In vitro Evaluation of Fungal Susceptibility and Inhibition of Virulence by Diosgenin.

Fungal infections are a public health problem that mainly affects immunosuppressed people, Candida spp. have been responsible for most sources of contamination and invasive fungal infections described around the world. The need arises to find new therapeutic approaches to combat growing infections. Plants and natural products have been considered a valuable source for discovering new molecules with active ingredients. Diosgenin is a sapogenin found in the families of Leguminosae and Dioscoreaceae, it is obtained mainly from the dioscin saponin through the hydrolysis method, it is a phytochemical that has been highlighted in the treatment of various diseases, as well as in combating microbial resistance. The present study aimed to evaluate the susceptibility of fungal strains to diosgenin, as well as verify the association with the reference drug and evaluate the inhibition of the virulence factor through morphological changes in the yeast state to the filamentous form of hyphae and pseudohyphae in strains of Candida albicans, Candida tropicalis and Candida krusei using the broth microdilution method and microculture technique. Antifungal assays revealed that diosgenin was not able to inhibit the growth of the tested strains. However, it was able to inhibit the fungal dimorphism of the strains evaluated, however further studies are recommended to verify its effectiveness against other virulence factors.

In vitro evaluation of the susceptibility of bacterial biofilms to hecogenin acetate.

Biofilms are a bacterial resistance strategy through which microorganisms organize themselves in the form of a colony fixed to a surface that is protected by a polymer matrix. Infectious diseases that result in biofilm formation have been considered a relevant public health problem due to the potential to increase patient morbidity and mortality, in addition to increasing the burden on health services. Such pathologies are treated with the use of antimicrobial drugs, the indiscriminate use of which has contributed to the process of bacterial resistance, demanding the need to invest in new alternatives to combat them. Based on this, the present work aimed to evaluate the anti-biofilm formation and eradication capacity of Hecogenin Acetate, a steroidal sapogenin of natural origin with important antibacterial properties. For this, we used strains of Streptococcus mutans INCQS 00,446 (ATCC 25,175), Enterococcus faecalis INCQS 00,018 (ATCC 14,506), Staphylococcus epidermidis INCQS 00,016 (ATCC 12,228), Staphylococcus aureus ATCC 25,923, Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 259,223. The formation, formation inhibition and treatment assays were carried out in microdilution plates and revealed using the crystal violet method. Readings were carried out using absorbance at wavelengths of 492 nm. All tests were performed in triplicate and statistical analyzes were performed using Graphpad Prism v.5.0 software. It was observed that the bacterial strains used have a relevant capacity for biofilm formation, with the Gram positive ones identified in the present study as the best former. In the results of the analyzes with bacterial biofilm, it was identified that Hecogenin Acetate had a relevant antibiofilm capacity, and could therefore serve as a basis for further research into the development of new antimicrobial drugs.

Anti-inflammatory Activity: In silico and In vivo of Sapogenins Present in Agave brittoniana subsp. brachypus (Trel.).

BACKGROUND: Agave brittoniana subsp. brachypus is an endemic plant of Cuba, which contains different steroidal sapogenins with anti-inflammatory effects. This work aims to develop computational models which allow the identification of new chemical compounds with potential anti-inflammatory activity. METHODS: The in vivo anti-inflammatory activity was evaluated in two rat models: carrageenaninduced paw edema and cotton pellet-induced granuloma. In each study, we used 30 Sprague Dawley male rats divided into five groups containing six animals. The products isolated and administrated were fraction rich in yuccagenin and sapogenins crude. RESULTS: The obtained model, based on a classification tree, showed an accuracy value of 86.97% for the training set. Seven compounds (saponins and sapogenins) were identified as potential antiinflammatory agents in the virtual screening. According to in vivo studies, the yuccagenin-rich fraction was the greater inhibitor of the evaluated product from Agave. CONCLUSION: The evaluated metabolites of the Agave brittoniana subsp. Brachypus showed an interesting anti-inflammatory effect.

Potentiation of Antibiotic Action and Efflux Pump Inhibitory Effect on Staphylococcus aureus Strains by Solasodine.

A worrisome fact is the increase in microbial resistance, which has as its main cause the indiscriminate use of antibiotics. Scientific studies have investigated bioactive compounds such as steroidal sapogenins, in the perspective of new beneficial alternatives for the control of bacterial resistance. Therefore, the objective of this work was to verify the antibacterial activity as well as the modifying action of antibiotics associated with solasodine and its ability to inhibit the efflux pump mechanism in strains of Staphylococcus aureus. Tests were performed to verify the minimum inhibitory concentration (MIC). In addition, the action-modifying potential of antibiotics and the inhibitory capacity of the efflux pump NorA and MepA through synergistic effects on the antibiotic and ethidium bromide were evaluated. Solasodine showed significant results for the standard bacteria with an MIC of 512 µg/mL, and when associated with the antibiotics gentamicin and nofloxacin for the multidrug-resistant bacteria S. aureus 10, Escherichia coli 06, and Pseudomonas aeruginosa 24, it showed a 50% reduction in MIC. The association of solasodine with the antibiotic ciprofloxacin against S. aureus K2068 (MepA) showed synergism, with a reduction in the MIC of the antibiotic from 64 µg/mL to 40 µg/mL, and also a reduction in the MIC when the antibiotic was used in conjunction with the efflux pump inhibitors. Solasodine may be acting on the mechanism of action of the antibiotic, as it has shown a potentiating effect when associated with antibiotics, inducing a reduction in the MIC against Gram-positive and Gram-negative bacteria. Therefore, this study demonstrated significant results for the potentiating action of solasodine when associated with antibiotics of clinical importance.

Role of peripheral and central sensitization in the anti-hyperalgesic effect of hecogenin acetate, an acetylated sapogenin, complexed with ß-cyclodextrin: Involvement of NFκB and p38 MAPK pathways.

Neuropathic pain develops due to injury to the somatosensory system, affecting the patient's quality of life. In view of the ineffectiveness of the current pharmacotherapy, substances obtained from natural products (NPs) are a promising alternative. One NP that has been discussed in the literature is hecogenin acetate (HA), a steroidal sapogenin with anti-inflammatory and antinociceptive activity. However, HA has low water solubility, which affects its bioavailability. Thus, the objective of this study was to evaluate the anti-hyperalgesic activity of pure and complexed hecogenin acetate (HA/ßCD) in an animal model of chronic neuropathic and inflammatory pain. The inclusion complex was prepared at a molar ratio of 1:2 (HA:ßCD) by the lyophilization method. For the induction of chronic inflammatory pain, the mice received an intraplantar injection of CFA (complete Freund's adjuvant), and were evaluated for mechanical hyperalgesia and for the levels of myeloperoxidase (MPO) in the skin of the paw after eight days of treatment. HA and HA/ßCD reduced mechanical hyperalgesia in relation to the vehicle group until the fourth and fifth hours, respectively, in the acute evaluation, with a superior effect of the complexed form over the pure form in the second and third hour after treatment (p < 0.001). In the chronic evaluation, HA and HA/ßCD reduced hyperalgesia in relation to the vehicle in the eight days of treatment (p < 0.001). Both pure (p < 0.01) and complexed (p < 0.001) forms reduced myeloperoxidase activity in the skin of the animals' paw. Groups of animals subjected to the same pharmacological protocol were submitted to the partial sciatic nerve ligation (PSNL) model and evaluated for mechanical and thermal hyperalgesia, and cold allodynia. HA and HA/ßCD reduced mechanical hyperalgesia until the fourth and sixth hours, respectively, and both reduced hyperalgesia in relation to the vehicle in the chronic evaluation (p < 0.001). HA and HA/ßCD also reduced thermal hyperalgesia and cold allodynia (p < 0.05 and p < 0.001, respectively). The analysis of the spinal cord of these animals showed a decrease in the levels of the pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 and a reduction in the phosphorylation of NFκB and p38MAPK, as well as a decrease in microglioses compared to the vehicle group. In addition, HA/ßCD reduced the nociception induced by intraplantar injection of agonist TRPA1 (p < 0.01) and TRPM8 (p < 0.05). Treatment for eight days with HA and HA/ßCD showed no signs of gastric or liver damage. HA and HA/ßCD were, therefore, shown to have antinociceptive effects in chronic pain models. Based on our exploration of the mechanisms of the action of HA, these effects are likely to be related to inhibited leukocyte migration, interaction with the TRPA1 and TRPM8 receptors, reduced pro-inflammatory cytokines levels, microglial expression and suppression of NF-κB p65 and p38 MAPK pathway signaling. Therefore, HA/ßCD has great potential for use in the treatment of chronic pain.

Optimization of the ultrasoud-assisted extraction of saponins from quinoa (Chenopodium quinoa Wild) using response surface methodology.

BACKGROUND: Quinoa grain has a bitter tasting layer in the pericarp called saponin, a triterpenoid glycoside with industrial potential. Traditionally, quinoa saponins are extracted with a large amount of water, which is why ultrasound technology constitutes an emerging technological alternative which is considered efficient and profitable compared to traditional extraction methods. The objective of this research was to determine the amplitude, time, and concentration of ethanol that guarantee a higher content of saponin through extraction assisted by ultrasound. METHODS: To find the optimal extraction conditions, the response surface methodology was used using the Box Behnken design with 5 central points, taking as a response the content of saponins (expressed in oleanolic acid as it is the most abundant sapogenin). RESULTS: According to the results obtained, the R2 values were in agreement with the adjusted R2, showing that the data fit the model well. The results showed that ethanol concentration has a significant effect (p < 0.05) on the saponin content in the extract. Optimization showed that the optimal extraction conditions were 70% ethanol, 59% amplitude and an exposure time of 12 min. These values were obtained experimentally to compare theoretical values and found residual error percentages less than 3%. The emulsifying activity was evaluated, reporting a value of 52,495 units of emulsion activity per milliliter (UAE/mL), and the foaming stability indicated that 87.54% of the initial foam was maintained after 5 min, indicating high stability. CONCLUSIONS: The parameters of ethanol concentration, amplitude and time were optimized in the extraction of saponins, assisted by ultrasound. Furthermore, the extract obtained had good foaming and emulsifying characteristics, suggesting its suitability for use in industry.

In vitro ovicidal and larvicidal activities of some saponins and flavonoids against parasitic nematodes of goats.

This study assessed the anthelmintic activity of plant-derived compounds against gastrointestinal nematodes of goats using the egg hatch and larval motility assays. The compounds tested were saponins (digitonin and aescin) and their respective sapogenins (aglycones), hecogenin acetate and flavonoids (catechin, hesperidin, isocordoin and a mixture of isocordoin and cordoin). Additionally, cytotoxicity of active substances was analysed on Vero cell through 3-4,5-dimethylthiazol-2-yl,2,5diphenyltetrazolium bromide (MTT) and propidium iodide (PI) tests. Significant reduction on the egg hatching (P 90%). Nevertheless, higher cytotoxicity was observed in the MTT assay, with IC50 of 0.20 mg mL-1 (aescin) and 0.0074 mg mL-1 (digitonin). Aescin and digitonin have a pronounced in vitro anthelmintic effect and the glycone portion of these saponins plays an important role in this activity.

Actividad espermicida de saponinas esteroidales y triterpénicas extraídas de diferentes plantas / Spermicidal activity of steroidal and triterpenoid saponins extracted from various plants

Introducción: la planificación familiar se ha planteado como un mecanismo para permitirle a las parejas decidir el momento adecuado de tener hijos. Sin embargo, estudios realizados por la Organización Mundial de la Salud han demostrado que esta no es solo importante en el interior de los núcleos familiares, porque los jóvenes generalmente inician su vida sexual desde muy temprana edad, lo cual hace necesario establecer estrategias para promover la salud sexual en este grupo etario. Objetivos: evaluar si la estructura molecular de las saponinas influye en la actividad espermicida que estas reportan e indagar si existen diferencias en el rendimiento de las saponinas triterpénicas y los esteroides como agentes espermicidas. Métodos: el proceso de obtención del extracto polar de cada una de las plantas se obtiene bajo el esquema general descrito sobre las saponinas esteroides. La evaluación previa de las muestras de semen se realizó bajo los protocolos propuestos por el manual de la Organización Mundial de la Salud de metodologías para el examen y procesamiento del semen humano de 2010. Resultados: las saponinas con núcleo triterpénico promueven una disminución significativa de la movilidad espermática (p< 0,05) al ser comparadas con aquellos tratamientos con extractos obtenidos a partir de plantas con núcleo esteroide. Además se observó diferencia entre una misma especie proveniente de diferentes lugares. Conclusión: las plantas seleccionadas para el estudio permiten observar diferencias entre los núcleos triterpénicas y esteroides.

Introduction: family planning is the mechanism allowing couples to choose the right moment to have children. However, studies conducted by the World Health Organization have shown that planning is not only important within households. Young people usually start their sex life at a very early age, which makes it necessary to establish strategies to promote sexual health in this age group. Objectives: determine whether the molecular structure of saponins influences their spermicidal activity, and contrast the performance of triterpenoid and steroidal saponins as spermicidal agents. Methods: polar extract of each plant was obtained by the general procedure described for steroidal saponins. Preliminary evaluation of semen samples was conducted following the methodologies recommended by the World Health Organization for the examination and processing of human semen (2010). Results: triterpenoid core saponins bring about a significant decrease in sperm mobility (p< 0.05) in comparison with treatments based on extracts from steroidal core plants. Differences were also found between specimens of the same species collected from different locations. Conclusion: the plants selected for the study show differences between the triterpenoid and steroidal cores.