diversity of Trypanosoma cruzi: an application of riboprinting and gradient gel electrophoresis methods

Analysis of restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Trypanosoma cruzi yields a typical `riboprint' profile that can vary intraspecifically. A selection of 21 stocks of T. cruzi and three outgroup taxa: T. rangeli, T. conorhini and Leishmania braziliensis were analysed by riboprinting to assess divergence within and between taxa. T. rangeli, T. conorhini and L. braziliensis could be easily differentiated from each other and from T. cruzi. Phenetic analysis of PCR-RFLP profiles indicated that, with one or two exceptions, stocks of T. cruzi could be broadly partitioned into two groups that formally corresponded to T. cruzi I and T. cruzi II respectively. To test if ribosomal 18S sequences were homogeneous within each taxon, gradient gel electrophoresis methods were employed utilising either chemical or temperature gradients. Upon interpretation of the melting profiles of riboprints and a section of the 18S independently amplified by PCR, there would appear to be at least two divergent 18S types present within T. cruzi. Heterogeneity within copies of the ribosomal 18S within a single genome has therefore been demonstrated and interestingly, this dimorphic arrangement was also present in the outgroup taxa. Presumably the ancestral duplicative event that led to the divergent 18S types preceded that of speciation within this group. These divergent 18S paralogues may have, or had, different functional pressures or rates of molecular evolution. Whether or not these divergent types are equally transcriptionally active throughout the life cycle, remain to be assessed.

Bacteria diversity and microbial biomass in forest, pasture and fallow soils in the southwestern Amazon basin

It is well-known that Amazon tropical forest soils contain high microbial biodiversity. However, anthropogenic actions of slash and burn, mainly for pasture establishment, induce profound changes in the well-balanced biogeochemical cycles. After a few years the grass yield usually declines, the pasture is abandoned and is transformed into a secondary vegetation called "capoeira" or fallow. The aim of this study was to examine how the clearing of Amazon rainforest for pasture affects: (1) the diversity of the Bacteria domain evaluated by Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE), (2) microbial biomass and some soil chemical properties (pH, moisture, P, K, Ca, Mg, Al, H + Al, and BS), and (3) the influence of environmental variables on the genetic structure of bacterial community. In the pasture soil, total carbon (C) was between 30 to 42 % higher than in the fallow, and almost 47 % higher than in the forest soil over a year. The same pattern was observed for N. Microbial biomass in the pasture was about 38 and 26 % higher than at fallow and forest sites, respectively, in the rainy season. DGGE profiling revealed a lower number of bands per area in the dry season, but differences in the structure of bacterial communities among sites were better defined than in the wet season. The bacterial DNA fingerprints in the forest were stronger related to Al content and the Cmic:Ctot and Nmic:Ntot ratios. For pasture and fallow sites, the structure of the Bacteria domain was more associated with pH, sum of bases, moisture, total C and N and the microbial biomass. In general microbial biomass in the soils was influenced by total C and N, which were associated with the Bacteria domain, since the bacterial community is a component and active fraction of the microbial biomass. Results show that the genetic composition of bacterial communities in Amazonian soils changed along the sequence forest-pasture-fallow.

Relationships between microbial activity and soil physical and chemical properties in native and reforested Araucaria angustifolia forests in the state of São Paulo, Brazil

Araucaria angustifolia (Bert.) O. Kuntze is the main component of the Mixed Ombrophilous forest and, in the State of São Paulo, it is associated with a high diversity of soil organisms, essential for the maintenance of soil quality, making the conservation of this ecosystem a major and pressing challenge. The objective of this study was to identify the physical and chemical properties that are most closely correlated with dehydrogenase enzyme activity, basal respiration and microbial biomass under native (NF) and replanted (RF) Araucaria angustifolia forests in three regions of the state of São Paulo, in winter and summer. The main differentiating factors between the areas were also determined. Each forest was represented by three true replications; at each site, from around the araucaria trees, 15 soil samples (0-20 cm) were collected to evaluate the soil physical, chemical and microbiological properties. At the same points, forest litter was sampled to assess mass and chemical properties. The following microbiological properties were evaluated: microbial biomass carbon (MBC), basal respiration (CO2-C), metabolic quotient (Q: CO2), dehydrogenase enzyme activity (DHA) as well as the physical properties (moisture, bulk density, macroporosity and total porosity), soil chemical properties [pH, organic carbon (org-C), P, Ca, K, Mg, Al, H+Al], litter dry mass, and C, N and S contents. The data were subjected to analysis of variance (TWO-WAY: ANOVA). A Canonical Discriminant Analysis (CDA) and a Canonical Correlation Analysis (CCA) were also performed. In the soil under NF, the values of K, P, soil macroporosity, and litter dry mass were higher and Q: CO2 and DHA lower, regardless of the sampling period, and DHA was lower in winter. In the RF areas, the levels of moisture, porosity and Q: CO2 were higher in both sampling periods, and DHA was higher in winter. The MBC was only higher under NF in the summer, while the litter contents of C, N and S were greater in winter. In winter, CCA showed a high correlation of DHA with CO2-C, pH and H+Al, while in the summer org-C, moisture, Mg, pH and litter C were more associated with DHA and CO2-C. The CDA indicated H+Al, available P, total porosity, litter S content, and soil moisture as the most discriminating variables between NF and RF, but moisture was the most relevant, in both seasons and CO2-C only in winter. The combined analysis of CCA and CDA showed that the contribution of the microbiological variables to a differentiation of the areas was small at both samplings, which may indicate that the period after reforestation was long enough to allow an almost complete recovery of the microbial activity.

Early Changes in Soil Metabolic Diversity and Bacterial Community Structure in Sugarcane under Two Harvest Management Systems

Preharvest burning is widely used in Brazil for sugarcane cropping. However, due to environmental restrictions, harvest without burning is becoming the predominant option. Consequently, changes in the microbial community are expected from crop residue accumulation on the soil surface, as well as alterations in soil metabolic diversity as of the first harvest. Because biological properties respond quickly and can be used to monitor environmental changes, we evaluated soil metabolic diversity and bacterial community structure after the first harvest under sugarcane management without burning compared to management with preharvest burning. Soil samples were collected under three sugarcane varieties (SP813250, SP801842 and RB72454) and two harvest management systems (without and with preharvest burning). Microbial biomass C (MBC), carbon (C) substrate utilization profiles, bacterial community structure (based on profiles of 16S rRNA gene amplicons), and soil chemical properties were determined. MBC was not different among the treatments. C-substrate utilization and metabolic diversity were lower in soil without burning, except for the evenness index of C-substrate utilization. Soil samples under the variety SP801842 showed the greatest changes in substrate utilization and metabolic diversity, but showed no differences in bacterial community structure, regardless of the harvest management system. In conclusion, combined analysis of soil chemical and microbiological data can detect early changes in microbial metabolic capacity and diversity, with lower values in management without burning. However, after the first harvest, there were no changes in the soil bacterial community structure detected by PCR-DGGE under the sugarcane variety SP801842. Therefore, the metabolic profile is a more sensitive indicator of early changes in the soil microbial community caused by the harvest management system.

Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis

Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA) and an eucalyptus arboretum (EAA). PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.