Isolation and Identification of Pigment-Producing Endophytic Fungi from the Amazonian Species Fridericia chica.

Pigments of fungal origin have aroused increasing interest in the food dye and cosmetic industries since the global demand for natural dyes has grown. Endophytic microorganisms are a source of bioactive compounds, and Amazonian plant species can harbor fungi with a wide range of biotechnological applications. Popularly known in Brazil as crajiru, Fridericia chica is a medicinal plant that produces a red pigment. In this study, a total of 121 fungi were isolated in potato dextrose agar from three plants. We identified nine pigment-producing endophytic fungi isolated from branches and leaves of F. chica. The isolates that showed pigment production in solid media were molecularly identified via multilocus analysis as Aspergillus welwitschiae, A. sydowii, Curvularia sp., Diaporthe cerradensis (two strains), Hypoxylon investiens, Neoscytalidium sp. (two strains) and Penicillium rubens. These isolates were subjected to submerged fermentation in two culture media to obtain metabolic extracts. The extracts obtained were analyzed in terms of their absorbance between 400 and 700 nm. The pigmented extract produced by H. investiens in medium containing yeast extract showed maximum absorbance in the red absorption range (UA700 = 0.550) and significant antioxidant and antimicrobial activity. This isolate can thus be considered a new source of extracellular pigment.

Dietary supplementation with Lippia sidoides essential oil improves organ integrity but the specific activity of antioxidant enzymes is dose-dependent in Danio rerio.

The nutritional quality of food can affect the health of animals. This study examined the effects of dietary supplementation with Lippia sidoides essential oil (LSEO) on the physiology of Danio rerio. Four hundred fourty-eight fish were divided into 28 tanks and subjected to different dietary treatments: a control group with no supplementation, a group with grain alcohol supplementation and five groups with LSEO at concentrations of 0.25%, 0.50%, 0.75%, 1.00% and 1.25%. After 15 days, histological and enzymatic analyses were conducted. The 0.25% LSEO group exhibited lower glutathione peroxidase and catalase activity compared to the 1.00% group. Additionally, fish in the 0.25% LSEO group showed improved liver, kidney and splenic integrity indices. These findings support the inclusion of 0.25% LSEO in the diet of D. rerio, suggesting potential benefits for fish physiology and encouraging further research on phytotherapeutics in fish diets.

Therapeutic Potential of Leaves from Fridericia chica (Bonpl.) L. G. Lohmann: Botanical Aspects, Phytochemical and Biological, Anti-Inflammatory, Antioxidant and Healing Action.

Plants of the species Fridericia chica (Bonpl.) L. G. Lohmann (Bignoniaceae), which are widely distributed in Brazil and named crajiru in the state of Amazonas, are known in folk medicine as a traditional medicine in the form of a tea for the treatment of intestinal colic, diarrhea, and anemia, among other diseases. The chemical analysis of extracts of the leaves has identified phenolic compounds, a class of secondary metabolites that provide defense for plants and benefits to the health of humans. Several studies have shown the therapeutic efficacy of F. chica extracts, with antitumor, antiviral, wound healing, anti-inflammatory, and antioxidant activities being among the therapeutic applications already proven. The healing action of F. chica leaf extract has been demonstrated in several experimental models, and shows the ability to favor the proliferation of fibroblasts, which is essential for tissue repair. The anti-inflammatory activity of F. chica has been clearly demonstrated by several authors, who suggest that it is related to the presence of 3-deoxyanthocyanidins, which is capable of inhibiting pro-inflammatory pathways such as the kappa B (NF-kB) nuclear transcription factor pathway. Another important effect attributed to this species is the antioxidant effect, attributed to phenolic compounds interrupting chain reactions caused by free radicals and donating hydrogen atoms or electrons. In conclusion, the species Fridericia chica has great therapeutic potential, which is detailed in this paper with the objective of encouraging new research and promoting the sum of efforts for the inclusion of herbal medicines in health systems around the world.

Ovicidal effect of essential oils of Lippia alba, Lippia sidoides and Lippia gracilis on the acanthocephalan Neoechinorhynchus buttnerae (Eoacanthocephala: Neoechinorhynchidae).

Acanthocephalosis in tambaqui Colossoma macropomum culture is a limiting factor for the production of the species, being the most recurrent, which makes it necessary to develop strategies to control parasitosis even in the early stages of parasite development. This study aimed to elucidate the morphological characteristics of eggs of the acanthocephalan Neoechinorhynchus buttnerae and the ovicidal effect of the essential oils of Lippia alba, Lippia sidoides, and Lippia gracilis. The parasites were collected from hosts naturally infected with N. buttnerae, and the eggs obtained from the female 'parasites' abdominal cavity were added to 12-well plates containing the essential oils at different dilutions. After the eggs (N = 300) were separated and measured (length and width) with the aid of an optical microscope coupled to a digital program, the viability of the treated eggs was monitored 24 h after treatments, L. gracilis essential oil was 100% effective at the highest concentration, whereas L. sidoides essential oil showed over 50% efficacy at 2 mg/ml, reaching 100% efficacy at all higher concentrations. Lippia. alba essential oil did not show satisfactory efficacy in preventing N. buttnerae egg hatching.

Physicochemical characterization and cosmetic applications of Passiflora nitida Kunth leaf extract

Abstract Passiflora nitida Kunth, an Amazonian Passiflora species, is little studied, although the specie's high biological potential. Herein the plant's pharmacognostic characterization, extract production, antioxidant potential evaluation, and application of this extract in cosmetic products is reported. The physical chemical parameters analyzed were particle size by sieve analysis, loss through drying, extractive yield, total ash content, laser granulometry, specific surface area and pore diameter (SBET), differential scanning calorimetry, thermogravimetry (TG), and wave dispersive X-Ray fluorescence (WDXRF). Total phenol/flavonoid content, LC-MS/MS analysis, DPPH and ABTS antioxidant radical assays, cytotoxicity, melanin, and tyrosinase inhibition in melanocytes test provided evidence to determine the content of the major constituent. P. nitida dry extract provided a fine powder with mesopores determined by SBET, with the TG curve showing five stages of mass loss. The antioxidant potential ranged between 23.5-31.5 mg∙mL-1 and tyrosinase inhibition between 400-654 µg∙mL-1. The species presented an antimelanogenic effect and an inhibitory activity of cellular tyrosinase (26.6%) at 25 µg/mL. The LC-MS/MS analysis of the spray-dried extract displayed the main and minor phenolic compounds constituting this sample. The results indicate that P. nitida extract has promising features for the development of cosmetic formulations

Induced aerocystitis and hemato-immunological parameters in Nile tilapia fed supplemented diet with essential oil of Lippia alba / Aerocistite induzida e parâmetros hemato-imunológicos em tilápia do Nilo suplementada com óleo essencial de Lippia alba

The present study evaluated the dietary supplementation with essential oil of Lippia alba on the hemato-immunological parameters of Nile tilapia (Oreochromis niloticus) submitted to acute inflammation induced by carrageenin injection in the swim bladder. For a period of 45 days, 96 fish were divided in four treatments in triplicate, as follows: fish fed supplemented diet with essential oil of L. alba (4 mL kg-1 dry ration) injected with carrageenin; fish fed supplemented diet with cereal alcohol injected with carrageenin; fish fed unsupplemented diet with essential oil injected with carrageenin; fish fed unsupplemented diet and noninjected. Cortisol levels, erythrogram, leukogram and the inflammatory infiltrate were analyzed 6 h after inflammatory stimulus. Carrageenin-injected fish showed acute inflammatory reaction in the swim bladder characterized by higher infiltrate of neutrophils and monocytes. The circulating neutrophils number was significantly higher in fish fed L. alba when compared to other treatments. No difference in cortisol levels was found. For dose, time and administration form tested, supplementation with essential oil of L. alba did not present anti-inflammatory activity. On the other hand, influence of dietary supplementation was observed on the neutrophils number after induced aerocystitis highlighting its immunomodulatory characteristic.(AU)

O presente estudo avaliou a suplementação dietária com óleo essencial de Lippia alba sobre os parâmetros hemato-imunológicos em tilápias do Nilo (Oreochromis niloticus) submetidas à inflamação aguda induzida por carragenina na bexiga natatória. Pelo período de 45 dias, 96 peixes foram divididos em quarto tratamentos em triplicata: a) peixes suplementados com óleo esencial de L. alba (4 mL kg-1 de ração) injetados com carragenina; b) peixes suplementados com álcool de cereais injetados com carragenina; peixes não suplementados com óleo essencial injetados com carragenina; c) peixes não suplementados não injetados. Os níveis de cortisol, eritrograma, leucograma e o infiltrado inflamatório foram analisados seis horas após o estímulo inflamatório. Peixes injetados com carragenina apresentaram reação inflamatória aguda na bexiga natatória caracterizada por maior infiltrado de neutrófilos e monócitos. O número de neutrófilos circulantes foi significativamente maior nos peixes suplementados com L. alba quando comparado aos outros tratamentos. Não houve diferença nos níveis de cortisol. Para a dose, o tempo e a forma de administração testada, a suplementação com óleo essencil de L. alba não apresentou atividade anti-inflamatória. Por outro lado, foi constatada influência da suplementação dietária no número de neutrófilos após a aerocistite enfatizando a sua característica imunomoduladora.(AU)

Blood biochemical parameters and melanomacrophage centers in Nile tilapia fed essential oils of clove basil and ginger.

Nile tilapia (Oreochromis niloticus) is the most farmed freshwater fish in the world, however, disease outbreaks are the main cause of losses in production. Due to this, there is an increasing interest in natural products for enhancing disease resistance, without causing physiological impairment. The aim of this study was to verify the effect of the essential oils of clove basil (Ocimum gratissimum) and ginger (Zingiber officinale) on physiological and immunological parameters of nile tilapia. After 35 and 55 days of supplementation (0.5%, 1.0% and 1.5%), blood collection was performed for determination of metabolites (glucose, cholesterol, triglycerides, serum total protein and immunoglobulins) and lysozyme activity. Spleen tissue was also collected at 55 days for analyzing melanomacrophages centers. At 35 days, cholesterol showed significant reduction in the treatments 0.5% and 1.5 ginger and 1.5% clove basil. The serum triglycerides showed significant reduction in these same treatments, in addition to 1.0% clove basil. At 55 days all the supplemented groups showed significant reduction for both parameters. The lysozyme activity at 35 days was significantly higher in fish supplemented with 1.0% ginger and showed no difference among the treatments at 55 days. Regarding the melanomacrophage centers, at 35 days increased number in fish supplemented with 0.5% clove basil was observed but with no significant difference in the total percent area of spleen occupied by centers. The levels of glucose, total serum protein and immunoglobulins did not show significant differences in both sampling times. The addition of essential oils showed to be useful for improving physiological status of the fish, without excessive activation of defense mechanisms.

Ethnobotanical study of antimalarial plants in the middle region of the Negro River, Amazonas, Brazil / Etnobotânica de plantas antimaláricas no médio Rio Negro, Amazonas, Brasil

ABSTRACT The middle Rio Negro region is an interesting place to seek novel antimalarial compounds because of the traditional knowledge of the Amazon population in conjunction with the high biodiversity of the region. The objective of this work was to study the use of antimalarial plants in Barcelos, state of Amazonas, Brazil. Fifty-two local specialists from eight different communities were interviewed over one year. The identified plants were collected with the assistance of local specialists, classified to species level and deposited in herbarium. In total, 55 antimalarial plants were identified in use in the region, of which 16 had not been previously reported in other published studies. Many factors could be involved with the use of antimalarial plants by the Barcelos population, such as the accessibility of these medicinal plants, efficiency and safety of using these plants, the accessibility to drugs or other medical treatments, plant bitterness, and the gender of the interviewees. Our results indicate that the population of Barcelos possesses an extensive knowledge on the use of a diverse array of antimalarial plants, and may contribute to the development of novel antimalarial compounds.

RESUMO O conhecimento tradicional da população amazônica, associado à grande biodiversidade da região, faz do médio Rio Negro um lugar propício para a pesquisa de novos remédios antimaláricos. O objetivo deste trabalho foi estudar o uso de plantas antimaláricas no município de Barcelos, Amazonas, Brasil. Ao longo de um ano foram entrevistados 52 especialistas de oito comunidades de Barcelos. As plantas indicadas foram coletadas com o auxílio dos especialistas, identificadas e depositadas em herbário. Foram mencionadas 55 plantas antimaláricas, das quais 16 nunca foram citadas em outros trabalhos previamente publicados. Muitos fatores podem estar associados ao uso destas plantas antimaláricas, tais quais o acesso a estas plantas, sua eficiência e segurança, o acesso a outros tratamentos médicos, o amargor das plantas e o gênero das pessoas entrevistadas. Nossos resultados indicam que a população de Barcelos é detentora de um rico conhecimento sobre o uso de plantas medicinais antimaláricas e pode contribuir para o desenvolvimento de novas drogas antimaláricas.

Antiparasitic activity, histopathology and physiology of Colossoma macropomum (tambaqui) exposed to the essential oil of Lippia sidoides (Verbenaceae).

In vivo and in vitro antiparasitic activity of the essential oil of Lippia sidoides and blood and histological alterations were assessed in Colossoma macropomum (tambaqui). Essential oil concentrations of 10, 20, 40, 80, 160 and 320mg/L were assayed in vitro against monogenoideans Anacanthorus spathulatus, Notozothecium janauachensis and Mymarothecium boegeri from fish gills. Lippia sidoides essential oil concentrations of 320 and 160mg/L were 100% effective against monogenoideans in 10min and 1h of exposure, respectively. However, the effectiveness of 100% concentrations of 80mg/L and 40mg/L occurred in 3 and 6h, respectively. In the in vivo tests, juvenile fish were submitted to 60min of baths with 10mg/L and 15min of baths with 20mg/L of the essential oil of L. sidoides. These therapeutic baths were not efficient against Ichthyophthirius multifiliis, and monogenoideans present in the gills of C. macropomum. In addition, 10 and 20mg/L of the essential oil of L. sidoides caused an anesthetic effect on the fish and did not influence total glucose and protein plasma levels; however, it decreased the number of total erythrocytes in fish exposed to the higher concentration of this essential oil. Severe alterations and irreversible damage were observed in the fish gills just after L. sidoides essential oil baths and after 24h of recovery. The most recurrent lesions found were hyperplasia and fusion of the lamellar epithelium, vasodilation, detachment of the gill epithelium and lamellar aneurism, epithelial breakdown with hemorrhage, congestion, edema and necrosis, proliferation of the mucous cells and chloride cells and lamellar hypertrophy. Therefore, since the essential oil of L. sidoides has in vitro antiparasitic activity and low concentrations of it have shown toxic effects, the bioactive potential of its main chemical components should be investigated, as well as more efficient forms of its administration in therapeutic baths in order to eliminate fish parasites.

Efficacy of 11 Brazilian essential oils on lethality of the cattle tick Rhipicephalus (Boophilus) microplus.

Herbal extracts have been investigated as an alternative for parasite control, aiming to slow the development of resistance and to obtain low-cost biodegradable parasiticides. The goal of this study was to evaluate the efficacy, in vitro, of 11 essential oils from Brazil on reproductive efficiency and lethality of the cattle tick Rhipicephalus (Boophilus) microplus. The effects of oils extracted from Curcuma longa, Zingiber officinale, Lippia alba, Lippia gracilis, Lippia origanoides, Lippia sidoides, Mentha arvensis, Mentha piperita, Croton cajucara (white and red), and Croton sacaquinha on ticks were investigated by the Immersion Test with Engorged Females (ITEF) and the modified Larval Packet Test (LPT). Distilled water and 2% Tween 80 were used as control treatments. Chemical analysis of the oils was done with gas chromatography coupled with mass spectrometry. Analysis of the in vitro tests using Probit (SAS program) allowed the calculation of lethal concentrations (LCs). Lower reproductive efficiency indexes and higher efficacy percentages in the ITEF were obtained with the oils extracted from C. longa (24 and 71%, respectively) and M. arvensis oils (27 and 73%, respectively). Lower LC50 was reached with C. longa (10.24 mg/mL), L. alba (10.78 mg/mL), M. arvensis (22.31 mg/mL), L. sidoides (27.67 mg/mL), and C. sacaquinha (29.88 mg/mL) oils. In the LPT, species from Zingiberaceae and Verbenaceae families caused 100% lethality at 25 mg/mL, except for L. sidoides. The most effective oils were from C. longa, L. gracilis, L. origanoides, L. alba, and Z. officinale. The LC50 and LC90 were, respectively: 0.54 and 1.80 mg/mL, 3.21 and 7.03 mg/mL, 3.10 and 8.44 mg/mL, 5.85 and 11.14 mg/mL, and 7.75 and 13.62 mg/mL. The efficacy was directly related to the major components in each essential oil, and the oils derived from Croton genus presented the worst performance, suggesting the absence of synergistic effect among its compounds. Since C. longa, containing 62% turmerone, was the one most efficient against ticks, this compound may be potentially used for tick control, but further research is needed, especially to assess toxicity of these compounds to the host. These new studies, together with the results presented here, may provide a strong rationale for designing pre-clinical and clinical studies with these agents.

In vitro susceptibility of Plasmodium falciparum Welch field isolates to infusions prepared from Artemisia annua L. cultivated in the Brazilian Amazon.

Artemisinin is the active antimalarial compound obtained from the leaves of Artemisia annua L. Artemisinin, and its semi-synthetic derivatives, are the main drugs used to treat multi-drug-resistant Plasmodium falciparum (one of the human malaria parasite species). The in vitro susceptibility of P. falciparum K1 and 3d7 strains and field isolates from the state of Amazonas, Brazil, to A. annua infusions (5 g dry leaves in 1 L of boiling water) and the drug standards chloroquine, quinine and artemisinin were evaluated. The A. annua used was cultivated in three Amazon ecosystems (várzea, terra preta de índio and terra firme) and in the city of Paulínia, state of São Paulo, Brazil. Artemisinin levels in the A. annua leaves used were 0.90-1.13% (m/m). The concentration of artemisinin in the infusions was 40-46 mg/L. Field P. falciparum isolates were resistant to chloroquine and sensitive to quinine and artemisinin. The average 50% inhibition concentration values for A. annua infusions against field isolates were 0.11-0.14 µL/mL (these infusions exhibited artemisinin concentrations of 4.7-5.6 ng/mL) and were active in vitro against P. falciparum due to their artemisinin concentration. No synergistic effect was observed for artemisinin in the infusions.

In vitro susceptibility of Plasmodium falciparum Welch field isolates to infusions prepared from Artemisia annua L. cultivated in the Brazilian Amazon

Artemisinin is the active antimalarial compound obtained from the leaves of Artemisia annua L. Artemisinin, and its semi-synthetic derivatives, are the main drugs used to treat multi-drug-resistant Plasmodium falciparum (one of the human malaria parasite species). The in vitro susceptibility of P. falciparum K1 and 3d7 strains and field isolates from the state of Amazonas, Brazil, to A. annua infusions (5 g dry leaves in 1 L of boiling water) and the drug standards chloroquine, quinine and artemisinin were evaluated. The A. annua used was cultivated in three Amazon ecosystems (várzea, terra preta de índio and terra firme) and in the city of Paulínia, state of São Paulo, Brazil. Artemisinin levels in the A. annua leaves used were 0.90-1.13% (m/m). The concentration of artemisinin in the infusions was 40-46 mg/L. Field P. falciparum isolates were resistant to chloroquine and sensitive to quinine and artemisinin. The average 50% inhibition concentration values for A. annua infusions against field isolates were 0.11-0.14 μL/mL (these infusions exhibited artemisinin concentrations of 4.7-5.6 ng/mL) and were active in vitro against P. falciparum due to their artemisinin concentration. No synergistic effect was observed for artemisinin in the infusions.

Effects of culture filtrates of endophytic fungi obtained from Piper aduncum L. on the growth of mycobacterium tuberculosis

Substances that inhibit the growth of Mycobacterium tuberculosis could potentially be used as antibiotics. These substances could also be added to test culture media to improve the speed of tuberculosis diagnosis. The aim of this work was to investigate the influence of culture filtrates of endophytic fungi isolated from P. aduncum L. on the growth of M. tuberculosis. To achieve this objective, the following methodology was used: a) endophytic fungi were isolated from the leaves and stems of P. aduncum L.; b) the isolated fungi were submitted to submerged bioprocessing; c) culture filtrates from the bioprocess were assayed to evaluate their effect on the growth of M. tuberculosis. We isolated 315 fungal types, which represented 85 morphologies, from different parts of P. aduncum L. The bioassays were performed on 82 culture filtrates and 6 plant extracts and resulted in the detection of 1 culture filtrate that stimulated the growth of M. tuberculosis and 15 that inhibited microbial growth. None of the phytochemical extracts had an effect on the growth of M. tuberculosis. In conclusion, we observed that the endophytic fungi isolated from P. aduncum L. (Piperaceae) produced extracellular metabolites (present in the culture filtrate) that affect the growth of M. tuberculosis. These compounds have the potential to be used as antimicrobials or in the diagnosis of tuberculosis.

In vivo and in vitro antimalarial activity of 4-nerolidylcatechol.

4-Nerolidylcatechol (4-NC) isolated from Piper peltatum L. (Piperaceae) was evaluated for in vitro antiplasmodial activity against Plasmodium falciparum (cultures of both standard CQR (K1) and CQS (3D7) strains and two Amazonian field isolates) and for in vivo antimalarial activity using the Plasmodium berghei-murine model. 4-NC exhibits significant in vitro and moderate in vivo antiplasmodial activity. 4-NC administered orally and subcutaneously at doses of 200, 400 and 600 mg/kg/day suppressed the growth of P. berghei by up to 63% after four daily treatments (days 1-4). Also, 4-NC exhibited important in vitro antiplasmodial activity against both standard and field P. falciparum strains in which 50% inhibition of parasite growth (IC(50) ) was produced at concentrations of 0.05-2.11 µg/mL and depended upon the parasite strain. Interestingly, healthy (non-infected) mice that received 4-NC orally presented (denatured) blood plasma which exhibited significant in vitro activity against P. falciparum. This is evidence that mouse metabolism allows 4-NC or active metabolites to enter the blood. Further chemical and pharmacological studies are necessary to confirm the potential of 4-NC as a new antimalarial prototype.

Piper peltatum: biomass and 4-nerolidylcatechol production.

Piper peltatum L. is used for the treatment of inflammation, malaria, and other ailments. 4-Nerolidylcatechol (4-NC) is a valuable natural product that has important anti-inflammatory, antimalarial, and antioxidant properties. 4-NC is a component of P. peltatum and P. umbellatum extracts, which are used in cosmetics. The aim of this work was to evaluate the production of plant biomass and the production of 4-NC in roots of cultivated P. peltatum over a full life cycle. Seedlings were produced in a greenhouse and then transplanted. The weight of dry plant parts (leaves, stems, roots, and inflorescences); numbers of stems, leaves, and inflorescences; and the leaf-to-stem ratio were evaluated at intervals of 60 days after transplanting (DAT). Extracts were prepared using 1:1 ethanol-chloroform and an ultrasound bath. Roots, leaves, and inflorescences contained 4-NC according to TLC photodensitometry analysis. Quantification of 4-NC in root extracts was performed using HPLC-DAD analysis. Per-hectare production of 4-NC by roots was estimated based on quantitative HPLC analysis and biomass data. Optimal per-hectare yields of 4-NC were obtained by harvesting roots between 350 and 400 DAT. In this period, the average yield was 27 kg 4-NC per hectare. Importantly, at the time of maximal overall production of root biomass (470 DAT), there was a decrease in the production of 4-NC (23.8 kg/ha), probably due to the onset of senescence.

New antimalarial and cytotoxic 4-nerolidylcatechol derivatives.

4-Nerolidylcatechol (1) was isolated from cultivated Pothomorphe peltata root on a multigram scale using straight-forward solvent extraction-column chromatography. New semi-synthetic derivatives of 1 were prepared and tested in vitro against multidrug-resistant Plasmodium falciparum K1 strain. Mono-O-methyl, mono-O-benzyl, O,O-dibenzyl and O,O-dibenzoyl derivatives 2-8 exhibited IC(50) in the 0.67-22.52 microM range. Mono-O-methyl ethers 6 and 7 inhibited the in vitro growth of human tumor cell lines HCT-8 (colon carcinoma), SF-295 (central nervous system), LH-60 (human myeloblastic leukemia) and MDA/MB-435 (melanoma). In general, derivatives 2-8 are more stable to light, air and pH at ambient temperatures than their labile, natural precursor 1. These derivatives provide leads for the development of a novel class of antimalarial drugs with enhanced chemical and pharmacological properties.