Effect of Extracts, Fractions, and Isolated Molecules of Casearia sylvestris to Control Streptococcus mutans Cariogenic Biofilm.

The effects of extracts, fractions, and molecules of Casearia sylvestris to control the cariogenic biofilm of Streptococcus mutans were evaluated. First, the antimicrobial and antibiofilm (initial and pre-formed biofilms) in prolonged exposure (24 h) models were investigated. Second, formulations (with and without fluoride) were assessed for topical effects (brief exposure) on biofilms. Third, selected treatments were evaluated via bacterium growth inhibition curves associated with gene expression and scanning electron microscopy. In initial biofilms, the ethyl acetate (AcOEt) and ethanolic (EtOH) fractions from Brasília (BRA/DF; 250 µg/mL) and Presidente Venceslau/SP (Water/EtOH 60:40 and Water/EtOH 40:60; 500 µg/mL) reduced ≥6-logs vs. vehicle. Only the molecule Caseargrewiin F (CsF; 125 µg/mL) reduced the viable cell count of pre-formed biofilms (5 logs vs. vehicle). For topical effects, no formulation affected biofilm components. For the growth inhibition assay, CsF yielded a constant recovery of surviving cells (≅3.5 logs) until 24 h (i.e., bacteriostatic), and AcOEt_BRA/DF caused progressive cell death, without cells at 24 h (i.e., bactericidal). CsF and AcOEt_BRA/DF damaged S. mutans cells and influenced the expression of virulence genes. Thus, an effect against biofilms occurred after prolonged exposure due to the bacteriostatic and/or bactericidal capacity of a fraction and a molecule from C. sylvestris.

Corrigendum: (-)-T-Cadinol, a Sesquiterpene Isolated From Casearia sylvestris (Salicaceae)-Displayed In Vitro Activity and Causes Hyperpolarization of the Membrane Potential of Trypanosoma cruzi.

[This corrects the article DOI: 10.3389/fphar.2021.734127.].

In Vitro Schistosomicidal Activities of the Leaf Extracts from Casearia sylvestris Varieties.

Schistosomiasis, a tropical disease caused by flatworms, may affect the liver, spleen, bladder, and intestine. Casearia sylvestris Swartz, a medicinal plant, displays antiprotozoal, antimicrobial, antifungal, and antiulcer activities. We have evaluated the in vitro schistosomicidal activity of two C. sylvestris varieties against Schistosoma mansoni adult worms at concentrations between 12.5 and 200 µg/mL. At 100 and 200 µg/mL, the ethanolic C. sylvestris var. sylvestris leaf extract enriched in casearin-like diterpenes eliminated 100 % of the parasites after incubation for 72 h and 48 h, respectively, whilst the same extract at 200 µg/mL eliminated 96 %, 100 %, and 100 % of the parasites after incubation for 24, 48, and 72 h, respectively. On the other hand, the hydroalcoholic C. sylvestris var. lingua leaf extract at 200 µg/mL eliminated 60.4 and 66.7 % of the parasites after incubation for 48 and 72 h, respectively. The presence of casearin-like diterpenes and glycosylated flavonoids was confirmed based on chromatographic techniques and mass spectrometry data.

(-)-T-Cadinol-a Sesquiterpene Isolated From Casearia sylvestris (Salicaceae)-Displayed In Vitro Activity and Causes Hyperpolarization of the Membrane Potential of Trypanosoma cruzi.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 6-8 million people worldwide, mainly from developing countries. The treatment is limited to two approved nitro-derivatives, nifurtimox and benznidazole, with several side effects and reduced efficacy. Casearia sylvestris has been used in folk medicine as an antiseptic and cicatrizing in skin diseases. In the present work, the hexane phase from the MeOH extract from the leaves of Casearia sylvestris afforded a fraction composed by the sesquiterpene T-cadinol, which was chemically characterized by NMR and HRMS. The activity of T-cadinol was evaluated against T. cruzi, and IC50 values of 18 (trypomastigotes) and 15 (amastigotes) µM were established. The relation between the mammalian toxicity and the antiparasitic activity resulted in a selectivity index >12. Based on this promising activity, the mechanism of action was investigated by different approaches using fluorescent-based techniques such as plasma membrane permeability, plasma membrane electric potential, mitochondrial membrane electric potential, reactive oxygen species, and the intracellular calcium (Ca2+) levels. The obtained results demonstrated that T-cadinol affected neither the parasite plasma membrane nor the electric potential of the membrane. Nevertheless, this compound induced a mitochondrial impairment, resulting in a hyperpolarization of the membrane potential, with decreased levels of reactive oxygen species. No alterations in Ca2+ levels were observed, suggesting that T-cadinol may affect the single mitochondria of the parasite. This is the first report about the occurrence of T-cadinol in C. sylvestris, and our data suggest this sesquiterpene as an interesting hit compound for future optimizations in drug discovery studies for Chagas disease.

Casearia sylvestris Essential Oil Degradation Products Generated by Leaf Processing.

Casearia sylvestris is an endemic tree of the Latin America that the essential oil (EO) has anti-inflammatory and gastroprotective actions. This study evaluates the chemical composition of the EO from the volatile fractions of in natura, fresh, and dried C. sylvestris var. sylvestris and var. lingua leaves. For both varieties, the dried leaves presented higher EO yield as compared to fresh leaves. The major EO chemical components were (E)-caryophyllene, α-humulene, germacrene D, bicyclogermacrene, spathulenol, caryophyllene oxide, and humulene epoxide II. In both varieties, the content of sesquiterpene hydrocarbons decreased and oxygenated sesquiterpenes increased on going from in natura to fresh and dried leaves, which indicated that leaf drying and hydrodistillation modified the volatile composition. The results also suggested that bicyclogermacrene and (E)-caryophyllene were oxidized during processing, to generate spathulenol and caryophyllene oxide, respectively. C. sylvestris varieties and in natura, fresh, and dried leaves differed in terms of the chemical composition of volatiles, which could affect the EO biological activities.

Employing optical microscopy technique to determine the tissue action of Hamamelis virginiana L. (Hamamelidaceae), Maytenus ilicifolia Mart. ex Reissek (Celastraceae) and Casearia sylvestris Sw. (Salicaceae) tinctures on skin-fixed histological sections of Wistar rats

Objective: In this study, we aimed to determine the action of the tinctures of Hamamelis virginiana, Maytenus ilicifolia, and Casearia sylvestris on tissues. For this, we investigated the histological sections of fixed skin tissue of Wistar rats for the changes in collagen and elastic fibers, epithelial cells, conjunctive cells, epidermal attachments, pigments, and granules using the optical microscopy technique. Since in the literature and published articles, the use of in vivo models, such as Wistar rats, is predominant to evaluate the healing action of herbal medicines. Methods: The tinctures of Hamamelis virginiana, Maytenus ilicifolia, and Casearia sylvestris, and ethanol 70% (blank) were used. The tinctures were obtained at 10% (w/v) through percolation using 70% ethanol (v/v) as the extraction liquid. This study was conducted in duplicate for each tincture and different incubation times (4, 24, and 48 h) at 37 °C in an oven. The slides used in this study (Wistar rat skin) were previously processed at the histology laboratory since the waste material was donated to conduct this experiment. The Research Ethics Committee approved the use of animals of the Life Sciences Center of the Pontifical Catholic University of Campinas (PUC-CAMPINAS) under the protocol approval number 367/08. Before initiating the staining process, the slides were removed from the oven, and the tincture deposited on the slides was removed. Weigert staining was performed subsequently. For semi-quantitative analysis, the histological sections were carefully observed, and the number of collagen and elastic fibers was evaluated based on the following scale: (+) normal fiber presence, (−) slight decrease, (− −) moderate decrease, and (− − −) intense decrease. The images were digitally captured to obtain the results using a photomicroscope. Results: The degradation of collagen fibers was best evidenced upon using Hamamelis virginiana tincture, which is concordant with the existing reports in the literature on its healing action via the precipitation of dermal proteins. Maytenus ilicifolia and Casearia sylvestris tinctures exhibited low proteolytic capacity as they only caused degradation of elastic fibers that are more delicate and very different from collagen in their constitution. Therefore, the application of the latter two as healing agents (which acts through precipitating proteins) is limited. Conclusions: This experimental histological model using the optical microscopy technique exhibits the advantage of agility and high efficiency as a simple and powerful approach.

Essential oils in pathogen resistance induction of Eucalyptus benthamii Maiden et Cambage / Óleos essenciais na indução de resistência a patógenos em Eucalyptus benthamii Maiden et Cambage

This study evaluated the essential oils of Melaleuca alternifolia, Casearia sylvestris and Eugenia uniflora as inducers of defense mechanisms in Eucalyptus benthamii seedlings. Four mL of each oil, with a concentration of 0.75% were sprayed in E. benthamii seedlings and two bioassays were performed, in the first, the essential oils were sprayed and after 30 days, sugars, proteins, peroxidases, phenylalanine ammonia (PAL), and phenols were evaluated and; in the second, seven days after the first analysis, the essential oils were again sprayed and after three days, the same variables were evaluated. The essential oils of M. alternifolia, C. sylvestris, and E. uniflora sprayed had no significant effects on E. benthamii seedlings after 30 days in terms of total sugars, proteins, peroxidase, PAL activity, and phenols. However, when M. alternifolia and E. uniflora essential oils were sprayed seven days after the first analysis with evaluation after 3 days, an increase in total sugars was observed. After these days, all essential oils promoted an increase in protein levels. The oils of E. uniflora and C. sylvestris also increased peroxidase levels. The PAL defense enzyme not showed increased when essential oils were used. The essential oils of M. alternifolia and C. sylvestris had potential as inducers of defense mechanisms on E. benthamii seedlings after 3 days of their application, what it demonstrated not be permanent.(AU)

Este trabalho teve como objetivo avaliar os óleos essenciais de Melaleuca alternifolia, Casearia sylvestris e Eugenia uniflora como indutores de mecanismos de defesa em mudas de Eucalyptus benthamii. Quatro mL de cada óleo, com concentração de 0,75%, foram pulverizados em mudas de E. benthamii e foram realizados dois bioensaios. No primeiro foram pulverizados os óleos essenciais e após 30 dias, açúcares, proteínas, peroxidases, fenilalanina amônia (PAL) , e fenóis foram avaliados e; na segunda, sete dias após a primeira análise, os óleos essenciais foram novamente pulverizados e após três dias, as mesmas variáveis foram avaliadas. Os óleos essenciais de M. alternifolia, C. sylvestris e E. uniflora pulverizados não tiveram efeito significativo sobre as mudas de E. benthamii após 30 dias em termos de açúcares totais, proteínas, peroxidase, atividade PAL e fenóis. Entretanto, quando os óleos essenciais de M. alternifolia e E. uniflora foram pulverizados sete dias após a primeira análise com avaliação após três dias, foi observado um aumento nos açúcares totais. Depois desses dias, todos os óleos essenciais promoveram aumento nos níveis de proteínas. Os óleos de E. uniflora e C. sylvestris também aumentaram os níveis de peroxidase. A enzima de defesa PAL não se mostrou aumentada quando óleos essenciais foram usados. Os óleos essenciais de M. alternifolia e C. sylvestris apresentaram potencial como indutores de mecanismos de defesa em mudas de E. benthamii após três dias de sua aplicação, o que demonstrou não ser permanente.(AU)

Essential oils in pathogen resistance induction of Eucalyptus benthamii Maiden et Cambage / Óleos essenciais na indução de resistência a patógenos em Eucalyptus benthamii Maiden et Cambage

ABSTRACT: This study evaluated the essential oils of Melaleuca alternifolia, Casearia sylvestris and Eugenia uniflora as inducers of defense mechanisms in Eucalyptus benthamii seedlings. Four mL of each oil, with a concentration of 0.75% were sprayed in E. benthamii seedlings and two bioassays were performed, in the first, the essential oils were sprayed and after 30 days, sugars, proteins, peroxidases, phenylalanine ammonia (PAL), and phenols were evaluated and; in the second, seven days after the first analysis, the essential oils were again sprayed and after three days, the same variables were evaluated. The essential oils of M. alternifolia, C. sylvestris, and E. uniflora sprayed had no significant effects on E. benthamii seedlings after 30 days in terms of total sugars, proteins, peroxidase, PAL activity, and phenols. However, when M. alternifolia and E. uniflora essential oils were sprayed seven days after the first analysis with evaluation after 3 days, an increase in total sugars was observed. After these days, all essential oils promoted an increase in protein levels. The oils of E. uniflora and C. sylvestris also increased peroxidase levels. The PAL defense enzyme not showed increased when essential oils were used. The essential oils of M. alternifolia and C. sylvestris had potential as inducers of defense mechanisms on E. benthamii seedlings after 3 days of their application, what it demonstrated not be permanent.

RESUMO: Este trabalho teve como objetivo avaliar os óleos essenciais de Melaleuca alternifolia, Casearia sylvestris e Eugenia uniflora como indutores de mecanismos de defesa em mudas de Eucalyptus benthamii. Quatro mL de cada óleo, com concentração de 0,75%, foram pulverizados em mudas de E. benthamii e foram realizados dois bioensaios. No primeiro foram pulverizados os óleos essenciais e após 30 dias, açúcares, proteínas, peroxidases, fenilalanina amônia (PAL) , e fenóis foram avaliados e; na segunda, sete dias após a primeira análise, os óleos essenciais foram novamente pulverizados e após três dias, as mesmas variáveis foram avaliadas. Os óleos essenciais de M. alternifolia, C. sylvestris e E. uniflora pulverizados não tiveram efeito significativo sobre as mudas de E. benthamii após 30 dias em termos de açúcares totais, proteínas, peroxidase, atividade PAL e fenóis. Entretanto, quando os óleos essenciais de M. alternifolia e E. uniflora foram pulverizados sete dias após a primeira análise com avaliação após três dias, foi observado um aumento nos açúcares totais. Depois desses dias, todos os óleos essenciais promoveram aumento nos níveis de proteínas. Os óleos de E. uniflora e C. sylvestris também aumentaram os níveis de peroxidase. A enzima de defesa PAL não se mostrou aumentada quando óleos essenciais foram usados. Os óleos essenciais de M. alternifolia e C. sylvestris apresentaram potencial como indutores de mecanismos de defesa em mudas de E. benthamii após três dias de sua aplicação, o que demonstrou não ser permanente.

Essential oils in pathogen resistance induction of Eucalyptus benthamii Maiden et Cambage / Óleos essenciais na indução de resistência a patógenos em Eucalyptus benthamii Maiden et Cambage

This study evaluated the essential oils of Melaleuca alternifolia, Casearia sylvestris and Eugenia uniflora as inducers of defense mechanisms in Eucalyptus benthamii seedlings. Four mL of each oil, with a concentration of 0.75% were sprayed in E. benthamii seedlings and two bioassays were performed, in the first, the essential oils were sprayed and after 30 days, sugars, proteins, peroxidases, phenylalanine ammonia (PAL), and phenols were evaluated and; in the second, seven days after the first analysis, the essential oils were again sprayed and after three days, the same variables were evaluated. The essential oils of M. alternifolia, C. sylvestris, and E. uniflora sprayed had no significant effects on E. benthamii seedlings after 30 days in terms of total sugars, proteins, peroxidase, PAL activity, and phenols. However, when M. alternifolia and E. uniflora essential oils were sprayed seven days after the first analysis with evaluation after 3 days, an increase in total sugars was observed. After these days, all essential oils promoted an increase in protein levels. The oils of E. uniflora and C. sylvestris also increased peroxidase levels. The PAL defense enzyme not showed increased when essential oils were used. The essential oils of M. alternifolia and C. sylvestris had potential as inducers of defense mechanisms on E. benthamii seedlings after 3 days of their application, what it demonstrated not be permanent.

Este trabalho teve como objetivo avaliar os óleos essenciais de Melaleuca alternifolia, Casearia sylvestris e Eugenia uniflora como indutores de mecanismos de defesa em mudas de Eucalyptus benthamii. Quatro mL de cada óleo, com concentração de 0,75%, foram pulverizados em mudas de E. benthamii e foram realizados dois bioensaios. No primeiro foram pulverizados os óleos essenciais e após 30 dias, açúcares, proteínas, peroxidases, fenilalanina amônia (PAL) , e fenóis foram avaliados e; na segunda, sete dias após a primeira análise, os óleos essenciais foram novamente pulverizados e após três dias, as mesmas variáveis foram avaliadas. Os óleos essenciais de M. alternifolia, C. sylvestris e E. uniflora pulverizados não tiveram efeito significativo sobre as mudas de E. benthamii após 30 dias em termos de açúcares totais, proteínas, peroxidase, atividade PAL e fenóis. Entretanto, quando os óleos essenciais de M. alternifolia e E. uniflora foram pulverizados sete dias após a primeira análise com avaliação após três dias, foi observado um aumento nos açúcares totais. Depois desses dias, todos os óleos essenciais promoveram aumento nos níveis de proteínas. Os óleos de E. uniflora e C. sylvestris também aumentaram os níveis de peroxidase. A enzima de defesa PAL não se mostrou aumentada quando óleos essenciais foram usados. Os óleos essenciais de M. alternifolia e C. sylvestris apresentaram potencial como indutores de mecanismos de defesa em mudas de E. benthamii após três dias de sua aplicação, o que demonstrou não ser permanente.

Antimicrobial and antibiofilm activities of Casearia sylvestris extracts from distinct Brazilian biomes against Streptococcus mutans and Candida albicans.

BACKGROUND: Dental caries is a biofilm-diet-dependent worldwide public health problem, and approaches against microorganisms in cariogenic biofilms are necessary. METHODS: The antimicrobial and antibiofilm activities of 12 Casearia sylvestris extracts (0.50 mg/mL) from different Brazilian biomes (Atlantic Forest, Cerrado, Caatinga, Pampa, and Pantanal) and varieties (sylvestris, lingua, and intermediate) were tested against two species found in cariogenic biofilms (Streptococcus mutans and Candida albicans). The extracts effective against S. mutans were used to evaluate the "adhesion strength" of this bacterium to the salivary pellicle and initial glucan matrix and the S. mutans-GtfB activity. Also, the antimicrobial activity against S. mutans of three fractions (methanol, ethyl acetate, and hexane; 0.25 mg/mL) from the extracts was evaluated. RESULTS: Three extracts from the Atlantic Forest variety sylvestris (FLO/SC, GUA/CE, PRE/SP) reduced ≥50% (> 3 logs) S. mutans viable population (p < 0.0001 vs. vehicle), while two extracts from the same biome and variety (PAC/CE, PRE/SP) decreased ≥50% of the viable counts of C. albicans (p < 0.0001 vs. vehicle). For S. mutans biofilms, three extracts (GUA/CE, PAC/CE, PRE/SP) reduced the biomass by ≥91% (p > 0.0001 vs. vehicle) and 100% of the microbial population (p < 0.0001 vs. vehicle). However, for the fungal biofilm, two extracts (PAC/CE, PRE/SP) reduced the viable counts by ≥52% (p < 0.0001 vs. vehicle), but none reduced biomass. The extracts with higher antimicrobial and antibiofilm activities presented higher content of clerodane-type diterpenes and lower content of glycosylated flavonoids than the less active extracts. The extracts had no effect on the removal of cells adhered to the pellicle (p > 0.05 vs. vehicle) while promoted the detachment of a larger number of S. mutans cells from GtfB-glucan matrix (p < 0.0031 vs. vehicle), and FLO/SC, GUA/CE and PRE/SP reduced the quantity of glucans (p < 0.0136 vs. vehicle). Only the ethyl acetate fractions reduced the microbial population of S. mutans (p < 0.0001 vs. vehicle), except for one (PAC/CE). Among the ethyl acetate fractions, three from var. lingua (two from Cerrado, and one from Cerrado/Caatinga) reduced ≥83% of the microbial population. CONCLUSIONS: C. sylvestris extracts from Atlantic Forest var. sylvestris and ethyl acetate fractions from Cerrado and Cerrado/Caatinga var. lingua may be used as a strategy against cariogenic microorganisms.

Acidic and hepatic derivatives of bioactive clerodane diterpenes casearins J and O.

Clerodane diterpenes from Casearia sylvestris are antiulcerogenic and anti-inflammatory. The finding that they may undergo acid degradation or hepatic metabolization led to an investigation of their degradation products. Purified clerodane diterpenes (casearins J and O) were subjected to in vitro assays to simulate their oral administration. Resulting derivatives were identified using chromatographic and spectrometric techniques. Nitric oxide synthesis by LPS-stimulated macrophages was assayed to verify whether structural modifications alter the anti-inflammatory activity of diterpenes. Nine compounds (1-9) were identified after acid degradation remaining 5.05% of casearin J. Besides the remaining casearin O (13.1%), eight compounds (10-17) were identified. The dialdehydes from each casearin were the major constituents. S9 rat liver treatment of casearins J and O generated two compounds identical to some of those produced by acid degradation, which remained 36.8% and 36.5% intact, respectively. Both casearins and its derivatives were not cytotoxicity at concentrations lower than 0.312 µg/mL (0.555 µM for casearin J and 0.516 µM for casearin O) and did not inhibit the nitric oxide production in this concentration. Thus, the structural modifications conducted did not alter the activity of casearins and the anti-inflammatory pathway of diterpenes probably is not involved on nitric oxide modulation.

In vitro and in vivo anti-Helicobacter pylori activity of Casearia sylvestris leaf derivatives.

ETHNOPHARMACOLOGICAL RELEVANCE: The number of bacterial strains that are resistant to multiple conventional antimicrobial agents is increasing. In this context, natural products have been widely used as a strategy to treat diseases caused by bacteria. Infections by Helicobacter pylori have attracted attention because they are directly related to severe gastric medical conditions. Casearia sylvestris Swartz, popularly known as guaçatonga, is largely employed to treat gastric disorders in Brazilian folk medicine. This plant species has aroused much interest mainly because it displays anti-inflammatory activity and can act as an antiulcer agent. AIM OF THE STUDY: To evaluate the in vitro and in vivo anti-H. pylori action of C. sylvestris leaf derivatives incorporated or not in a nanostructured drug delivery system. MATERIALS AND METHODS: The essential oil (obtained by hydrodistillation) and ethanolic extract (obtained by maceration) were obtained from C. sylvestris leaves. The ethanolic extract was submitted to fractionation through solid phase extraction and column chromatography, to yield the ethanolic fractions. Hydrolyzed casearin J was achieved by submitting isolated casearin J to acid hydrolysis. The derivatives were chemically characterized by nuclear magnetic resonance (NMR), gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS) analyses. A nanostructured lipid system was used as drug delivery system. To assess the in vitro antibacterial activity of C. sylvestris leaf essential oil, ethanolic extract, and derivatives, microdilution, biofilm, and time-kill assays were performed against H. pylori ATCC 43504. Finally, the in vivo action was investigated by employing male Wistar rats experimentally infected with H. pylori. RESULTS: Many C. sylvestris leaf derivatives presented significant in vitro activity against H. pylori. Among the derivatives, fraction 2 (F2) was the most effective. In vivo tests showed that both the ethanolic extract and F2 decreased the ulcerative lesion size, but only the ethanolic extract eradicated H. pylori from the gastric lesions. Incorporation of plant derivatives in nanostructured lipid system blunted the in vitro action, as demonstrated by the microdilution assay. However, this incorporation improved the ethanolic extract activity against biofilms. CONCLUSION: C. sylvestris leaf derivatives are effective against H. pylori both in vitro and in vivo. According to phytochemical analyses, these derivatives are rich in terpenoids, which could be related to the anti-H. pylori action. Synergism could also underlie C. sylvestris efficacy judging from the fact that the sub-fractions and isolated compounds had lower activity than the extract. Incorporation in a nanostructured lipid system did not improve the activity of the compounds in our in vivo protocol.

Casearin D inhibits ERK phosphorylation and induces downregulation of cyclin D1 in HepG2 cells.

Cancer is a public health problem which represents the second cause of death in the world. In this framework, it is necessary to identify novel compounds with antineoplastic potential. Plants are an important source for discovering novel compounds with pharmacological potential. In this study, we aimed to investigate the antiproliferative potential of isolated compounds from Casearia sylvestris on tumor cell lines. Crude extract effectively reduced cell viability of 4 tumor cell lines (HepG2, A549, U251-MG, and HT-144) after 48h treatment. HepG2 and HT-144 were the most responsive cells. Three fractions (aqueous ethanol, n-hexane and ethyl acetate) were tested against HepG2 and HT-144 cells and we observed that compounds with antiproliferative activity were concentrated in n-hexane and ethyl acetate fractions. The casearins A, G and J were isolated from n-hexane fraction, while casearin D was obtained from ethyl acetate fraction. We demonstrated that casearin D significantly inhibited the clonogenic capacity of HepG2 cells after 24h exposure indicating its antiproliferative activity. In addition, G1/S transition cell cycle arrest in HepG2 cells was also observed. These effects are related, at least in part, to ability of the casearin D in reducing ERK phosphorylation and cyclin D1 expression levels.

Avaliação da atividade antibacteriana do hidróxido de cálcio associado a diferentes veículos em blocos de dentina contaminados por Enterococcus faecalis / Evaluation of the antibacterial activity of calcium hydroxide associated different vehicles in dentin blocks contaminated with Enterococcus faecalis

Introdução: A infecção endodôntica ocasionada por Enterococcus faecalis é um problema sério no tratamento de dentes comprometidos. É de preocupação do cirurgião dentista um completo saneamento do sistema de canais radiculares pela aplicação de medicação antisséptica entre sessões. Dentre estas medicações, a pasta de hidróxido de cálcio tem sido associada a diferentes veículos para potencializar sua ação. Objetivos: Diante disto, este estudo objetivou avaliar e comparar a eficiência da atividade antimicrobiana in vitro de diferentes pastas de hidróxido de cálcio frente ao E. faecalis. Material e Métodos: Os testes foram executados em 49 blocos de dentina infectados com E. faecalis e tratados com pastas de hidróxido de cálcio em diferentes veículos por uma semana. A eficiência das pastas foi avaliada pela microscopia confocal de varredura a laser. Para comparação entre as pastas foi empregado o teste de Kruskal-Wallis e pelo teste de Dunn para comparações individuais com nível de significância estabelecido em 5%. Resultados: A aplicação das diferentes pastas proporcionou uma significativa alteração na proporção de bactérias viáveis e não viáveis encontradas no biovolume celular total dos blocos de dentina. Conclusão: A pasta que revelou melhor desempenho antimicrobiano foi aquela cujo veículo foi água destilada. A pasta de hidróxido de cálcio associada ao extrato propilenoglicólico de guaçatonga não apresentou desempenho antimicrobiano sobre células de E. faecalis. (AU)

Introduction: The endodontic infection caused by Enterococcus faecalis is a serious problem in the treatment of compromised teeth. It is a concern to the dental surgeon a complete sanitation of the root canal system by applying antiseptic medication between sessions. Among these medications, the calcium hydroxide paste has been linked to different vehicles to enhance its action. Objectives: Thus, this study aimed to evaluate and compare the efficiency of in vitro antimicrobial activity of different pastes of calcium hydroxide against E. faecalis. Material and Methods: Tests were performed in 49 blocks dentin infected with E. faecalis and treated with calcium hydroxide pastes in different vehicles for a week. The efficiency of the pastes was evaluated by confocal laser scanning. The Kruskal-Wallis test was used to compare the pastes and the Dunn test was used for individual comparisons with a significance level set at 5%. Results: The application of different pastes provided a significan change in the proportion of viable and non-viable bacteria found in the total cell biovolume of the blocks of dentin. Conclusion: The paste that revealed the best antimicrobial performance was the one whose vehicle was distilled water. The calcium hydroxide paste associated with the extract of guaçatonga in propylene glycol showed no antimicrobial performance on cells of E. faecalis. (AU)

Toxicity study about a medicinal plant Casearia sylvestris: A contribution to the Brazilian Unified Health System (SUS).

ETHNOPHARMACOLOGICAL RELEVANCE: Casearia sylvestris S.w (Salicaceae) is catalogued by the Brazilian Unified Health System as a plant of interest for the Brazilian population with the purpose of treating inflammatory disorders, such as pain and gastrointestinal disorders based on the folk use and some literature about efficacy; however, no toxicological studies concerned the safety of extract fluid of this plant have been reported. AIM OF THE STUDY: The present study was carried out to evaluate the acute and subchronic toxicity of the hydroethanolic extract fluid (FE) obtained from leaves of C. sylvestris in Wistar rats. MATERIALS AND METHODS: In the acute toxicity test three female Wistar rats were treated with a single dose of FE (2000 mg/kg) administered by oral gavage and observed for 14 days in order to identify signs of toxicity or death. In subchronic toxicity study animals received, by daily gavage three doses 60, 120 and 240 mg/kg of the FE of the plant for 28 and 90 days. The animals were observed daily for clinical signs and mortality. Body weight and food consumption were measured weekly and at the end of treatment were analysed hematological, biochemical and histopathological parameters. Also was analysed the cellularity of bone marrow and spleen. Moreover, phytochemical analysis by HPLC-PDA-ESI(+)/MS and CG/MS/EI was carried out to qualify the constituents of the extract. RESULTS: The results of acute study indicated that the LD50 is higher than 2000 mg/kg and at 28 and 90 day oral toxicity showed that there were no toxic effects detected in any of the parameters evaluated: body weight and relative organ weight, general behavioral changes, haematological and biochemical parameters and histopathological examination. The analysis by HPLC-PDA-ESI(+)/MS and CG/MS/EI identified the flavonoids rutin, quercetin and luteolin and also chlorogenic on the extract. CONCLUSION: Based on this study the hydroethanolic fluid extract of C. sylvestris could be safe even when used over a long period for therapeutic uses proposed by the Brazilian Unified Health System.

Development of a comprehensive method for analyzing clerodane-type diterpenes and phenolic compounds from Casearia sylvestris Swartz (Salicaceae) based on ultra high performance liquid chromatography combined with chemometric tools.

This work describes the development and optimization of an analytical method utilizing liquid chromatography and chemometrics to evaluate and differentiate two varieties of Casearia sylvestris Swartz (Salicaceae) from São Paulo State (Brazil) based on their secondary metabolite profiles. Previously, analytical studies only concerned the analysis of clerodane-type diterpenes. Therefore, considering the importance of including phenolic compounds in such analysis, we used design of experiments to simultaneously extract and detect the largest number of compounds from both chemical classes. This new strategy allowed a comprehensive chromatographic analysis of C. sylvestris, and the results for the two varieties exhibited an interesting distribution according to their original ecosystems, suggesting a strong correlation to the main metabolites found in each species group. Besides their inherent morphological differences, C. sylvestris variety lingua, mainly found in Cerrado areas, predominantly contains phenolic compounds, while C. sylvestris variety sylvestris, mainly found in Atlantic Forest areas, contains mostly clerodane-type diterpenes. Finally, it was also possible to observe differences in the secondary metabolite composition within each group depending on the place where samples were collected.

Aspectos toxicológicos da planta medicinal Casearia sylvestris Swartz: revisão de literatura / Toxicology of the medicinal plant Casearia sylvestris Swartz: a literature review

In the West, the consumption of medicinal plants is increasing strongly, and up to 40% of the population use medicinal plants and phytotherapic preparations regularly, in the belief that they are innocuous and/or safe. However, in most cases there is no scientific proof of their pharmacological and toxicological properties. The aim of this study was to carry out a bibliographical review of the toxicological properties of Casearia sylvestris Swartz and collect data providing a basis for its rational use. Some studies validate classical therapeutic indications of C. sylvestris, such as to treat diarrhea and snakebites, justifying its empirical use. Nevertheless, there are few reports about its toxicological properties and these present only limited findings, describing results as 50% lethal dose (LD50) and the acute toxicity of its constituents. Currently, research is insufficient to ensure safety of popular preparations based on C. sylvestris. Therefore, it is necessary to make a fuller assessment of its deleterious profile, especially with respect to the toxic potential of its constituents, as well as its capacity to harm target organs and organic systems...

No Ocidente, o consumo de plantas medicinais vem aumentando substancialmente, onde cerca de 40% da população utiliza regularmente plantas medicinais e preparações fitoterápicas sob o rótulo de serem produtos inócuos ou seguros. Porém, na maioria das vezes, não há comprovação científica de suas propriedades farmacológicas e toxicológicas. O objetivo deste trabalho foi realizar um levantamento bibliográfico sobre as propriedades toxicológicas da Casearia sylvestris Swartz e coletar dados que fundamentem seu uso racional. Alguns estudos validam indicações terapêuticas clássicas da C. sylvestris como antidiarréico e no tratamento de ferimentos ofídicos, fundamentando seu uso empírico. Porém, poucos trabalhos relatam suas propriedades toxicológicas, havendo apenas abordagens limitadas em torno da Dose Letal 50% e da toxicidade aguda de seus constituintes. Até o presente momento, as pesquisas são insuficientes para garantir a segurança de preparações populares à base de C. sylvestris. Portanto, há necessidade de avaliar melhor seu perfil deletério, principalmente, no que diz respeito ao potencial tóxico de seus constituintes, assim como a sua capacidade lesiva sobre órgãos-alvos e sistemas orgânicos...

Cardiovascular protective effects of Casearia sylvestris Swartz in Swiss and C57BL/6 LDLr-null mice undergoing high fat diet.

ETHNOPHARMACOLOGICAL RELEVANCE: Although Casearia sylvestris Swartz is used in Brazilian folk medicine to treat obesity, no study has been conducted to evaluate the effects of this species in an experimental model of dyslipidemia and atherosclerosis. So, the aim of this study was to evaluate possible hypolipemiant and antiatherogenic activity of the methanolic extract obtained from Casearia sylvestris (MECS) using Swiss and C57BL/6 LDLr-null mice undergoing high fat diet (HFD). MATERIAL AND METHODS: Dyslipidemia and atherogenesis were induced by the administration of commercial HFD for 4 weeks. The MECS was administered orally at doses of 250 and 500mg/kg, once a day, for two weeks, starting from the 2nd week of HFD. The gain in body weight and systolic blood pressure (SBP) were measured weekly over the four week study. At the end of the experiments the levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol (VLDL-C) were measured by colorimetric method. Aldosterone, vasopressin and angiotensin converting enzyme (ACE) activity were also evaluated in collected serum. The renal function, atherogenic index serum (AIS) and in vitro antiplatelet activity were investigated. Additionally, histopathological analyzes were performed to determine the intima-media thickness (IMT) and intima media ratio (IMR) in aorta samples. RESULTS: The HFD induced dyslipidemia and major structural changes in the aortic wall, including raising of the systolic blood pressure in LDLr-null mice. In addition, we observed an increase in lipid peroxidation accompanied by a reduction of serum nitrite. The treatment with MECS was able to prevent the increase of SBP, TC, LDL-C, VLDL-C and triglycerides levels and increase HDL-C in Swiss and LDLr-null mice. These effects were accompanied by a significant reduction in oxidative stress. Moreover, AIS, IMT and IMR were significantly reduced in MECS-treated mice, and the extract was able to reduce platelet aggregation in vitro. CONCLUSION: This study demonstrated that MECS reduces the serum lipids and oxidative stress when orally administered to Swiss and LDLr-null mice. In addition, it was able to prevent arterial thickening induced by HFD and to inhibit platelet aggregation in vitro.

Liberação de componentes do extrato de Casearia sylvestris Swartz empregando membranas de látex natural como suporte / Analysis of Casearia sylvestris Swartz extract release from natural rubber latex membranes

A membrana de látex natural (NRL - Natural Rubber Latex), manipulada a partir do látex extraído da seringueira Hevea brasiliensis, tem apresentado grande potencial de aplicação no campo da biomedicina e dos biomateriais. Graças a sua biocompatibilidade e baixa taxa de rejeição, ela tem sido utilizada para próteses e enxertos, atuando como estimulante da angiogênese, adesão celular e barreira física a agentes infecciosos. Além dessas aplicações, as membranas são utilizadas como matriz de sistemas de liberação para avaliar o comportamento da liberação de fármacos e extratos de origem vegetal que apresentam propriedades medicinais. O sistema extrato-membrana tem como objeto de estudo uma nova abordagem dessas substâncias no tratamento de feridas visando à cicatrização e regeneração do tecido envolvido. Casearia sylvestris, conhecida popularmente como guaçatonga, pertence à família Salicaceae, conhecida na medicina popular pelos seus efeitos antiulcerogênicos, cicatrizantes, antiofídicos, anti-inflamatórios e antissépticos, propriedades já comprovadas por estudos. Trabalhos recentes demonstraram que a liberação controlada de fármacos e extratos utilizando membranas de látex natural é uma alternativa interessante e promissora para aplicações biomédicas. Assim, o objetivo deste projeto foi estudar a liberação do extrato de Casearia sylvestris incorporado à membrana de látex em soluções com diferentes valores de pH, com o propósito de estudar seu comportamento e sua liberação de forma controlada. A taxa de liberação do extrato do sistema extrato-membrana foi monitorada e analisada utilizando-se o método de espectroscopia ótica (UV). O pH básico age desestabilizando a membrana indicando que o polímero possui a habilidade de proteger o extrato de ser liberado em valores de pH ácidos, direcionando sua aplicação para o sítio de melhor absorção. Além disso, a liberação segue uma função bi-exponencial...

Natural Rubber Latex (NRL) membrane, made from latex extracted from the rubber tree Hevea brasiliensis, has shown great potential for use in the biomedicine and biomaterials area. Thanks to its biocompatibility and low rejection rate, it has been used for implants and grafts, acting as a stimulant of angiogenesis and cell adhesion and as a barrier against infectious agents. Besides these applications, the membranes are used as model release systems, to assess the release behavior of drugs and plant extracts that exhibit medicinal properties. The extract-membrane system represents a new approach to studying these substances, as aids to wound healing and tissue regeneration. Casearia sylvestris, popularly known as guaçatonga, belongs to the family Salicaceae, known in popular medicine for its anti-ulcerogenic, wound healing, anti-ophidian, antiinflammatory and antiseptic properties, all of which are proven by scientific studies. Recent studies have also shown that the controlled release of drugs and extracts from natural latex membranes is an interesting and promising process for biomedical applications. The objective of this project was thus to study the release of Casearia sylvestris extract incorporated into natural rubber membranes. The main concern was to study and optimize the controlled release of the extract at various pHs. The rate of release was monitored and analyzed by the method of optical spectroscopy (UV). Basic pHacts to destabilize the membrane, indicating that the polymer has the ability to protect the extract from being released at acidic pH values. The controlled release follows a bi-exponential function...

Antiparasitic activity and effect of casearins isolated from Casearia sylvestris on Leishmania and Trypanosoma cruzi plasma membrane.

Leishmaniasis and Chagas disease are infectious diseases caused by parasite Leishmania sp. and Trypanosoma cruzi, respectively, and are included among the most neglected diseases in several underdeveloped and developing countries, with an urgent demand for new drugs. Considering the antiparasitic potential of MeOH extract from leaves of Casearia sylvestris Sw. (Salicaceae), a bioguided fractionation was conducted and afforded four active clerodane diterpenes (casearins A, B, G, and J). The obtained results indicated a superior efficacy of tested casearins against trypomastigotes of T. cruzi, with IC50 values ranging from 0.53 to 2.77 µg/ml. Leishmania infantum promastigotes were also susceptible to casearins, with IC50 values in a range between 4.45 and 9.48 µg/ml. These substances were also evaluated for mammalian cytotoxicity against NCTC cells resulting in 50% cytotoxic concentrations (CC50) ranging from 1.46 to 13.76 µg/ml. Additionally, the action of casearins on parasite membranes was investigated using the fluorescent probe SYTOX Green. The obtained results demonstrated a strong interaction of casearins A and B to the plasma membrane of T. cruzi parasites, corroborating their higher efficacy against these parasites. In contrast, the tested casearins induced no alteration in the permeability of plasma membrane of Leishmania parasites, suggesting that biochemical differences between Leishmania and T. cruzi plasma membrane might have contributed to the target effect of casearins on trypomastigotes. Thus, considering the importance of studying novel and selective drug candidates against protozoans, casearins A, B, G, and J could be used as tools to future drug design studies.