Garcinia macrophylla: a Promising Underutilized Source of Bioactive Compounds in the Amazonia - A Review.

Native species from the Amazonia are still unknown or underutilized and few information about their chemical and biological properties are available in the literature. Among the underutilized plant species in the Amazonia, Garcinia macrophylla can be seen as a promising source of bioactive compounds with relevant biological properties. The stem bark and leaves were the main investigated plant parts, mainly concerning the antioxidant, antibacterial, cytotoxicity and antidiabetic properties. However, the bioactive compounds and biological properties of the edible fruits were not yet reported. Systematic investigations covering the Amazonia biome, concerning plants and vegetables as strategic resources are of paramount importance for the sustainable development of the forest. Therefore, this review gathered the available information in the literature concerning general aspects, chemical profile and biological properties of G. macrophylla, for the first time, which highlighted that systematic and robust in vitro and in vivo research, are still needed to elucidate the phytochemical profiles and associated relevant biological properties.

Genomic screening and molecular dynamics simulations of cyanovirin-N homologs from cyanobacteria phylum.

The phylum cyanobacteria are one of the most ancient groups of organisms on the planet and are well recognized due to its wide distribution, ecological role, and biotechnological potential. Cyanobacterial lectins are being extensively explored due to their antiviral activity, mainly because of their capacity of inhibiting HIV strains from infecting human cells by gp120 and gp41 binding. Cianovirin-N from Nostoc ellipsosporum was the first lectin isolated with this property. Since then, various homologs have been discovered and characterized. In this article, we present results of a genomic screening to find cyanovirin-N homologs (CVNH) in all cyanobacteria genomes available in the GenBank, resulting in 155 CVNH proteins with 63 presenting significant identity differences of cyanovirin-N. Homology modeling and molecular dynamics were employed to characterize 18 unexplored models and their functional capacity of binding to Manα(1-2)Man. Results presented here support the hypothesis of multiple ligand recognition for the CVNH family and may help to understand the function of these lectins for the producer cyanobacteria. Additionally, the theoretical results observed here justify carrying out experimental investigations that can expand the therapeutic potential of cyanobacterial lectins.

Optimization of Light Intensity and NaNO3 Concentration in Amazon Cyanobacteria Cultivation to Produce Biodiesel.

The objective of this study, for the first time, was to optimize Amazonian cyanobacterial culture conditions for improving cell productivity and lipid content, by analyzing the effect of light intensity and nitrogen concentration, for empirically evaluating biodiesel quality parameters. The strains Synechocystis sp. CACIAM05, Microcystis aeruginosa CACIAM08, Pantanalinema rosaneae CACIAM18, and Limnothrix sp. CACIAM25, were previously identified by morphological and molecular analysis (16S rRNA) and were selected based on their production of chlorophyll a and dry cell weight. Then, factorial planning (22) with central points was applied, with light intensity and NaNO3 concentration as independent variables. As response variables, cell productivity and lipid content were determined. Statistical analysis indicated that for all strains, the independent variables were statistically significant for cell productivity. Analysis of the fatty acid composition demonstrated diversity in the composition of the fatty acid profile from the experimental planning assays of each strain. The Biodiesel Analyzer software predicted the biodiesel quality parameters. CACIAM05 and CACIAM25 obtained better parameters with low levels of light intensity and NaNO3 concentration, whereas CACIAM08 and CACIAM18 obtained better parameters with low NaNO3 concentrations and high luminous intensity.

Genomic screening of new putative antiviral lectins from Amazonian cyanobacteria based on a bioinformatics approach.

Lectins are proteins of nonimmune origin, which are capable of recognizing and binding to glycoconjugate moieties. Some of them can block the interaction of viral glycoproteins to the host cell receptors acting as antiviral agents. Although cyanobacterial lectins have presented broad biotechnological potential, little research has been directed to Amazonian Cyanobacterial diversity. In order to identify new antiviral lectins, we performed genomic analysis in seven cyanobacterial strains from Coleção Amazônica de Cianobactérias e Microalgas (CACIAM). We found 75 unique CDS presenting one or more lectin domains. Since almost all were annotated as hypothetical proteins, we used homology modeling and molecular dynamics simulations to evaluate the structural and functional properties of three CDS that were more similar to known antiviral lectins. Nostoc sp. CACIAM 19 as well as Tolypothrix sp. CACIAM 22 strains presented cyanovirin-N homologues whose function was confirmed by binding free energy calculations. Asn, Glu, Thr, Lys, Leu, and Gly, which were described as binding residues for cyanovirin, were also observed on those structures. As for other known cyanovirins, those residues in both our models also made favorable interactions with dimannose. Finally, Alkalinema sp. CACIAM 70d presented one CDS, which was identified as a seven-bladed beta-propeller structure with binding sites predicted for sialic acid and N-acetylglucosamine. Despite its singular structure, our analysis suggested this molecule as a new putative antiviral lectin. Overall, the identification and the characterization of new lectins and their homologues are a promising area in antiviral research, and Amazonian cyanobacteria present biotechnological potential to be explored in this regard.

Therapeutic effect of andiroba oil (Carapa guianensis Aubl.) against oral mucositis: an experimental study in golden Syrian hamsters.

OBJECTIVES: The aim of this study was to investigate the healing activity of andiroba (Carapa guianensis Aubl.) against oral mucositis (OM) induced by 5-fluorouracil in golden Syrian hamsters. MATERIALS AND METHODS: A total of 122 animals were randomized and divided into six groups: andiroba oil 100%, andiroba oil 10%, andiroba oil 10% refined, no treatment group, all n = 28; and negative control (NC) and cyclophosphamide (CPA) groups, both n = 5. OM was induced by intraperitoneal administration of 60 mg/kg 5-FU on days 0, 5 and 10 followed by mechanical trauma on the oral mucosa on days 1 and 2. From day 1 to day 15, the animals of the andiroba group were treated three times a day. On days 4, 8, 12 and 15, the mucosa was photographed and removed for clinical and histopathological analysis. The bone marrow of the femur was removed and the micronucleus test was performed to evaluate the cytotoxicity and genotoxicity. The data were subjected to analysis of variance, followed by the Tukey and Bonferroni test. RESULTS: Treatment with 100% andiroba oil reduced the degree of OM compared to that reported in the other groups (p < 0.05). Andiroba oil at both concentrations was not cytotoxic, but treatment with 100% andiroba oil showed a genotoxic potential (p < 0.001). CONCLUSIONS: Frequent administration of andiroba oil accelerated the healing process in an experimental model of 5-fluorouracil-induced OM. However, the genotoxicity of andiroba in other cell systems and under other conditions are being tested. CLINICAL RELEVANCE: The use of andiroba in topical form may be associated with reduced intensity of OM. Seek therapeutic alternatives to minimize the pain and suffering that these side effects cause cancer patients is an important scientific step.

Anti-dengue virus activity of scytovirin and evaluation of point mutation effects by molecular dynamics and binding free energy calculations.

The absence of a specific treatment against DENV has led to intensive research into developing strategies for curing the infection. One lectin with high antiviral activity is scytovirin, which was isolated from the cyanobacterium Scytonema varium and has proven activity against HIV and Zaire Ebola Virus. To achieve the results presented here, we tested the affinity of full-length scytovirin, SD1 and SD2 separately, and six SD1 mutants for DENV glycoprotein E carbohydrate by Molecular Dynamics (MD) simulations and binding free energy calculations. It was possible to identify the key residues for protein-ligand interaction such as Glu10, Ala11, Pro17, Ans18, Arg30, Thr41, Ser42 and Arg43, which also has importance action against HIV. All binding free energy calculations showed negative values to ΔGbind of protein-DENV carbohydrate complexation. Additionally, these results are similar to the values of scytovirin and HIV gp120 carbohydrate complexation (-32.20 kcal/mol). Furthermore, we found that SD1 individually has more affinity to the carbohydrate and the Asn9, Glu10, Asn18, Arg30 and Arg43 demonstrated an important role in this matter. We also found that mutant G48R has better affinity (-34.10 kcal/mol) for the DENV carbohydrate than the wild type protein (-27.15 kcal/mol).

In silico analysis of the cyanobacterial lectin scytovirin: new insights into binding properties.

Scytovirin is a lectin isolated from the cyanobacterium Scytonema varium that has shown activity against HIV, SARS coronavirus and Zaire Ebola virus. Its 95 amino acids are divided into two structural domains (SD), the first spanning amino acids 1-48 (SD1) and the second 49-95 (SD2). Interestingly, the domains are nearly identical but differ in their affinities for carbohydrates. With the aim of enhancing understanding of the binding properties of scytovirin, we performed molecular dynamics (MD) simulations of scytovirin complexed with Man4. We set up three systems: (i) Man4 bound to both domains (SD1 + SD2) using the full-length protein; (ii) Man4 bound to an incomplete protein, containing only SD1 and (iii) Man4 bound to an incomplete protein containing only SD2. Contrary to other reports, binding free energy results suggest that Man4 can bind simultaneously to SD1 and SD2 binding regions, but SD1 individually has the best values of energy and the best affinity for Man4. Decomposition of the binding free energy showed that the residues that interact with Man4 were different in the three systems, suggesting that the binding mechanism of Man4 varies between full-length protein, SD1 and SD2. The results presented here may help to formulate strategies to use scytovirin and promote mutagenesis studies to improve the antiviral activity of scytovirin.

The lipidome, genotoxicity, hematotoxicity and antioxidant properties of andiroba oil from the Brazilian Amazon.

Andirobeira is an Amazonian tree, the seeds of which produce a commercially valuable oil that is used in folk medicine and in the cosmetic industry. Andiroba oil contains components with anti-inflammatory, cicatrizing and insect-repellant actions. However, virtually nothing is known of the safety of this oil for humans. The aim of this work was therefore to investigate the hematotoxicity, genotoxicity and mutagenicity of andiroba oil using the comet and micronucleus assays, and to assess its antioxidant properties and lipidome as a means of addressing safety issues. For the experiments, andiroba oil was administered by gavage for 14 consecutive days in nulliparous female Swiss mice randomly distributed in four groups: negative control and three doses of oil (500, 1000 and 2000 mg/kg/day). These doses were chosen based on recommendations of the OECD guideline no. 474 (1997). GC/MS was used to investigate the free fatty acid, cholesterol and triterpene content of andiroba oil in a lipidomic analysis. No clinical or behavioral alterations were observed throughout the period of treatment, and exposure to andiroba oil at the doses and conditions used here did not result in hematotoxic, genotoxic or mutagenic effects. Tests in vitro showed that oil sample 3 from southwestern of Brazilian Amazon had a high antioxidant capacity that may protect biological systems from oxidative stress, although this activity remains to be demonstrated in vivo.

Jambu Flower Extract (Acmella oleracea) Increases the Antioxidant Potential of Beer with a Reduced Alcohol Content.

Craft beers with different sensory perceptions have received the attention of more demanding consumers. In this sense, the application of plant extracts as brewing adjuncts is being increasingly studied. Allied with these perspectives is the consumption of lower alcoholic beverages, which also represents the desire for a market niche that has been growing gradually. The present work aimed to produce craft lager beer with the addition of plant extract and reduced alcohol content by partial replacement of malt with malt bagasse. The physical-chemical analyzes of the beer produced showed that it was possible to reduce the alcohol content by 40.5% compared to the control sample. In addition, an extract of Acmella oleracea (Jambu) obtained by supercritical extraction was added to increase the beer's antioxidant capacity. The ABTS, DPPH, and ORAC methods evaluated the antioxidant capacity. These assays were performed again after six months of storage. The quantification and identification of the significant substance in the extract (spilanthol) were performed using Gas Chromatography (GC-FID), Thin Layer Chromatography (TLC), and Attenuated Total Reflectance Infrared Spectroscopy (FTIR-ATR). The results showed significant increases in antioxidant activity compared to the sample without extract. This positive aspect opens a perspective for using jambu flower extract as a prominent antioxidant adjunct in beer.

Crude Enzyme Concentrate of Filamentous Fungus Hydrolyzed Chitosan to Obtain Oligomers of Different Sizes.

Chitosan is a non-cytotoxic polysaccharide that, upon hydrolysis, releases oligomers of different sizes that may have antioxidant, antimicrobial activity and the inhibition of cancer cell growth, among other applications. It is, therefore, a hydrolysis process with great biotechnological relevance. Thus, this study aims to use a crude enzyme concentrate (CEC) produced by a filamentous fungus to obtain oligomers with different molecular weights. The microorganism was cultivated in a liquid medium (modified Czapeck-with carboxymethylcellulose as enzyme inducer). The enzymes present in the CEC were identified by LC-MS/MS, with an emphasis on cellobiohydrolase (E.C 3.2.1.91). The fungus of the Aspergillus genus was identified by amplifying the ITS1-5.8S-ITS2 rDNA region and metaproteomic analysis, where the excreted enzymes were identified with sequence coverage greater than 84% to A. nidulans. Chitosan hydrolysis assays compared the CEC with the commercial enzyme (Celluclast 1.5 L®). The ability to reduce the initial molecular mass of chitosan by 47.80, 75.24, and 93.26% after 2.0, 5.0, and 24 h of reaction, respectively, was observed. FTIR analyses revealed lower absorbance of chitosan oligomers' spectral signals, and their crystallinity was reduced after 3 h of hydrolysis. Based on these results, we can conclude that the crude enzyme concentrate showed a significant technological potential for obtaining chitosan oligomers of different sizes.

Adsorption of Cu (II) Ions Present in the Distilled Beverage (Sugar Cane Spirit) Using Chitosan Derived from the Shrimp Shell.

Cachaça (sugar cane spirit) is a typically Brazilian distilled beverage. Copper ions can be present in craft beverages despite their acceptance in the national and international market. This study aims to evaluate the efficiency of chitosan as an adsorbent in removing copper (II) from cachaça. The structural characteristics of the obtained chitosan and the effect of adsorbed copper were evaluated by Fourier Transform Infrared Spectroscopy (ATR-FTIR), viscosimetry, X-ray diffraction (XRD), and scanning electron microscopy (SEM). The deacetylation reaction from chitin (shrimp shell) resulted in chitosan with a deacetylation degree of 88.9% (potentiometric titration) and 86.9% (FTIR), low crystallinity, and an estimated molecular weight of 162.96 kDa. The copper reduction rate was 84.09% evaluated by spectrophotometric titration and microwave-induced plasma optical emission spectrometry (MIP-OES). The amine groups of chitosan had adsorption affinity with copper ions, and the kinetic analysis showed a better fit of the data by the Elovich equation, suggesting that the chemosorption mechanism controlled the kinetic process. The results suggest that chitosan has the potential to improve the quality and safety of cachaça.

Ultrasound-assisted lipid extractions, enriched with sterols and tetranortriterpenoids, from Carapa guianensis seeds and the application of lipidomics using GC/MS.

This study describes the optimized stages of lipid extraction assisted by ultrasound to increase the concentrations of limonoids and steroids from andiroba seeds, identified as Carapa guianensis Aublet, and the lipidome analyzed by TLC and GC/MS. After boiling and peeling, crushed seeds were subjected to extractions with n-hexane (G1), acetone (G2), and methanol (G3) in an ultrasonic bath for 30 minutes at 50 °C. These extracts were analyzed by analytical TLC; aliquots of each extract and a marketable oil, used as a reference, were transesterified followed by silylation with BSTFA + 1% TMCS, and analyzed by GC/MS. The reference oil and the biomass of the seeds did not present significant differences in the profiles of free fatty acids (FFA). However, differences were observed in the profiles of tetranortriterpenoids (TnTT) (limonoids) in the seeds. Afterward, the analysis of the extracts G1, G2, and G3 detected free steroids: campesterol, stigmasterol, and ß-sitosterol; and limonoids: 7-deacetyl-7-oxogedunin, 6α-acetoxy-gedunin, deacetylgedunin, and epoxyacetylgedunin. The use of lipidomic techniques associated with ultrasound-assisted extraction was applied for the first time to enhance the triterpenoids and steroids, mainly in G3, describing a faster and more economical process, and allowing a one-step lipidome analysis of the andiroba seeds.

Andiroba oil and nanoemulsion (Carapa guianensis Aublet) reduce lesion severity caused by the antineoplastic agent doxorubicin in mice.

Doxorubicin (DOX) is an anthracycline antibiotic used in the fight against many types of cancer. Although it is quite effective for this purpose, its clinical use is limited by its severe side effects, highlighting the relevance of efforts to identify substances that act to minimize these effects. In this work, we sought to verify the ability of andiroba oil (AO) and a nanoemulsion of andiroba oil (AN) to lessen the side effects of DOX. The animals were separated into 7 groups with 6 animals each: mice treated with AO (2000 mg/kg), AN (2000 mg/kg), the antineoplastic agent DOX (40 mg/kg), AO+DOX, AN+DOX and solvent controls was used of negative control (corn oil and nanoemulsion surfactant). AO and AN were administered for 14 consecutive days orally by gavage and on the 13th day, applied DOX by intraperitoneal route (i.p.), in order to evaluate the protective potential of andiroba. The animals were euthanized on the 15th day. Hematological, biochemical, histological, and immunohistochemical parameters were analyzed. Andiroba reduced several aspects of the severity of lesions caused by DOX, decreasing hematotoxicity and the severity of histological changes in the liver and kidneys, and reducing the frequency of apoptotic cell death. In many cases, AN showed greater efficacy than AO alone, reflecting the feasibility of using this nanotechnology to improve the pharmacokinetics of lipid compounds in the body. The study sheds new light on the therapeutic benefits of andiroba and suggests new ways for investigating how the quantity and quality of lipid compounds affect exposed organisms.

The multivariate statistical selection of fungal strains isolated from Neoteredo reynei, with the high hydrolytic potential to deconstruct cellulose.

The aim of this study was to select isolated filamentous fungi, naturally occurring in the digestive tract of Neoteredo reynei, with high potential to hydrolyze cellulolytic biomass. The selection of the fungi strains, which produce cellulases, was performed by adding carboxymethylcellulose (CMC), microcrystalline cellulose (MCC) or glucose in the media containing peptone and yeast-extract. In this case, the glucose was used as a reference standard for the growth of the mycelia. The abilities of the fungal strains, to hydrolyze cellulose, on the solid media (MCC or CMC), were evaluated. Two methods were used: the congo-red and the speed of mycelial growth. The measurements of the diameters were performed at 24 h intervals, and the speed of the mycelial growth was calculated after 72 h of cultivation. The molecular and morphological identification of the fungi were applied to the isolated strains. Statistical analysis, including ANOVA and Tukey test, and multivariate analysis were applied as tools to select the strains. Twelve strains were isolated and the results of the identification were 2 strains of Hypocrea lutea, 2 strains of Trichoderma reesei, 2 strains of Aspergillus niger; 2 genera of Aspergillus sp., 2 genera of Trichoderma sp., 1 genus of Fusarium sp. and 1 genus of Gliocadium sp. The discrimination analysis methods such as HCA (Hierarchical Cluster Analysis) and PCA (Principal Component Analysis) indicated three strains with the highest potential to hydrolyze cellulose. In this study, the selection strategy was successful, resulting in the classification of strains from the genera Trichoderma and Hypocrea. This is the first time that this kind of studying was applied to select the potential of the cellulolytic fungal strains, isolated from N. reynei, using the methods of the growth of the mycelial diameter and the statistical discrimination.

In silico characterization of a cyanobacterial plant-type isoaspartyl aminopeptidase/asparaginase.

Asparaginases are found in a range of organisms, although those found in cyanobacteria have been little studied, in spite of their great potential for biotechnological application. This study therefore sought to characterize the molecular structure of an L-asparaginase from the cyanobacterium Limnothrix sp. CACIAM 69d, which was isolated from a freshwater Amazonian environment. After homology modeling, model validation was performed using a Ramachandran plot, VERIFY3D, and the RMSD. We also performed molecular docking and dynamics simulations based on binding free-energy analysis. Structural alignment revealed homology with the isoaspartyl peptidase/asparaginase (EcAIII) from Escherichia coli. When compared to the template, our model showed full conservation of the catalytic site. In silico simulations confirmed the interaction of cyanobacterial isoaspartyl peptidase/asparaginase with its substrate, ß-Asp-Leu dipeptide. We also observed that the residues Thr154, Thr187, Gly207, Asp218, and Gly237 were fundamental to protein-ligand complexation. Overall, our results suggest that L-asparaginase from Limnothrix sp. CACIAM 669d has similar properties to E. coli EcAIII asparaginase. Our study opens up new perspectives for the biotechnological exploitation of cyanobacterial asparaginases.

Microbiologic oxidation of isosafrole into piperonal.

The biotransformation of isosafrole by Cladosporium sphaerospermum yielded piperonal, which is a compound of great commercial importance in the flavor and fragrance industries. The experiments were performed in 500-mL conical flasks containing 100 mL of Czapek-modified medium in an orbital shaker with controlled agitation and temperature. Spores of C. sphaerospermum were used as inocula, and after 96 h of incubation the substrate was added to the culture. Samples of 2 mL were withdrawn at 24-h intervals and analyzed by gas chromatography, (GC) and/or GC/MS spectroscopy.

Production of L-asparaginase by filamentous fungi.

L-asparaginase production was investigated in the filamentous fungi Aspergillus tamarii and Aspergillus terreus. The fungi were cultivated in medium containing different nitrogen sources. A. terreus showed the highest L-asparaginase (activity) production level (58 U/L) when cultivated in a 2% proline medium. Both fungi presented the lowest level of L-asparaginase production in the presence of glutamine and urea as nitrogen sources. These results suggest that L-asparaginase production by of filamentous fungi is under nitrogen regulation.

A dehydrorotenoid produced by callus tissue culture and wild plant roots of Boerhaavia coccinea

Calli cultures were established from leaves and stem of B. coccinea plantlet produced in vitro and analysed for isoflavonoid content. The quantification of 6,9,11-trihydroxy-6a,12a-dehydrorotenoid isolated from the roots of Boerhaavia coccinea P. Miller collected from its natural environment, and the same metabolite produced in callus tissue culture of the same plant are described in this paper. The rotinary quantitative HPLC analysis indicated that callus culture produced the same isoflavonoid compound found in the roots of intact wild growing plant. The amount of the secondary metabolite produced in vitro was 955.35 µg/g of dry cell weight, 2.5 times more than the highest amount concentration produced by the wild growing plant in its natural environment.

Cultura de calos foram estabelecidos de folhas e galhos finos de plântula de B. coccinea produzida in vitro e analisada para isoflavonóide. A quantificação do 6,9,11-triidroxi-6a,12a-desidro-rotenóide isolado das raízes de B. coccinea P Miller, coletada em seu habitat natural, e do mesmo rotenóide produzido na cultura de células estão descritos neste artigo. A análise rotineira em CLAE mostrou que a cultura de calos produziu o mesmo isoflavonóide encontrado nas raízes da planta do campo. A quantidade do metabólito secundário produzido in vitro foi de 955.35 µg/g de massa seca de callus, atingindo uma concentração de 2,5 vezes maior do que a quantidade do metabólito produzido pela planta em seu meio ambiente natural.

Production of L-asparaginase by filamentous fungi

L-asparaginase production was investigated in the filamentous fungi Aspergillus tamarii and Aspergillus terreus. The fungi were cultivated in medium containing different nitrogen sources. A. terreus showed the highest L-asparaginase (activity) production level (58 U/L) when cultivated in a 2 percent proline medium. Both fungi presented the lowest level of L-asparaginase production in the presence of glutamine and urea as nitrogen sources. These results suggest that L-asparaginase production by of filamentous fungi is under nitrogen regulation.