Mechanism of action and synergistic effect of Eugenia uniflora extract in Candida spp.

The limited arsenal of antifungal drugs have prompted the search for novel molecules with biological activity. This study aimed to characterize the antifungal mechanism of action of Eugenia uniflora extract and its synergistic activity with commercially available antifungal drugs on the following Candida species: C. albicans, C. tropicalis, C. glabrata, C. parapsilosis and C. dubliniensis. In silico analysis was performed to predict antifungal activity of the major compounds present in the extract. Minimal inhibitory concentrations (MICs) were determined in the presence of exogenous ergosterol and sorbitol. Yeast cells were grown in the presence of stressors. The loss of membrane integrity was assessed using propidium iodide staining (fluorescence emission). Synergism between the extract and antifungal compounds (in addition to time kill-curves) was determined. Molecular docking revealed possible interactions between myricitrin and acid gallic and enzymes involved in ergosterol and cell wall biosynthesis. Candida cells grown in the presence of the extract with addition of exogenous ergosterol and sorbitol showed 2 to 8-fold increased MICs. Strains treated with the extract revealed greater loss of membrane integrity when compared to their Fluconazole counterparts, but this effect was less pronounced than the membrane damage caused by Amphotericin B. The extract also made the strains more susceptible to Congo red and Calcofluor white. A synergistic action of the extract with Fluconazole and Micafungin was observed. The E. uniflora extract may be a viable option for the treatment of Candida infections.

Phytochemical profile, in vitro activities, and toxicity of optimized Eugenia uniflora extracts / Perfil fitoquímico, actividades in vitro y toxicidad de extractos optimizados de Eugenia uniflora

In this study, we investigated the influence of mixture design on the chemical profile of Eugenia unifloraleaves, evaluating the antioxidant and antimicrobial activities, the toxic and hemolytic potential, with the focus on the improvement of the polyphenol's extraction for incorporation of the extract in semi-solid forms with antifungal action. The chemical analysis was evaluated by UV-Vis and HPLC. The antimicrobial, antioxidant, and hemolytic activities were monitored. The flavonoid content ranged from 2.63-7.98 %w/w and tannins from 5.42-18.29 %w/w. The extract consisted of gallic acid (0.09-1.29%; w/w), ellagic acid (0.09-0.37%; w/w), and myricitrin (0.18-1.20%; w/w). The most successful solvent system with the highest level of active extract was water: ethanol: propylene glycol. The extracts showed fungicidal properties (3.9 µg/mL), high antioxidant activity (IC50: 9.50 µg/mL), and low toxicity. These solvent mixtures can improve the in vitro bioactivities when compared to pure solvents and this result demonstrates the importance of mixture designs as useful tools for creating high-quality herbal products and elucidate the potential of E. uniflora glycolic extracts as active herbal pharmaceutical ingredients in topical delivery systems.

En este estudio investigamos la influencia del diseño de mezclas en el perfil químico de hojas de Eugenia uniflora, evaluando las actividades antioxidantes y antimicrobianas, el potencial tóxico y hemolítico, con el foco en la mejora de la extracción de polifenoles para la incorporación del extracto en formas semi-sólidas con acción antifúngica. El análisis químico se evaluó mediante UV-Vis y HPLC. Se monitorizaron las actividades antimicrobianas, antioxidantes y hemolíticas. El contenido de flavonoides osciló entre 2,63 y 7,98% p/p and taninos de 5,42-18,29% p/p. El extracto consistió en ácido gálico (0.09-1.29%; p/p), ácido elágico (0.09-0.37%; p/p) y miricitrina (0.18-1.20%; p/p). El sistema de disolventes más exitoso con el nivel más alto de extracto activo fue agua: etanol: propilenglicol. Los extractos mostraron propiedades fungicidas (3.9 µg/mL), alta actividad antioxidante (IC50: 9.50 µg/mL) y baja toxicidad. Estas mezclas de disolventes pueden mejorar las bioactividades in vitro en comparación con los disolventes puros y este resultado demuestra la importancia de los diseños de mezclas como herramientas útiles para crear productos a base de hierbas de alta calidad y dilucidar el potencial de los extractos glicólicos de E. uniflora como ingredientes farmacéuticos a base de hierbas en sistemas de entrega activos.

Extract from Opuntia ficus-indica cladode delays the Aedes aegypti larval development by inducing an axenic midgut environment.

This study evaluated the effects of acute exposure of Aedes aegypti third instar (L3 ) larvae to the saline extract of Opuntia ficus-indica cladodes on the biological cycle and fertility of the emerging adults. For this, larvae were treated for 24 h with the extract at » LC50 (lethal concentration to kill 50% of larvae), ½ LC50 or LC50 ; the development and reproduction of the emerged adults were evaluated after a recovery period of 9 days. The resistance of proteins in the extract to hydrolysis by L3 digestive enzymes and histomorphological alterations in the larval midgut were also investigated. The extract contained lectin, flavonoids, cinnamic derivatives, terpenes, steroids, and reducing sugars. It showed a LC50 of 3.71% for 48 h. The data indicated mean survival times similar in control and extract treatments. It was observed development delay in extract-treated groups, with a lower number of adults than in control. However, the females that emerged laid similar number of eggs in control and treatments. Histological evaluation revealed absence of bacterial and fungal microorganisms in the food content in midguts from larvae treated with cladode extract. Electrophoresis revealed that three polypeptides in the extract resisted to hydrolysis by L3 digestive proteases for 90 min. The lectin activity was not altered even after 24-h incubation with the enzymes. In conclusion, the extract from O. ficus-indica can delay the development of Ae. aegypti larvae, which may be linked to induction of an axenic environment at larval midgut and permanence of lectin activity even after proteolysis.

Development of an analytical method for determination of polyphenols and total tannins from leaves of Syzygium cumini L. Skeels.

Syzygium cumini L. Skeels belongs to Myrtaceae family. This species has been recognized by its antidiabetic, anti-inflammatory, and antimicrobial activities. Despite ever-increasing scientific interest for this species there is no pharmacopeia method for characterization and standardization of S. cumini yet. So, toward this aim, the objective of this work was to develop an efficient analytical methodology able to determine polyphenols and tannins content from leaves hydroethanolic extract of S. cumini using Folin-Ciocalteu method by ultraviolet absorption spectrophotometry (UV-Vis). The analytical methodology was developed for the first time in the literature for leaves of this specie shown to be fast and low-cost with results expressed through tannic acid equivalent (TAE). Moreover, the methodology presented selectivity with maximum absorption at 706 nm wavelength, linearity with R2>0.99; limit of detection 0.275 µg TAE mL-1 and 0.102 µg TAE mL-1; limit of quantification 1.046 µg TAE mL-1 and 0.912 µg TAE mL-1 for total polyphenols and total tannins, respectively. Furthermore, the methodology was accurate with recover value greater than 98%, as well as exact, reproductive, and robust with coefficient of variation values less than 15% for both compounds. All the results are found within the fixed limits according to RDC 166/2017.

Influence of Eugenia uniflora Extract on Adhesion to Human Buccal Epithelial Cells, Biofilm Formation, and Cell Surface Hydrophobicity of Candida spp. from the Oral Cavity of Kidney Transplant Recipients.

This study evaluated the influence of the extract of Eugenia uniflora in adhesion to human buccal epithelial cells (HBEC) biofilm formation and cell surface hydrophobicity (CSH) of Candida spp. isolated from the oral cavity of kidney transplant patients. To evaluate virulence attributes in vitro, nine yeasts were grown in the presence and absence of 1000 µg/mL of the extract. Adhesion was quantified using the number of Candida cells adhered to 150 HBEC determined by optical microscope. Biofilm formation was evaluated using two methodologies: XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and crystal violet assay, and further analyzed by electronic scan microscopy. CSH was quantified with the microbial adhesion to hydrocarbons test. We could detect that the extract of E. uniflora was able to reduce adhesion to HBEC and CSH for both Candida albicans and non-Candida albicansCandida species. We also observed a statistically significant reduced ability to form biofilms in biofilm-producing strains using both methods of quantification. However, two highly biofilm-producing strains of Candida tropicalis had a very large reduction in biofilm formation. This study reinforces the idea that besides growth inhibition, E. uniflora may interfere with the expression of some virulence factors of Candida spp. and may be possibly applied in the future as a novel antifungal agent.

Chromatographic profiles of extractives from leaves of Eugenia uniflora

ABSTRACT Eugenia uniflora L., Myrtaceae, popularly known as "pitanga", is used in traditional medicine due the properties attributed to its chemical content, these being mainly hydrolysable tannins and flavonoids. This study provides a qualitative and quantitative evaluation of chemical profile from leaves of E. uniflora. The HPLC analysis was carried out on a C18 column (4.6 mm × 250 mm, 5 µm) by gradient elution with methanol and water (acidified with trifluoracetic acid); and silica gel Plates 60-F 254 with 10-12 µm and 5-6 µm particles, respectively for TLC and High HPTLC analysis. The chromatographic data obtained from HPLC, TLC and HPTLC presented bands and peaks related to flavonoids (myricitrin and derivatives) and tannins (gallic and ellagic acids), which were observed from different samples. The chromatographic similarities enabled the building of a typical fingerprint for the herbal material. The similarity analysis of the sample data by Pearson correlation showed R values >0.9 among peaks (HPLC) and bands (HPTLC). In addition, the analytical methodology developed by HPLC enabled the satisfactory quantification of marker substances [ellagic acid = 0.22% and 0.20% (m/m); gallic acid = 0.20% and 0.43%; myricitrin = 0.42 and 1.74% (m/m) in herbal drug and crude extract, respectively]. The procedure was also validated in accordance with the assays required by Brazilian legislation. Thus, the HPTLC and HPLC methods developed in this study provide helpful and simple tools for the quality evaluation both qualitatively and quantitatively of raw materials and extractives from leaves of E. uniflora.

Effect of the Ethyl Acetate Fraction of Eugenia uniflora on Proteins Global Expression during Morphogenesis in Candida albicans.

Candida albicans is able to switch from yeast to hyphal growth and this is an essential step for tissue invasion and establishment of infection. Due to the limited drug arsenal used to treat fungal infections and the constant emergence of resistant strains, it is important to search for new therapeutic candidates. Therefore, this study aimed to investigate by proteomic analysis the role of a natural product (Eugenia uniflora) in impairing hypha formation in C. albicans. We also tested the potential action of E. uniflora to prevent and treat oral candidiasis induced in a murine model of oral infection and the ability of polymorphonuclear neutrophils to phagocytize C. albicans cells treated with the ethyl acetate fraction of the extract. We found that this fraction greatly reduced hypha formation after morphogenesis induction in the presence of serum. Besides, several proteins were differentially expressed in cells treated with the fraction. Surprisingly, the ethyl acetate fraction significantly reduced phagocytosis in C. albicans (Mean 120.36 ± 36.71 yeasts/100 PMNs vs. 44.68 ± 19.84 yeasts/100 PMNs). Oral candidiasis was attenuated when C. albicans cells were either pre-incubated in the presence of E. uniflora or when the fraction was applied to the surface of the oral cavity after infection. These results were consistent with the reduction in CFU counts (2.36 vs. 1.85 Log10 CFU/ml) and attenuation of tissue damage observed with histopathological analysis of animals belonging to treated group. We also observed shorter true hyphae by direct examination and histopathological analysis, when cells were treated with the referred natural product. The E. uniflora ethyl acetate fraction was non-toxic to human cells. E. uniflora may act on essential proteins mainly related to cellular structure, reducing the capacity of filamentation and attenuating infection in a murine model, without causing any toxic effect on human cells, suggesting that it may be a future therapeutic alternative for the treatment of Candida infections.

Spectrophotometric Quantification of Flavonoids in Herbal Material, Crude Extract, and Fractions from Leaves of Eugenia uniflora Linn.

BACKGROUND: The traditional use of Eugenia uniflora L. ("Pitanga") is reported due to several properties, which have often been related to its flavonoid content. OBJECTIVE: The aim was to evaluate analytical procedures for quantification of total flavonoids content (TFCs) by ultraviolet-visible (UV-Vis) spectrophotometry in the herbal material (HM), crude extract (CE), and fractions from leaves of E. uniflora. MATERIALS AND METHODS: The method for quantification of flavonoids after complexation with aluminum chloride (AlCl3) was evaluated: amount of sample (0.25-1.5 g); solvent (40%-80% ethanol); reaction time and AlCl3 concentration (2.5%-7.5%). The procedures by direct dilution (DD) and after acid hydrolysis (AH) were used and validated for HM and CE and applied to the aqueous fraction (AqF), hexane fraction, and ethyl acetate fractions (EAF). RESULTS: The ideal conditions of analysis were ethanol 80% as solvent; 0.5 g of sample; λmax of 408 (DD) and 425 nm (AH); 25 min after addition of AlCl3 5%. The procedures validated for standards and samples showed linearity (R2 > 0.99) with limit of detection and limit of quantification between 0.01 and 0.17 mg/mL (rutin and quercetin); and 0.03 and 0.09 mg/mL (quercetin), for DD and AH, respectively. The procedures were accurate (detect, practice, and repair < 5% and recovery >90%), and stable under robustness conditions (luminosity, storage, reagents, and equipment). The TFCs in AqF and EAF were 0.65 g% and 17.72 g%, calculated as rutin. CONCLUSIONS: UV-Vis methods for quantification of TFC in HM, CE, and fractions from leaves of E. uniflora were suitably validated. Regarding the analysis of fractions, the EAF achieved enrichment of about nine times in the content of flavonoids. SUMMARY: The total flavonoids content (TFCs) of herbal material, crude extract, and fractions from Eugenia uniflora can be quantified by ultraviolet-visibleThe spectrophotometric methods (direct dilution and acid hydrolysis) were reproducible and able to quantify TFC in raw material and derivatives from leaves of E. unifloraHigher flavonoids content was observed in ethyl acetate fractions after enrichment. Abbreviations Used: HM: Herbal material, CE: Crude extract, AqF: Aqueous fraction, HF: Hexanic fraction, EAF: Ethyl acetate fraction, TFC: Total flavonoids content, HCl: Hydrochloric acid, DD: Direct dilution, AH: After hydrolysis, RSD: Relative standard, A.U.: Absorption units.

Chromatographic and Spectrophotometric Analysis of Phenolic Compounds from Fruits of Libidibia ferrea Martius.

BACKGROUND: Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz (Fabaceae) is a tree which is native to Brazil, widely known as "Jucá," where its herbal derivatives are used in folk medicine with several therapeutic properties. The constituents, which have already been described in the fruit, are mainly hydrolysable tannins (gallic acid [GA] and ellagic acid [EA]). OBJECTIVE: The aim of this study was to investigate the phenolic variability in the fruit of L. ferrea by ultraviolet/visible (UV/VIS) and chromatographic methods (high-performance liquid chromatography [HPLC]/high-performance thin layer chromatography [HPTLC]). MATERIALS AND METHODS: Several samples were collected from different regions of Brazil and the qualitative (fingerprints by HPTLC and HPLC) and quantitative analysis (UV/VIS and HPLC) of polyphenols were performed. RESULTS: The HPTLC and HPLC profiles allowed separation and identification of both major analytical markers: EA and GA. The chemical profiles were similar in a number of spots or peaks for the samples, but some differences could be observed in the intensity or area of the analytical markers for HPTLC or HPLC, respectively. Regarding the quantitative analysis, the polyphenolic content by UV/VIS ranged from 13.99 to 37.86 g% expressed as GA or from 10.75 to 29.09 g% expressed as EA. The contents of EA and GA by liquid chromatography-reversed phase (LC-RP) method ranged from 0.57 to 2.68 g% and from 0.54 to 3.23 g%, respectively. CONCLUSION: The chemical profiles obtained by HPTLC or HPLC, as well as the quantitative analysis by spectrophotometry or LC-RP method, were suitable for discrimination of each herbal sample and can be used as tools for the comparative analysis of the fruits from L. ferrea. SUMMARY: The polyphenols of fruits of Libidibia ferrea can be quantified by UV/VIS and HPLCThe HPLC method was able to detect the gallic and ellagic acids in several samples of fruits of Libidibia ferreaThe phenolic profiles of fruits from Libidibia ferrea by HPTLC and HPLC were reproductible. Abbreviations used: HPTLC: high performance thin layer chromatography, HPLC: high performance liquid chromatography, UV-Vis: spectrophotometry.

Reversed-phase-liquid chromatography method for separation and quantification of gallic acid from hydroalcoholic extracts of Qualea grandiflora and Qualea parviflora.

BACKGROUND: Qualea parviflora and Qualea grandiflora (Vochysiaceae), commonly known in Brazil as "pau-terra" and "pau-terrinha," respectively, have been widely used in the treatment of ulcer and gastritis. These therapeutic effects are attributed to various compounds present in the plants, including phenolic compounds such as gallic acid, due to their important antioxidant activity. OBJECTIVE: The aim of the present study was to validate a high performance liquid chromatography with diode array detection (HPLC-DAD) method for the quantitative determination of gallic acid in the stem bark of Q. parviflora and Q. grandiflora hydroalcoholic extracts. MATERIALS AND METHODS: The chromatography analysis was successfully achieved on a Dionex column, Acclaim(®) 120 (250 mm × 4.60 mm, 5 µm) with a gradient elution of water and methanol at a flow rate of 0.8 mL/min and ultraviolet detection at 280 nm. RESULTS: The validation data, including linearity, precision, specificity, accuracy and robustness of this method demonstrated good reliability and sensitivity. CONCLUSION: The method is able to quantify gallic acid in the stem bark of both species. What is more, the chromatographic peaks showed good resolution and there are also the advantages of easy sample preparation and a short time between each injection.

Validation of a spectrophotometric methodology for the quantification of polysaccharides from roots of Operculina macrocarpa (jalapa)

The roots from Operculina macrocarpa (L.) Urb., Convolvulaceae, are widely used in Brazilian traditional medicine as a laxative and purgative. The biological properties of this drug material have been attributed to its polysaccharides content. Thus, the aim of this study was to evaluate the polysaccharide content in drug material from O. macrocarpa by spectrophotometric quantitative analysis. The root was used as plant material and the botanical identification was performed by macro and microscopic analysis. The plant material was used to validate the spectrophotometric procedures at 490 nm for the quantification of the reaction product from drug polysaccharides and phenol-sulfuric acid solution. The analytical procedure was evaluated in order to comply with the necessary legal requirements by the determination of the following parameters: specificity, linearity, selectivity, precision, accuracy and robustness. This study provides with a simple and valid analytical procedure (linear, precise, accurate and reproducible), which can be satisfactorily used for quality control and standardization of herbal drug from O. macrocarpa.

Total phytosterol content in drug materials and extracts from roots of Acanthospermum hispidum by UV-VIS spectrophotometry

Acanthospermum hispidum DC., Asteraceae, is widely used in folk medicine in Brazil to treat respiratory diseases; this biological property has been attributed to its phytosterol content. This study evaluated the spectrophotometric assay method to quantify the total phytosterol content in raw materials and extracts from roots of A. hispidum. The procedure was based on the quantification at 625 nm after the Liebermann-Burchard reaction. The method was evaluated for linearity, repeatability, intermediate precision, accuracy and robustness. The date indicated that the procedure is a valid analytical tool for materials and herbal derivatives from A. hispidum. .

Development and evaluation of emulsions from Carapa guianensis (Andiroba) oil.

Carapa guianensis, a popular medicinal plant known as "Andiroba" in Brazil, has been used in traditional medicine as an insect repellent and anti-inflammatory product. Additionally, this seed oil has been reported in the literature as a repellent against Aedes aegypti. The aim of this work is to report on the emulsification of vegetable oils such as "Andiroba" oil by using a blend of nonionic surfactants (Span 80® and Tween 20®), using the critical hydrophilic-lipophilic balance (HLB) and pseudo-ternary diagram as tools to evaluate the system's stability. The emulsions were prepared by the inverse phase method. Several formulations were made according to a HLB spreadsheet design (from 4.3 to 16.7), and the products were stored at 25°C and 4°C. The emulsion stabilities were tested both long- and short-term, and the more stable one was used for the pseudo-ternary diagram study. The emulsions were successfully obtained by a couple of surfactants, and the HLB analysis showed that the required HLB of the oil was 16.7. To conclude, the pseudo-ternary diagram identified several characteristic regions such as emulsion, micro-emulsion, and separation of phases.