Extraction of Antioxidant Compounds from Brazilian Green Propolis Using Ultrasound-Assisted Associated with Low- and High-Pressure Extraction Methods.

The demand for bee products has been growing, especially regarding their application in complementary medicine. Apis mellifera bees using Baccharis dracunculifolia D.C. (Asteraceae) as substrate produce green propolis. Among the examples of bioactivity of this matrix are antioxidant, antimicrobial, and antiviral actions. This work aimed to verify the impact of the experimental conditions applied in low- and high-pressure extractions of green propolis, using sonication (60 kHz) as pretreatment to determine the antioxidant profile in the extracts. Total flavonoid content (18.82 ± 1.15-50.47 ± 0.77 mgQE·g-1), total phenolic compounds (194.12 ± 3.40-439.05 ± 0.90 mgGAE·g-1) and antioxidant capacity by DPPH (33.86 ± 1.99-201.29 ± 0.31 µg·mL-1) of the twelve green propolis extracts were determined. By means of HPLC-DAD, it was possible to quantify nine of the fifteen compounds analyzed. The results highlighted formononetin (4.76 ± 0.16-14.80 ± 0.02 mg·g-1) and p-coumaric acid (

Evaluation of the release, stability and antioxidant activity of Brazilian red propolis extract encapsulated by spray-drying, spray-chilling and using the combination of both techniques.

Red propolis, originary from Northeast Brazil, has a unique composition and a great commercial interest. However, due to the presence of ethanol and its remarkable sensory characteristic, its application in food products is challenging. Thus, the aim of this work was to microencapsulate the red propolis extract by spray-drying, spray-chilling, and combining both techniques. The particles loaded with propolis extracts were characterised and evaluated according to the stability of phenolic compounds, flavonoids and formononetin, during 60 days of storage. In addition, the formononetin release was also monitored during the oral, gastric, and intestinal phases in the in vitro digestion process. All produced particles presented matrix-type with size, distribution, shape, hygroscopicity, and dispersibility parameters that varied according to the carrier and encapsulation process applied. The techniques used to fabricate the particles efficiently obtained powdered propolis extract and protected the extract's bioactive compounds, total flavonoids and formononetin throughout the analysed period. The gastrointestinal release study presented distinctive releases in all phases (oral, gastric, and intestinal). The spray-dried particles, for example, released formononetin mainly in the oral stage. While the spray-chilled particles were primarily released in the intestinal phase, and coated particles were released gradually throughout the assay, reaching maximum relief in the intestinal phase. In conclusion, using microencapsulation techniques by spray-drying, spray-chilling, and their combination developed particles with different levels of protection during storage, releases and characteristics, which resulted in a range of possible applications in the food, feed, cosmetic, and pharmaceutical industries.

Optimization of enzymatic parameters for the production of formononetin from red clover (Trifolium pratense L.) through a response surface methodology.

Trifolium pratense, is a forage found worldwide, but it is negatively impacted by the clover root borer, Hylastinus obscurus. Methanolic extraction has been reported for isolating formononetin from vegetal tissues, with an antifeeding effect on H. obscurus. However, this methodology is time-consuming and also extracts other secondary metabolites, whereas enzymatic assays can provide higher specificity. Hence, the objective of this work was to determine the optimal conditions in pH, temperature, and incubation time for the activity of isoflavone synthase via a response surface model. Once these parameters were optimized, the concentration of formononetin in cultivars and experimental lines of T. pratense was evaluated enzymatically. The results showed that the best condition for developing the enzymatic assay was pH 9.1 with an incubation at 34.5 °C for 155 min. The formononetin content fluctuated between 0.74 and 1.96 mg/g of fresh weight, where Precoz-3, Precoz-1, and Superqueli-INIA presented the highest production.

Development of a New Formulation Based on In Situ Photopolymerized Polymer for the Treatment of Spinal Cord Injury.

Spinal Cord Injury (SCI) promotes a cascade of inflammatory events that are responsible for neuronal death and glial scar formation at the site of the injury, hindering tissue neuroregeneration. Among the main approaches for the treatment of SCI, the use of biomaterials, especially gelatin methacryloyl (GelMA), has been proposed because it is biocompatible, has excellent mechanical properties, favoring cell adhesion and proliferation. In addition, it can act as a carrier of anti-inflammatory drugs, preventing the formation of glial scars. The present work presents the development and in situ application of a light-curing formulation based on GelMA containing a natural extract rich in anti-inflammatory, antioxidant and neuroprotective substances (hydroalcoholic extract of red propolis-HERP) in an experimental model of SCI in rats. The formulations were prepared and characterized by time of UV exposition, FTIR, swelling and degradation. The hydrogels containing 1 mg/mL of HERP were obtained by the exposure to UV radiation of 2 µL of the formulation for 60 s. The locomotor evaluation of the animals was performed by the scale (BBB) and demonstrated that after 3 and 7 days of the injury, the GelMA-HERP group (BBB = 5 and 7) presented greater recovery compared to the GelMA group (BBB = 4 and 5). Regarding the inflammatory process, using histomorphological techniques, there was an inflammation reduction in the groups treated with GelMA and GelMA-HERP, with decreases of cavitation in the injury site. Therefore, it is possible to conclude that the use of GelMA and GelMA-HERP hydrogel formulations is a promising strategy for the treatment of SCI when applied in situ, as soon as possible after the injury, improving the clinical and inflammatory conditions of the treated animals.

Formononetin: Biological effects and uses - A review.

Formononetin (FORM) is an isoflavone from the group of phytoestrogens that exhibits a broad spectrum of physiological effects beneficial to health through dependent and independent mechanisms of estrogen. This article aimed to present FORM main functions and future prospects for applications in different areas. Scientific publications and patents dated between 1998 and 2019 were analyzed. FORM has potential as an active compound of interest to product development for the industries of food, medicine, and cosmetics, among others. Moreover, in the medical area, this active compound has shown potential in the prevention and treatment of several diseases, including chronic ones, such as cancer, obesity, and neurodegenerative diseases.

Red Propolis and Its Dyslipidemic Regulator Formononetin: Evaluation of Antioxidant Activity and Gastroprotective Effects in Rat Model of Gastric Ulcer.

Propolis has various pharmacological properties of clinical interest, and is also considered a functional food. In particular, hydroalcoholic extracts of red propolis (HERP), together with its isoflavonoid formononetin, have recognized antioxidant and anti-inflammatory properties, with known added value against dyslipidemia. In this study, we report the gastroprotective effects of HERP (50-500 mg/kg, p.o.) and formononetin (10 mg/kg, p.o.) in ethanol and non-steroidal anti-inflammatory drug-induced models of rat ulcer. The volume, pH, and total acidity were the evaluated gastric secretion parameters using the pylorus ligature model, together with the assessment of gastric mucus contents. The anti-Helicobacter pylori activities of HERP were evaluated using the agar-well diffusion method. In our experiments, HERP (250 and 500 mg/kg) and formononetin (10 mg/kg) reduced (p < 0.001) total lesion areas in the ethanol-induced rat ulcer model, and reduced (p < 0.05) ulcer indices in the indomethacin-induced rat ulcer model. Administration of HERP and formononetin to pylorus ligature models significantly decreased (p < 0.01) gastric secretion volumes and increased (p < 0.05) mucus production. We have also shown the antioxidant and anti-Helicobacter pylori activities of HERP. The obtained results indicate that HERP and formononetin are gastroprotective in acute ulcer models, suggesting a prominent role of formononetin in the effects of HERP.

Peptides and isoflavones in gastrointestinal digests contribute to the anti-inflammatory potential of cooked or germinated desi and kabuli chickpea (Cicer arietinum L.).

It is largely unknown how processing affects bioactive potential of chickpea proteins to prevent bowel inflammatory diseases. The aim was to investigate the anti-inflammatory activity of protein concentrates from germinated and cooked chickpeas (GC and CC, respectively) and its relationship with protein and isoflavone composition before and after in vitro gastrointestinal digestion and absorption. Anti-inflammatory activity of GC digests was almost 2-fold higher than CC digests (p < 0.05), which was associated to greater content of peptides, formononetin and biochanin A (p < 0.05). Anti-inflammatory activity of phenolic fraction in digests was 7-fold higher than the protein fraction (p < 0.05). The most active peptide fraction from GC digest (IC50 = 93 µg/mL) contained a total of 24 peptides derived from legumin and vicilin. In conclusion, this study stands out the potential of germinated chickpea proteins concentrates to exert anti-inflammatory effects in the lower gut which may contribute to the prevention of bowel inflammatory diseases.

Differential formononetin content in cultivars and experimental lines of red clover (Trifolium pratense L. ) plants affect the feeding behaviour of Hylastinus obscurus (Coleoptera: Curculionidae) / Contenido diferencial de formononetina en cultivares y líneas experimentales de plantas de trébol rosado (Trifolium pratense L. ) afecta la conducta alimenticia de Hylastinus obscurus (Coleoptera: Curculionidae)

Red clover (Trifolium pratense L.) is a perennial plant widely used as a forage resource for several animals. This plant is the exclusive host of Hylastinus obscurus (Marsham) which causes irreparable damages to the root system affecting their persistence. It has been reported that the presence of the isoflavonoid formononetin in roots of red clover could act as an antifeedant on H. obscurus. There are not studies related to the formononetin content in red clover roots to the antifeedant effect elicited by experimental lines and cultivar of red clover. Six red clover genotypes were investigated in both formononetin content and their respective antifeedant action. The results showed to Sabtoron High and Superqueli-INIA with both the highest formononetin content in red clover roots and antifeedant effect, allowing to suggest that this secondary metabolites could be used as a chemical factor for red clover plants. Moreover, a rapid methodology for searching red clover genotypes with high formononetin content is reported.

El trébol rosado (Trifolium pratense L.) es una planta perenne ampliamente utilizada como fuente de forraje de variados animales. Esta planta es el exclusivo hospedero de Hylastinus obscurus (Marsham) el cual causa irreparables daños al sistema radical afectando seriamente su persistencia. Se ha reportado que la presencia del isoflavonoide formononetina en raíces del trébol rosado podría actuar como antialimentario sobre H. obscurus. Actualmente no existen estudios que relacionen el contenido de formononetina en raíces de trébol rosado con el efecto antialimentario elicitado por líneas experimentales y cultivares de trébol rosado. Seis genotipos de esta leguminosa fueron evaluados en cuanto a su contenido de formononetina y actividad antialimentaria. Los resultados mostraron que los cultivares Sabtoron High y Superqueli-INIA presentaron altos niveles de formononetina en sus raíces y efecto antialimentario sobre H. obscurus, lo que permite sugerir que este metabolito secundario podría ser usado como factor químico para incrementar la persistencia de plantas de trébol rosado. Además, se informa una metodología rápida para la búsqueda de genotipos con altos contenidos de formononetina.

Hydroxypropyl-ß-cyclodextrin-containing hydrogel enhances skin formononetin permeation/retention.

OBJECTIVES: This study was aimed to investigate the in vitro permeation potential of hydrogel formulations containing the isoflavones formononetin and biochanin A and cyclodextrins in different combinations. METHODS: The permeation assay was performed using porcine skin discs on Franz diffusion cells model. The isoflavone contents of the formulations were quantified in the different layers of the skin using a validated HPLC-PDA method. KEY FINDINGS: The isoflavones individually incorporated into the formulations showed high permeation potential, especially formononetin, after the incorporation of hydroxypropyl-ß-cyclodextrin that enhanced its permeation in the epidermis and dermis. Biochanin A showed 2.7 times of permeation capacity in the epidermis and dermis mainly after incorporation of cyclodextrins in the formulations. Formononetin showed reduction in its permeation when incorporated in the formulations together to biochanin A, showing the absence of synergism. CONCLUSIONS: Our results indicated a noticeable skin permeation promoting effect of HPßCD in formononetin formulation. Furthermore, formononetin and biochanin A can permeate the skin being mostly retained in the epidermis and dermis, revealing its potential use in cosmetic preparations intended to prevent skin aging.

Arbuscular mycorrhizal fungi and colonization stimulant in cotton and maize / Fungos micorrízicos arbusculares e estimulante da colonização na cultura do algodoeiro e do milho

The objective of this study was to evaluate the effects of inoculation with different arbuscular mycorrhizal fungi native to Cerrado, associated with the application of colonization stimulant (7-hydroxy, 4-methoxy-isoflavone) at the initial growth of cotton and maize plants in a greenhouse. The experiment was carried out in a completely randomized design and 7x2 factorial scheme, with five species of arbuscular mycorrhizal fungi and combined inoculation (uniting all species in equal proportion) and native fungi, in the presence and absence of colonization stimulant in maize and cotton crops. Shoots dry matter (SDM), roots dry matter (RDM), mycorrhizal colonization and accumulation of calcium, zinc and phosphorus in the SDM were evaluated at flowering. Inoculation of arbuscular mycorrhizal fungi and the use of colonization stimulant at low density of arbuscular mycorrhizal fungi propagules was beneficial to plant development. Inoculation with mycorrhizal fungi demonstrated different effects for the various parameters evaluated in cotton and maize, and the application of colonization stimulant promoted higher mycorrhizal colonization and initial growth in cotton, when associated with native AMF, and in corn, when associated with inoculation with Dentiscutata heterogama, Gigaspora margarita and Rhizophagus clarus.(AU)

O objetivo deste estudo foi avaliar os efeitos da inoculação com diferentes fungos micorrízicos arbusculares nativos do Cerrado, associados à aplicação de estimulante de colonização (7-hidroxi, 4-metoxi-isoflavona) no crescimento inicial de plantas de algodão e milho em casa de vegetação. O experimento foi conduzido em delineamento inteiramente casualizado e esquema fatorial 7x2, com cinco espécies de fungos micorrízicos arbusculares e inoculação combinada (unindo todas as espécies em proporção igual) e fungos nativos, na presença e ausência de estimulante de colonização em culturas de milho e algodão. Avaliou-se a massa de matéria seca (SDM), matéria seca de raízes (RDM), colonização micorrízica e acúmulo de cálcio, zinco e fósforo na SDM. A inoculação com fungos micorrízicos arbusculares e o uso de estimulante de colonização em baixa densidade de propágulos de fungos micorrízicos arbusculares foi benéfico para o desenvolvimento da planta. A inoculação com fungos micorrízicos mostrou efeitos diferentes para os vários parâmetros avaliados no algodão e no milho, e a aplicação de estimulante de colonização promoveu maior colonização micorrízica e crescimento inicial no algodão quando associado à fungos micorrízicos arbusculares nativa e no milho quando associado à inoculação com Dentiscutata heterogama, Gigaspora margarita e Rhizophagus clarus.(AU)

Arbuscular mycorrhizal fungi and colonization stimulant in cotton and maize / Fungos micorrízicos arbusculares e estimulante da colonização na cultura do algodoeiro e do milho

The objective of this study was to evaluate the effects of inoculation with different arbuscular mycorrhizal fungi native to Cerrado, associated with the application of colonization stimulant (7-hydroxy, 4-methoxy-isoflavone) at the initial growth of cotton and maize plants in a greenhouse. The experiment was carried out in a completely randomized design and 7x2 factorial scheme, with five species of arbuscular mycorrhizal fungi and combined inoculation (uniting all species in equal proportion) and native fungi, in the presence and absence of colonization stimulant in maize and cotton crops. Shoots dry matter (SDM), roots dry matter (RDM), mycorrhizal colonization and accumulation of calcium, zinc and phosphorus in the SDM were evaluated at flowering. Inoculation of arbuscular mycorrhizal fungi and the use of colonization stimulant at low density of arbuscular mycorrhizal fungi propagules was beneficial to plant development. Inoculation with mycorrhizal fungi demonstrated different effects for the various parameters evaluated in cotton and maize, and the application of colonization stimulant promoted higher mycorrhizal colonization and initial growth in cotton, when associated with native AMF, and in corn, when associated with inoculation with Dentiscutata heterogama, Gigaspora margarita and Rhizophagus clarus.

O objetivo deste estudo foi avaliar os efeitos da inoculação com diferentes fungos micorrízicos arbusculares nativos do Cerrado, associados à aplicação de estimulante de colonização (7-hidroxi, 4-metoxi-isoflavona) no crescimento inicial de plantas de algodão e milho em casa de vegetação. O experimento foi conduzido em delineamento inteiramente casualizado e esquema fatorial 7x2, com cinco espécies de fungos micorrízicos arbusculares e inoculação combinada (unindo todas as espécies em proporção igual) e fungos nativos, na presença e ausência de estimulante de colonização em culturas de milho e algodão. Avaliou-se a massa de matéria seca (SDM), matéria seca de raízes (RDM), colonização micorrízica e acúmulo de cálcio, zinco e fósforo na SDM. A inoculação com fungos micorrízicos arbusculares e o uso de estimulante de colonização em baixa densidade de propágulos de fungos micorrízicos arbusculares foi benéfico para o desenvolvimento da planta. A inoculação com fungos micorrízicos mostrou efeitos diferentes para os vários parâmetros avaliados no algodão e no milho, e a aplicação de estimulante de colonização promoveu maior colonização micorrízica e crescimento inicial no algodão quando associado à fungos micorrízicos arbusculares nativa e no milho quando associado à inoculação com Dentiscutata heterogama, Gigaspora margarita e Rhizophagus clarus.

Biological Activities of Red Propolis: A Review.

BACKGROUND: The red propolis (RdProp) is a resin produced by Apis mellifera bees, which collect the reddish exudate on the surface of its botanic source, the species Dalbergia ecastophyllum, popularly known in Brazil as "rabo de bugio". Considered as the 13th type of Brazilian propolis, this resin has been gaining prominence due to its natural composition, rich in bioactive substances not found in other types of propolis. OBJECTIVE: This review aims to address the most important characteristics of RdProp, its botanical origin, the main constituents, its biological properties and the patents related to this natural product. METHOD: By means of the SciFinder, Google Patents, Patus® and Spacenet, scientific articles and patents involving the term "red propolis" were searched until August 2017. RESULTS: A number of biological properties, including antimicrobial, anti-inflammatory, antiparasitic, antitumor, antioxidant, metabolic and nutraceutical activities are attributed to RdProp, demonstrating the great potential of its use in the food, pharmaceutical and cosmetics industries. CONCLUSION: The available papers are associated to pharmacological potential of RdProp, but the molecular mechanisms or bioactive compounds responsible for each activity have not yet been fully elucidat ed. The RdProp patents currently found are directed to components for the pharmaceutical industry (EP2070543A1; WO2014186851A1; FR3006589A1; CN1775277A; CN105797149A; CN1879859A), cosmetic (JP6012138B2; JP2008247830A; JP6012138B2) and food (JP5478392B2; CN101380052A; WO2006038690A1).

Arbuscular mycorrhizal fungi and colonization stimulant in cotton and maize / Fungos micorrízicos arbusculares e estimulante da colonização na cultura do algodoeiro e do milho

ABSTRACT: The objective of this study was to evaluate the effects of inoculation with different arbuscular mycorrhizal fungi native to Cerrado, associated with the application of colonization stimulant (7-hydroxy, 4’-methoxy-isoflavone) at the initial growth of cotton and maize plants in a greenhouse. The experiment was carried out in a completely randomized design and 7x2 factorial scheme, with five species of arbuscular mycorrhizal fungi and combined inoculation (uniting all species in equal proportion) and native fungi, in the presence and absence of colonization stimulant in maize and cotton crops. Shoots dry matter (SDM), roots dry matter (RDM), mycorrhizal colonization and accumulation of calcium, zinc and phosphorus in the SDM were evaluated at flowering. Inoculation of arbuscular mycorrhizal fungi and the use of colonization stimulant at low density of arbuscular mycorrhizal fungi propagules was beneficial to plant development. Inoculation with mycorrhizal fungi demonstrated different effects for the various parameters evaluated in cotton and maize, and the application of colonization stimulant promoted higher mycorrhizal colonization and initial growth in cotton, when associated with native AMF, and in corn, when associated with inoculation with Dentiscutata heterogama, Gigaspora margarita and Rhizophagus clarus.

RESUMO: O objetivo deste estudo foi avaliar os efeitos da inoculação com diferentes fungos micorrízicos arbusculares nativos do Cerrado, associados à aplicação de estimulante de colonização (7-hidroxi, 4’-metoxi-isoflavona) no crescimento inicial de plantas de algodão e milho em casa de vegetação. O experimento foi conduzido em delineamento inteiramente casualizado e esquema fatorial 7x2, com cinco espécies de fungos micorrízicos arbusculares e inoculação combinada (unindo todas as espécies em proporção igual) e fungos nativos, na presença e ausência de estimulante de colonização em culturas de milho e algodão. Avaliou-se a massa de matéria seca (SDM), matéria seca de raízes (RDM), colonização micorrízica e acúmulo de cálcio, zinco e fósforo na SDM. A inoculação com fungos micorrízicos arbusculares e o uso de estimulante de colonização em baixa densidade de propágulos de fungos micorrízicos arbusculares foi benéfico para o desenvolvimento da planta. A inoculação com fungos micorrízicos mostrou efeitos diferentes para os vários parâmetros avaliados no algodão e no milho, e a aplicação de estimulante de colonização promoveu maior colonização micorrízica e crescimento inicial no algodão quando associado à fungos micorrízicos arbusculares nativa e no milho quando associado à inoculação com Dentiscutata heterogama, Gigaspora margarita e Rhizophagus clarus.

Isoflavone formononetin from red propolis acts as a fungicide against Candida sp

Abstract A bioassay-guided fractionation of two samples of Brazilian red propolis (from Igarassu, PE, Brazil, hereinafter propolis 1 and 2) was conducted in order to determine the components responsible for its antimicrobial activity, especially against Candida spp. Samples of both the crude powdered resin and the crude ethanolic extract of propolis from both locations inhibited the growth of all 12 tested Candida strains, with a minimum inhibitory concentration of 256 µg/mL. The hexane, acetate and methanol fractions of propolis 1 also inhibited all strains with minimum inhibitory concentration values ranging from 128 to 512 µg/mL for the six bacteria tested and from 32 to 1024 µg/mL for the yeasts. Similarly, hexane and acetate fractions of propolis sample 2 inhibited all microorganisms tested, with minimum inhibitory concentration values of 512 µg/mL for bacteria and 32 µg/mL for yeasts. The extracts were analyzed by HPLC and their phenolic profile allowed us to identify and quantitate one phenolic acid and seven flavonoids in the crude ethanolic extract. Formononetin and pinocembrin were the major constituents amongst the identified compounds. Formononetin was detected in all extracts and fractions tested, except for the methanolic fraction of sample 2. The isolated isoflavone formononetin inhibited the growth of all the microorganisms tested, with a minimum inhibitory concentration of 200 µg/mL for the six bacteria strains tested and 25 µg/mL for the six yeasts. Formononetin also exhibited fungicidal activity against five of the six yeasts tested. Taken together our results demonstrate that the isoflavone formononetin is implicated in the reported antimicrobial activity of red propolis.

Isoflavone formononetin from red propolis acts as a fungicide against Candida sp.

A bioassay-guided fractionation of two samples of Brazilian red propolis (from Igarassu, PE, Brazil, hereinafter propolis 1 and 2) was conducted in order to determine the components responsible for its antimicrobial activity, especially against Candida spp. Samples of both the crude powdered resin and the crude ethanolic extract of propolis from both locations inhibited the growth of all 12 tested Candida strains, with a minimum inhibitory concentration of 256µg/mL. The hexane, acetate and methanol fractions of propolis 1 also inhibited all strains with minimum inhibitory concentration values ranging from 128 to 512µg/mL for the six bacteria tested and from 32 to 1024µg/mL for the yeasts. Similarly, hexane and acetate fractions of propolis sample 2 inhibited all microorganisms tested, with minimum inhibitory concentration values of 512µg/mL for bacteria and 32µg/mL for yeasts. The extracts were analyzed by HPLC and their phenolic profile allowed us to identify and quantitate one phenolic acid and seven flavonoids in the crude ethanolic extract. Formononetin and pinocembrin were the major constituents amongst the identified compounds. Formononetin was detected in all extracts and fractions tested, except for the methanolic fraction of sample 2. The isolated isoflavone formononetin inhibited the growth of all the microorganisms tested, with a minimum inhibitory concentration of 200µg/mL for the six bacteria strains tested and 25µg/mL for the six yeasts. Formononetin also exhibited fungicidal activity against five of the six yeasts tested. Taken together our results demonstrate that the isoflavone formononetin is implicated in the reported antimicrobial activity of red propolis.

Isoflavone formononetin from red propolis acts as a fungicide against Candida sp

A bioassay-guided fractionation of two samples of Brazilian red propolis (from Igarassu, PE, Brazil, hereinafter propolis 1 and 2) was conducted in order to determine the components responsible for its antimicrobial activity, especially against Candida spp. Samples of both the crude powdered resin and the crude ethanolic extract of propolis from both locations inhibited the growth of all 12 tested Candida strains, with a minimum inhibitory concentration of 256 µg/mL. The hexane, acetate and methanol fractions of propolis 1 also inhibited all strains with minimum inhibitory concentration values ranging from 128 to 512 µg/mL for the six bacteria tested and from 32 to 1024 µg/mL for the yeasts. Similarly, hexane and acetate fractions of propolis sample 2 inhibited all microorganisms tested, with minimum inhibitory concentration values of 512 µg/mL for bacteria and 32 µg/mL for yeasts. The extracts were analyzed by HPLC and their phenolic profile allowed us to identify and quantitate one phenolic acid and seven flavonoids in the crude ethanolic extract. Formononetin and pinocembrin were the major constituents amongst the identified compounds. Formononetin was detected in all extracts and fractions tested, except for the methanolic fraction of sample 2. The isolated isoflavone formononetin inhibited the growth of all the microorganisms tested, with a minimum inhibitory concentration of 200 µg/mL for the six bacteria strains tested and 25 µg/mL for the six yeasts. Formononetin also exhibited fungicidal activity against five of the six yeasts tested. Taken together our results demonstrate that the isoflavone formononetin is implicated in the reported antimicrobial activity of red propolis. (AU)

Antinociceptive and anti-inflammatory effects of Brazilian red propolis extract and formononetin in rodents.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis has been used as a folk medicine for centuries around the world due to its wide spectrum of biological activities. The red propolis, a new Brazilian variety of this apimaterial, has presented an unusual chemical composition, including isoflavones such as formononetin and biochanin A. Since both the green and red varieties of propolis are traditionally used as medicine and commercialized with no label differentiation, the study of the activities of red propolis extract has become important in order to clarify whether this product has the same activities as commercial ones. In this work, we demonstrated the potential action of the hydroalcoholic extract of red propolis (HERP) and its biomarker, formononetin, as antinociceptive and anti-inflammatory drugs on experimental models. MATERIALS AND METHODS: The HERP was chemically characterised by HPLC/DAD analyses. The biological activities of the HERP (3, 10, and 30mg/kg) and formononetin (10mg/kg) were evaluated using the antinociceptive (acetic acid, formalin, and glutamate injections) and anti-inflammatory (carrageenan-induced hindpaw oedema and peritonitis) models in mice after oral administration. The open field test was also performed. RESULTS: Formononetin, one of the main biomarker of red propolis, was identified in the HERP (21.62mg/g). Pretreatment with the HERP (10 and 30mg/kg) and formononetin (10mg/kg) produced reduction (P<0.001) in the number of abdominal writhes, but the HERP was more effective (P<0.001) than formononetin. In the formalin test, all HERP doses (3, 10, and 30mg/kg, P<0.001) inhibited the late phase (inflammatory pain) of formalin-induced licking, but the inhibition of neurogenic pain was observed only when the higher doses (10 and 30mg/kg; P<0.05) were used. Formononetin caused inhibition (P<0.001) only in the second phase of formalin-induced nociception similarly at all HERP doses in the same phase of the test. The responses in glutamate-induced model presented crescent inhibition (P<0.05) with 10 and 30mg/kg of HERP. Also, formononetin inhibited (P<0.001) the nociception induced by glutamate similarly to 30mg/kg of HERP. There were no significant differences in the open field test after HERP administration, but formononetin decrease the spontaneous motor behaviour. Regarding the anti-inflammatory assessment, the HERP (10 and 30mg/kg, P<0.05) and formononetin (P<0.001) treatments caused a significant inhibition of the oedema response. All doses of HERP (3, 10, and 30mg/kg, P<0.05) and formononetin (P<0.001) also inhibited the carrageenan-induced leukocyte migration. In both cases, the results for the HERP at 30mg/kg and formononetin were similar. CONCLUSIONS: The HERP and formononetin presented significant anti-inflammatory activity. Moreover, the HERP presented antinociceptive action on inflammatory and neurogenic pain without motor side effects, possibly due to the action of other constituents present in the extract. These results, together, support the popular usage of this natural product.

Antioxidant responses in bean (Phaseolus vulgaris) roots colonized by arbuscular mycorrhizal fungi.

• Degradation of reactive oxygen species in arbuscular mycorrhizas (AM) may be an efficient mechanism to attenuate the activation of plant defenses. Here, we evaluated the activities of superoxide dismutase (SOD), guaiacol-peroxidase (GPX) and catalase (CAT) in bean (Phaseolus vulgaris) mycorrhizal roots at different conditions and stages of symbiosis development. • Bean plants were inoculated with Glomus clarum (Gc) or G. intraradices (Gi), under low (LP) and high P (HP) concentrations, and grown under glasshouse conditions. In a second experiment, bean seeds were treated with formononetin and inoculated with Gc under LP and HP conditions. The activities of SOD, GPX and CAT were evaluated. • SOD was induced only in roots colonized by Gc, at a late stage of the symbiosis development under LP, and at an early stage under HP. GPX was induced in roots colonized by Gc at an early time point and suppressed later under LP. In general, CAT was induced in roots colonized by Gc under LP. CAT activities in roots were dependent on P and formononetin treatment. • The possible roles of SOD, GPX and CAT in AM are discussed.