Stability of a Natural-Based Cream Containing Lecythis pisonis Extract.

The outer pericarps of Lecythis pisonis Camb. are by-products with a high content of antioxidant substances. The goal of the present study was to analyze the incorporation of L. pisonis pericarp extract in an all-natural formulation. Physical-chemical and organoleptic characteristics and Challenge tests were evaluated before and after the stability test of the cosmetic formulation. The instability of the formulation increased with the increase of the concentration of the extract. The physical-chemical and organoleptic parameters did not present alterations after the stability test. The extract of the outer pericarp of L. pisonis seems to be compatible with the cream formulation. However, the concentration should be analyzed to not interfere with the stability of the product. The natural formulation developed is under the legal requirements of the National Agency of Sanitary Monitoring of Brazil (Agência Nacional de Vigilância Sanitária), presenting an efficient conservation system.

A multiplex RT-PCR method to detect papaya meleira virus complex in adult pre-flowering plants.

Papaya sticky disease (PSD), which can destroy orchards, was first attributed to papaya meleira virus (PMeV). However, the discovery of papaya meleira virus 2 (PMeV2) associated with PSD plants impose the need to detect this viral complex. We developed a multiplex RT-PCR (mPCR) technique capable of detecting two viruses in a single assay from pre-flowering plant samples, which is a useful tool for early diagnosis of PSD. We also determined the limit of detection (LOD) using asymmetric plasmid dilutions of both PMeV and PMeV2, which revealed that a higher titer of one virus prevents detection of the other. Thus, this technique is an alternative method for detecting PMeV and PMeV2 in a single reaction.

Transcriptome analysis provides insights into the delayed sticky disease symptoms in Carica papaya.

KEY MESSAGE: Global gene expression analysis indicates host stress responses, mainly those mediated by SA, associated to the tolerance to sticky disease symptoms at pre-flowering stage in Carica papaya. Carica papaya plants develop the papaya sticky disease (PSD) as a result of the combined infection of papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), or PMeV complex. PSD symptoms appear only after C. papaya flowers. To understand the mechanisms involved in this phenomenon, the global gene expression patterns of PMeV complex-infected C. papaya at pre-and post-flowering stages were assessed by RNA-Seq. The result was 633 and 88 differentially expressed genes at pre- and post-flowering stages, respectively. At pre-flowering stage, genes related to stress and transport were up-regulated while metabolism-related genes were down-regulated. It was observed that induction of several salicylic acid (SA)-activated genes, including PR1, PR2, PR5, WRKY transcription factors, ROS and callose genes, suggesting SA signaling involvement in the delayed symptoms. In fact, pre-flowering C. papaya treated with exogenous SA showed a tendency to decrease the PMeV and PMeV2 loads when compared to control plants. However, pre-flowering C. papaya also accumulated transcripts encoding a NPR1-inhibitor (NPR1-I/NIM1-I) candidate, genes coding for UDP-glucosyltransferases (UGTs) and several genes involved with ethylene pathway, known to be negative regulators of SA signaling. At post-flowering, when PSD symptoms appeared, the down-regulation of PR-1 encoding gene and the induction of BSMT1 and JA metabolism-related genes were observed. Hence, SA signaling likely operates at the pre-flowering stage of PMeV complex-infected C. papaya inhibiting the development of PSD symptoms, but the induction of its negative regulators prevents the full-scale and long-lasting tolerance.

Induction of NAD (P)H: Quinone reductase 1 (QR1) and antioxidant activities in vitro of 'Toranja Burarama' (Citrus maxima [Burm.] Merr.).

Toranja 'Burarama', Citrus maxima (Burm.) Merr. (Citrus grandis), is a new citrus discovered in the State of Espírito Santo, Brazil. As several varieties of citrus are known to possess antioxidant and cancer chemopreventive properties, the aim of the study was to evaluate in vitro if this Toranja possess these properties. The antioxidant activity, the potential to induce quinone reductase 1, and the influence on cell viability were measured. ESI(-)FT-ICR MS analysis was also performed and identified flavonoids, coumarins, and fatty acids in the extract. The ethyl acetate and methanolic extracts of the peels presented the highest antioxidant activity in vitro by DPPH (IC50  = 298.3 ± 2.6 µg/ml and 303.8 ± 0.4 µg/ml), ABTS assay (IC50  = 298.2 ± 6.4 µg/ml and 296.4 ± 2.5 µg/ml), and FRAP (IC50  = 234.6 ± 1.8 µg/ml and 398.1 ± 3.8 µg/ml). The ethyl acetate extract of the peel induced quinone reductase 1 activity in Hepa1c1c7 cells, indicating that C. maxima exhibited cancer chemopreventive properties.

Chemical profile of pineapple cv. Vitória in different maturation stages using electrospray ionization mass spectrometry.

BACKGROUND: Pineapple is the fruit of Ananas comosus var. comosus plant, being cultivated in tropical areas and has high energy content and nutritional value. Herein, 30 samples of pineapple cv. Vitória were analyzed as a function of the maturation stage (0-5) and their physico-chemical parameters monitored. In addition, negative-ion mode electrospray ionization mass spectrometry [ESI(-)FT-ICR MS] was used to identify and semi-quantify primary and secondary metabolites present in the crude and phenolic extracts of pineapple, respectively. RESULTS: Physico-chemical tests show an increase in the total soluble solids (TSS) values and in the TSS/total titratable acidity ratio as a function of the maturity stage, where a maximum value was observed in stage 3 (¾ of the fruit is yellow, which corresponds to the color of the fruit peel). ESI(-)FT-ICR MS analysis for crude extracts showed the presence mainly of sugars as primary metabolites present in deprotonated molecule form ([M - H]- and [2 M - H]- ions) whereas, for phenolic fractions, 11 compounds were detected, being the most abundant in the third stage of maturation. This behavior was confirmed by quantitative analysis of total polyphenols. CONCLUSION: ESI-FT-ICR MS was efficient in identifying primary (carbohydrates and organic acids) and secondary metabolites (13 phenolic compounds) presents in the crude and phenolic extract of the samples, respectively. © 2017 Society of Chemical Industry.

A Capsid Protein Fragment of a Fusagra-like Virus Found in Carica papaya Latex Interacts with the 50S Ribosomal Protein L17.

Papaya sticky disease is caused by the association of a fusagra-like and an umbra-like virus, named papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), respectively. Both viral genomes are encapsidated in particles formed by the PMeV ORF1 product, which has the potential to encode a protein with 1563 amino acids (aa). However, the structural components of the viral capsid are unknown. To characterize the structural proteins of PMeV and PMeV2, virions were purified from Carica papaya latex. SDS-PAGE analysis of purified virus revealed two major proteins of ~40 kDa and ~55 kDa. Amino-terminal sequencing of the ~55 kDa protein and LC-MS/MS of purified virions indicated that this protein starts at aa 263 of the deduced ORF1 product as a result of either degradation or proteolytic processing. A yeast two-hybrid assay was used to identify Arabidopsis proteins interacting with two PMeV ORF1 product fragments (aa 321-670 and 961-1200). The 50S ribosomal protein L17 (AtRPL17) was identified as potentially associated with modulated translation-related proteins. In plant cells, AtRPL17 co-localized and interacted with the PMeV ORF1 fragments. These findings support the hypothesis that the interaction between PMeV/PMeV2 structural proteins and RPL17 is important for virus-host interactions.

Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms.

Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections.

Virucidal and antiviral activities of pomegranate (Punica granatum) extract against the mosquito-borne Mayaro virus.

BACKGROUND: The arthropod-borne Mayaro virus (MAYV) causes "Mayaro fever," a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. There are currently no licensed drugs against most mosquito-transmitted viruses. Punica granatum (pomegranate) fruits cultivated in Brazil have been subjected to phytochemical investigation for the identification and isolation of antiviral compounds. In the present study, we explored the antiviral activity of pomegranate extracts in Vero cells infected with Mayaro virus. METHODS: The ethanol extract and punicalagin of pomegranate were extracted solely from the shell and purified by chromatographic fractionation, and were chemically identified using spectroscopic techniques. The cytotoxicity of the purified compounds was measured by the dye uptake assay, while their antiviral activity was evaluated by a virus yield inhibition assay. RESULTS: Pomegranate ethanol extract (CC50 = 588.9, IC50 = 12.3) and a fraction containing punicalagin as major compound (CC50 = 441.5, IC50 = 28.2) were shown to have antiviral activity (SI 49 and 16, respectively) against Mayaro virus, an alphavirus. Immunofluorescence analysis showed the virucidal effect of pomegranate extract, and transmission electron microscopy (TEM) revealed damage in viral particles treated with this extract. CONCLUSIONS: The P. granatum extract is a promising source of antiviral compounds against the alphavirus MAYV and represents an excellent candidate for future studies with other enveloped RNA viruses.

Difference between the cell wall roughnesses of mothers and daughters of Saccharomyces cerevisiae subjected to high pressure stress.

High hydrostatic pressure (HHP) stress generates cellular responses similar to those to other stresses that yeasts endure in fermentation tanks. Structural and spatial compaction of molecules, as well as weakening and stretching of plasma membranes and cell walls, are often observed and have a significant influence on the fermentative process. Atomic force microscopy (AFM) yields accurate data on the morphological characteristics of yeast cell walls, providing important insights for the development of more productive yeast strains. Saccharomyces cerevisiae cell wall assessment using AFM in the intermittent contact reading mode using a silicon cantilever, before and after application of a pressure of 100 MPa for 30 min, demonstrated that mother and daughter cells have different responses. Daughter cells were more sensitive to the effects of HHP, presenting lower average Ra (arithmetic roughness), Rz (ten-point average roughness), and Rq (root-mean-square roughness) after exposure to high pressure. Better adaptation to stress in mother cells leads to higher cell wall resistance and, therefore, to better protection.

Chemical composition and anti-Mayaro virus activity of Schinus terebinthifolius fruits.

Brazilian traditional medicine has explored the antiviral properties of many plant extracts, including those from the Brazilian pepper tree, Schinus terebinthifolius. In the present study, we investigated the chemical composition and anti-mayaro virus (MAYV) activity of S. terebinthifolius fruit. Extensive virucidal activity (more than 95%) was detected for the ethyl acetate extract and the isolated biflavonoids. From the ethyl acetate extract of Schinus terebinthifolius fruits, two bioflavonoids were isolated ((2S, 2″S)-2,3,2″,3″-tetrahydroamentoflavone and agathisflavone), which showed strong virucidal activity against Mayaro virus. Furthermore, several other compounds like terpenes and phenolics were identified by hyphenated techniques (GC-MS, LC-MS and HPLC-UV), as well as by mass spectrometry. Immunofluorescence assay confirmed antiviral activity and transmission electron microscopy revealed damage in viral particles treated with biflavonoids. The data suggest the direct action of the extract and the biflavonoids on the virus particles. The biflavonoids tetrahydroamentoflavone and agathisflavone had strong virucidal activity and reduced MAYV infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00698-z.

Synthesis of Eugenol Derivatives and Evaluation of their Antifungal Activity Against Fusarium solani f. sp. piperis.

BACKGROUND: Fusarium solani f. sp. piperis is a phytopathogen that causes one of the most destructive diseases in black pepper crops, resulting in significant economic and crop production losses. Consequently, the control of this fungal disease is a matter of current and relevant interest in agriculture. OBJECTIVE: The objective was to synthesize eugenol derivatives with antifungal activity. METHODS: In this study, using bimolecular nucleophilic substitution and click chemistry approaches, four new and three known eugenol derivatives were obtained. The eugenol derivatives were characterized and their antifungal and cytotoxic effects were evaluated. RESULTS: Eugenol derivative 4 (2-(4-allyl-2-methoxyphenoxy)-3-chloronaphthalene-1,4-dione) was the most active against F. solani f. sp. piperis and showed acceptable cytotoxicity. Compound 4 was two-fold more effective than tebuconazole in an antifungal assay and presented similar cytotoxicity in macrophages. The in silico study of ß-glucosidase suggests a potential interaction of 4 with amino acid residues by a cation-π interaction with residue Arg177 followed by a hydrogen bond with Glu596, indicating an important role in the interactions with 4, justifying the antifungal action of this compound. In addition, the cytotoxicity after metabolism was evaluated as a mimic assay with the S9 fraction in HepG2 cells. Compound 4 demonstrated maintenance of cytotoxicity, showing IC50 values of 11.18 ± 0.5 and 9.04 ± 0.2 µg mL-1 without and with the S9 fraction, respectively. In contrast, eugenol (257.9 ± 0.4 and 133.5 ± 0.8 µg mL-1), tebuconazole (34.94 ± 0.2 and 26.76 ± 0.17 µg mL-1) and especially carbendazim (251.0 ± 0.30 and 34.7 ± 0.10 µg mL-1) showed greater cytotoxicity after hepatic biotransformation. CONCLUSION: The results suggest that 4 is a potential candidate for use in the design of new and effective compounds that could control this pathogen.

Effects of the Papaya meleira virus on papaya latex structure and composition.

Spontaneous latex exudation is the main symptom of papaya sticky (meleira) disease caused by the Papaya meleira virus (PMeV), a double-stranded RNA (dsRNA) virus. This paper describes different effects of PMeV on papaya latex. Latex samples were subjected to different histochemical tests to evaluate their chemical composition. Additionally, the integrity of the latex particles was assessed by transmission and scanning electron microscopy analysis. Biochemical and micro- and macro-element measurements were performed. PMeV dsRNA extraction was performed to evaluate the interaction of the virus with the latex particles. Sticky diseased latex was positive for alkaloid biosynthesis and showed an accumulation of calcium oxalate crystals. PMeV also increased H(2)O(2) synthesis within sticky diseased laticifers. The protein, sugar and water levels were altered, probably due to chemical changes. The morphology of the latex particles was further altered; PMeV particles seemed to be bound to the latex particles. The alkaloid and H(2)O(2) biosynthesis in the papaya laticifers indicate a papaya defense response against PMeV. However, such efforts failed, as the virus affected the plant latex. The effects described here suggest some advantages of the infection process, including facilitating the movement of the virus within the papaya plant.

New approach for papaya latex storage without virus degradation.

Papaya meleira virus (PMeV) is the causal agent of papaya (Carica papaya L.) sticky disease, which has been detected through analysis of its double-stranded RNA (dsRNA) genome from plant latex. In this work we demonstrate that PMeV dsRNA is protected during 25 days when latex is diluted in citrate buffer pH 5.0 (1:1 v/v) and maintained at -20ºC. At the same temperature, some protection was observed for pure latex or latex diluted in ultra-pure water. Conversely, the dsRNA was almost completely degraded after 25 days when maintained at 25ºC, indicating the need for freezing. The proper procedures to collect and store papaya latex described here will contribute to efficient and large scale use of molecular diagnosis of PMeV.

Phytochemical profile of genotypes of Euterpe edulis Martius - Juçara palm fruits.

Juçara fruit (Euterpe edulis) has received attention due to its similarities to Euterpe oleracea (Açaí). The aim of this study was to evaluate the cytotoxicity, bioactive compounds, antioxidant capacities and chemopreventive activities of the fruit pulps of six populations of E. edulis (J1-J6) and one population of E. espiritosantense from different ecological regions. ESI(-)-FT-ICR-MS was used to evaluate the pulp composition. The varieties J1 and J4 presented higher polyphenol contents, while J2 and J5 showed higher anthocyanin contents. ESI-FT-ICR MS identified cyanidin-3-rutinoside (J1, J2, J3, J4, J5, J7), protocatechuic acid, methylhydroxybenzoate hexoside and rutin (J1 to J7) and malvidin-glicoside (J2 to J5). The J2, J3, J4, J5 and J6 samples inhibited inducible nitric oxide synthase (iNOS). The chemoprevention biomarker quinone reductase was significantly induced by J6. Pulp from plants J3, J4, J6 and J7 significantly reduced the inflammatory cytokine TNF-α, and J6 was selected as having the most potential for cultivation and consumption.

Diaspididae (Hemiptera: Coccoidea) of Espírito Santo, Brazil.

Twenty-seven species of armored scale insects (Hemiptera: Diaspididae) are newly recorded from Espírito Santo, Brazil, and information on the host plants and geographic distribution of the 31 species of Diaspididae that have been identified in the State is provided. New plant host records are reported for 11 of the diaspidid species studied and results are discussed with respect to development of agriculture in this and similar areas with objectives of modernization and diversification.

Antimicrobial activity and potential use of monoterpenes as tropical fruits preservatives.

Banana, papaya and pineapple are the most consumed tropical fruits in the world, being Brazil one of the main producers. Fungi Colletotrichum musae, Colletotrichum gloeosporioides and Fusarium subglutinans f.sp. ananas cause severe post harvest diseases and losses in fruits quality. The aim of this work was to evaluate the effectiveness of five monoterpenes to inhibit the mycelial growth and conidia germination of these three phytopathogens. The monoterpenes citral, citronellal, L-carvone, isopullegol and α-pinene were diluted in ethanol to final concentrations from 0.2 to 1%. All monoterpenes were found to inhibit the growth of the three studies fungi in a dose-dependent manner. Citral was the most effective of the oils tested and showed potent fungicidal activity at concentrations above 0.5%. Also, in vivo evaluation with these tropical fruits demonstrated the efficiency of citral to inhibit fungal growth. These results indicate the potential use of citral as a natural pesticide control of post-harvest fruit diseases.

Anti-Escherichia coli activity of extracts from Schinus terebinthifolius fruits and leaves.

Ethanol extracts obtained from Schinus terebinthifolius Raddi fruits and leaves were active against Escherichia coli with MIC of 78 µg mL-1 for both extracts. Phytochemical analyses revealed a major presence of phenolic acids, tannins, fatty acids and acid triterpenes in the leaves and phenolic acids, fatty acids, acid triterpenes and biflavonoids in the fruits. Major compounds isolated from the plant, such as the acid triterpene schinol, the phenolic acid derivative ethyl gallate and the biflavonoids agathisflavone and tetrahydroamentoflavone, showed very little activity against E. coli. Bioautography of the ethanol extracts on silica gel plate showed inhibition zones for E. coli. They were removed from the plate and the compounds identified as a mixture of myristic, pentadecanoic, palmitic, heptadecanoic, stearic, nonadecanoic, eicosanoic, heneicosanoic and behenic fatty acids.

Label-free quantitative proteomic analysis of pre-flowering PMeV-infected Carica papaya L.

Papaya meleira virus (PMeV) infects papaya (Carica papaya L.) and leads to Papaya Sticky Disease (PSD) or "Meleira", characterized by a spontaneous exudation of latex from fruits and leaves only in the post-flowering developmental stage. The latex oxidizes in contact with air and accumulates as a sticky substance on the plant organs, impairing papaya fruit's marketing and exportation. To understand pre-flowering C. papaya resistance to PMeV, an LC-MS/MS-based label-free proteomics approach was used to assess the differential proteome of PMeV-infected pre-flowering C. papaya vs. uninfected (control) plants. In this study, 1333 proteins were identified, of which 111 proteins showed a significant abundance change (57 increased and 54 decreased) and supports the hypothesis of increased photosynthesis and reduction of 26S-proteassoma activity and cell-wall remodeling. All of these results suggest that increased photosynthetic activity has a positive effect on the induction of plant immunity, whereas the reduction of caspase-like activity and the observed changes in the cell-wall associated proteins impairs the full activation of defense response based on hypersensitive response and viral movement obstruction in pre-flowering C. papaya plants. BIOLOGICAL SIGNIFICANCE: The papaya (Carica papaya L.) fruit's production is severely limited by the occurrence of Papaya meleira virus (PMeV) infection, which causes Papaya Sticky Disease (PSD). Despite the efforts to understand key features involved with the plant×virus interaction, PSD management is still largely based on the observation of the first disease symptoms in the field, followed by the elimination of the diseased plants. However, C. papaya develops PSD only after flowering, i.e. about six-months after planting, and the virus inoculum sources are kept in field. The development of PMeV resistant genotypes is impaired by the limited knowledge about C. papaya resistance against viruses. The occurrence of a resistance/tolerance mechanism to PSD symptoms development prior to C. papaya flowering is considered in this study. Thus, field-grown and PMeV-infected C. papaya leaf samples were analyzed using proteomics, which revealed the modulation of photosynthesis-, 26S proteasome- and cell-wall remodeling-associated proteins. The data implicate a role for those systems in C. papaya resistance to viruses and support the idea of a partial resistance induction in the plants at pre-flowering stage. The specific proteins presented in the manuscript represent a starting point to the selection of key genes to be used in C. papaya improvement to PMeV infection resistance. The presented data also contribute to the understanding of virus-induced disease symptoms development in plants, of interest to the plant-virus interaction field.

Chemical profile of mango (Mangifera indica L.) using electrospray ionisation mass spectrometry (ESI-MS).

Mangifera indica L., mango fruit, is consumed as a dietary supplement with purported health benefits; it is widely used in the food industry. Herein, the chemical profile of the Ubá mango at four distinct maturation stages was evaluated during the process of growth and maturity using negative-ion mode electrospray ionisation Fourier transform ion cyclotron resonance mass spectrometry (ESI(-)FT-ICR MS) and physicochemical characterisation analysis (total titratable acidity (TA), total soluble solids (TSS), TSS/TA ratio, and total polyphenolic content). Primary (organic acids and sugars) and secondary metabolites (polyphenolic compounds) were mostly identified in the third maturation stage, thus indicating the best stage for harvesting and consuming the fruit. In addition, the potential cancer chemoprevention of the secondary metabolites (phenolic extracts obtained from mango samples) was evaluated using the induction of quinone reductase activity, concluding that fruit polyphenols have the potential for cancer chemoprevention.

Monitoring the physicochemical degradation of coconut water using ESI-FT-ICR MS.

Fresh and aged coconut water (CW) samples were introduced directly into the electrospray ionisation (ESI) source, and were combined with the Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) technique to characterise in situ chemical compounds produced during natural ageing (from 0 to 15 days) at room temperature (23 °C). The ESI-FT-ICR MS readings were acquired and the data were correlated to conventional methodologies: pH, total titratable acidity (TA), total soluble solids, microbial analyses, and ultraviolet visibility (UV-vis) spectroscopy analysis. In general, the pH and TA values changed after 3 days of storage making the CW unsuitable for consumption. The ESI(-)-FT-ICR data also showed a clear and evident change in the chemical profile of CW after 3 days of ageing in the m/z 150-250 and 350-450 regions. Initially, the relative intensity of the natural markers (the m/z 215 and 377 ions-sugar molecules) decreases as a function of ageing time, with the last marker disappearing after 3 days of ageing. New chemical species were then identified such as: citric (m/z 191), galacturonic (m/z 193), gluconic (m/z 195), and saccharic (m/z 209) acids. ESI(-)-FT-ICR MS is a powerful tool to predict the physicochemical properties of CW, such as the pH and TA, where species such as fructose, glucose, sucrose, and gluconic acid can be used as natural markers to monitor the quality of the fruits.