N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes.

Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 µg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.

N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes

Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 μg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.

A proline derivative-enriched methanol fraction from Sideroxylon obtusifolium leaves (MFSOL) stimulates human keratinocyte cells and exerts a healing effect in a burn wound model.

It was previously demonstrated that the methanol fraction of Sideroxylon obtusifolium (MFSOL) promoted anti-inflammatory and healing activity in excisional wounds. Thus, the present work investigated the healing effects of MFSOL on human keratinocyte cells (HaCaT) and experimental burn model injuries. HaCaT cells were used to study MFSOL's effect on cell migration and proliferation rates. Female Swiss mice were subjected to a second-degree superficial burn protocol and divided into four treatment groups: Vehicle, 1.0% silver sulfadiazine, and 0.5 or 1.0% MFSOL Cream (CrMFSOL). Samples were collected to quantify the inflammatory mediators, and histological analyses were performed after 3, 7, and 14 days. The results showed that MFSOL (50 µg/mL) stimulated HaCaT cells by increasing proliferation and migration rates. Moreover, 0.5% CrMFSOL attenuated myeloperoxidase (MPO) activity and also stimulated the release of interleukin (IL)-1ß and IL-10 after 3 days of treatment. CrMFSOL (0.5%) also enhanced wound contraction, promoted improvement of tissue remodeling, and increased collagen production after 7 days and VEGF release after 14 days. Therefore, MFSOL stimulated human keratinocyte (HaCaT) cells and improved wound healing via modulation of inflammatory mediators of burn injuries.

A proline derivative-enriched methanol fraction from Sideroxylon obtusifolium leaves (MFSOL) stimulates human keratinocyte cells and exerts a healing effect in a burn wound model

It was previously demonstrated that the methanol fraction of Sideroxylon obtusifolium (MFSOL) promoted anti-inflammatory and healing activity in excisional wounds. Thus, the present work investigated the healing effects of MFSOL on human keratinocyte cells (HaCaT) and experimental burn model injuries. HaCaT cells were used to study MFSOL's effect on cell migration and proliferation rates. Female Swiss mice were subjected to a second-degree superficial burn protocol and divided into four treatment groups: Vehicle, 1.0% silver sulfadiazine, and 0.5 or 1.0% MFSOL Cream (CrMFSOL). Samples were collected to quantify the inflammatory mediators, and histological analyses were performed after 3, 7, and 14 days. The results showed that MFSOL (50 μg/mL) stimulated HaCaT cells by increasing proliferation and migration rates. Moreover, 0.5% CrMFSOL attenuated myeloperoxidase (MPO) activity and also stimulated the release of interleukin (IL)-1β and IL-10 after 3 days of treatment. CrMFSOL (0.5%) also enhanced wound contraction, promoted improvement of tissue remodeling, and increased collagen production after 7 days and VEGF release after 14 days. Therefore, MFSOL stimulated human keratinocyte (HaCaT) cells and improved wound healing via modulation of inflammatory mediators of burn injuries.

Violacein antimicrobial activity on Staphylococcus epidermidis biofilm.

The aim of this study was to evaluate the antimicrobial potential of violacein (VIO) on Staphylococcus epidermidis biofilm. The minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were determined, as well as the effect of VIO exposure time on microbial viability in mature biofilm. Violacein showed good antibiofilm action, inhibiting biofilm formation and eradicating mature biofilm of S. epidermidis at concentrations of 20 µg.mL-1 and 160 µg.mL-1, respectively. At concentrations equal to MBEC and 2x MBEC, the biofilm was eradicated in 3 h and 2h30min of incubation, respectively.When evaluating VIO modulating effect on the action of clinically-used drugs (vancomycin, cefepime, ciprofloxacin and meropenem), especial synergism was observed in the violacein-ciprofloxacin association, it can completely erradicated the mature biofilm at the concentration of 1/2xMBEC and 1/4xMBEC, respectively. VIO shows good antimicrobial action on S. epidermidis biofilm and has the potential to synergistically modulate the activity of clinically-used antimicrobials.

Antichagasic effect of violacein from Chromobacterium violaceum.

AIMS: Violacein (VIO), a bacterial pigment produced by Chromobacterium violaceum, was examined to evaluate the antichagasic activity and its action mechanism against Trypanosoma cruzi Y strain. METHODS AND RESULTS: Violacein was tested against the epimastigote, trypomastigote and amastigote forms of T. cruzi Y strain (benznidazole-resistant strain). VIO inhibited all T. cruzi developmental forms, including amastigotes, which is implicated in the burden of infection in the chronic phase of Chagas disease (CD). VIO induced cell death in T. cruzi through apoptosis, as determined by flow cytometry analyses with specific molecular probes and morphological alterations, such as involvement of reactive oxygen species and changes in mitochondrial membrane potential and cell shrinkage. CONCLUSION: The results suggest antichagasic activity of VIO against T. cruzi Y strain with apoptotic involvement. SIGNIFICANCE AND IMPACT OF THE STUDY: The treatment of CD has limited efficacy and side effects that restrict patient tolerability and compliance. The VIO molecule could be used as a model for therapeutic alternatives for this disease.

Efeito do óleo essencial do Croton nepetifolius Baill na penetração cervical em ovelhas mestiças de Dorper e Santa Inês / Effect of the essential oil of Croton nepetifolius Baill on cervical penetration in crossbred Dorper and Santa Inês ewes

A fim de se avaliar o uso do óleo essencial de Croton nepetifolius Baill (OECn) na penetrabilidade cervical em ovelhas mestiças, 40 ovelhas foram distribuídas ao acaso em quatro grupos (n=10): controle, misoprostol, OECn50 e OECn100 (50 e 100µg do OECn, respectivamente). Após a sincronização do estro, utilizando CIDR e eCG (200UI), a profundidade de penetração da cérvix foi mensurada utilizando-se uma pipeta de inseminação artificial de bovino graduada, no período de zero até 72h após a retirada do CIDR. Os resultados foram expressos em média ± erro-padrão, submetidos à ANOVA seguida do teste de Tukey, enquanto os dados, em porcentagem, foram submetidos aos testes de Fisher ouqui-quadrado. Nenhuma diferença significativa (P>0,05) foi encontrada quanto ao grau de penetrabilidade cervical. Quanto ao tempo de passagem, os grupos misoprostol e OECn100 apresentaram um menor tempo de penetrabilidade às 60h(1,7±0,6 e 1,5±0,6min, respectivamente), quando comparados ao grupo controle (4,1±0,6min), que não diferiu significativamente do grupo OECn50 (2,3±0,6min). Portanto, o óleo essencial de Croton nepetifolius Baill pode ser utilizado para encurtar o tempo de penetrabilidade cervical em ovelhas submetidas à sincronização estral.(AU)

In order to evaluate the use of the essential oil of Croton nepetifolius Baill (EOCn) on cervical penetration in crossbred ewes, 40 ewes were randomly allocated into four groups (n= 10): CONTROL, MISOPROSTOL, EOCn50 and EOCn100 (50 e 100µg of the EOCn, respectively). After estrus synchronization, using CIDR and eCG (200IU), depth of cervical penetration was measured using artificial insemination gun for bovine species which was graduated and used from 0 to 72h after CIDR removal. Results were expressed as mean ± standard error mean, submitted to ANOVA and Tukey test while data in percentage were submitted to Fisher or Chi-Square test. No significant difference (P> 0.05) was observed at grade of cervical penetration. Concerning trespassing time, MISOPROTOL and EOCn100 groups presented a lower trespassing time at 60h (1.7±0.6 and 1.5±0.6min, respectively) than CONTROL group (4.1±0.6min), which did not differ significantly from EOCn50 (2.3±0.6min) group. Therefore, the essential oil of Croton nepetifolius Baill can be used to shorten the cervical penetration time in estrus synchronized ewes.(AU)

In vitro antitumor effect of a lignan isolated from Combretum fruticosum, trachelogenin, in HCT-116 human colon cancer cells.

The use of natural products in therapeutics has been growing over the years. Lignans are compounds with large pharmaceutical use, which has aroused interest in the search for new drugs to treat diseases. The present study evaluated the cytotoxicity of (-)-trachelogenin, a dibenzylbutyrolactone type lignan isolated from Combretum fruticosum, against several tumor and non-tumor cell lines using the MTT assay and its possible mechanism of action. (-)-Trachelogenin showed IC50 values ranging of 0.8-32.4µM in SF-295 and HL-60 cell lines, respectively and IC50 values >64µM in non-tumor cell lines. (-)-trachelogenin persistently induced autophagic cell death, with cytoplasmic vacuolization and formation of autophagosomes mediated by increasing LC3 activation and altering the expression levels of Beclin-1.

Violacein antimicrobial activity on Staphylococcus epidermidis and synergistic effect on commercially available antibiotics.

AIMS: The study aimed to assess whether violacein has antimicrobial activity on Staphylococcus epidermidis and synergistically modulates the action of commercially available antimicrobial drugs. METHODS AND RESULTS: Violacein showed excellent antimicrobial activity on biofilm-forming and nonbiofilm-forming S. epidermidis strains (ATCC 35984) (ATCC 12228), with bacteriostatic (MIC = 20 µg ml-1 and 10 µg ml-1 respectively) and bactericidal effects (MBC = 20 µg ml-1 for both strains), observed in short periods of exposure. The violacein bactericidal concentration led to S. epidermidis death after 2-3 h of exposure. Additionally, violacein synergistically modulated the activity of different antimicrobial classes on S. epidermidis ATCC 12228 (81·8%; n = 9) and on S. epidermidis ATCC 35984 (54·5%; n = 6), reducing the MIC of these antibiotics by up to 16-fold. CONCLUSION: Violacein shows excellent antimicrobial activity on S. epidermidis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Violacein shows the potential for the development of a new drug for the treatment of infections caused by S. epidermidis.

Comparative study of the antihypertensive effects of hexane, chloroform and methanol fractions of essential oil of Alpinia zerumbet in rats Wistar / Estudo comparativo do efeito anti-hipertensivo das frações hexânixa, clorofórmica e metanólica do óleo essencial da Alpinia zerumbet

ABSTRACT The aim of this study was to characterize components of the EOAz and its hexane (HFEOAz), chloroform (CFEOAz) and methanol (MFEOAz) fractions, and its antihypertensive effect. EOAz was extracted from leaves by hydrodistillation. Aliquot was subjected to selective desorption with silica gel column and eluted with hexane, chloroform and methanol. The components of the EOAz and fractions were analyzed by gas chromatography coupled with mass spectrometry and nuclear magnetic resonance spectroscopy of hydrogen. Experiments of vascular reactivity were performed with isolated aortic rings of male Wistar rats. Antihypertensive effect was evaluated in hypertensive rats submitted to the inhibition of synthesis of nitric oxide. Blood pressure was measured indirectly by tail plethysmography. MFEOAz showed the lowest EC50 (150.45 µg/mL), 1,8-cineole (27.81%) and terpinen-4-ol (57.35%) as main components. Single administration by nasogastric tube of EOAz, fractions and captopril significantly reduced the blood pressure of hypertensive rats, when compared to animals of the negative control group with distilled water. In conclusion, the potency of the MFEOAz was higher than that of EOAz and other fractions. The antihypertensive effect of EOAz and fractions was similar, higher than the negative control and lower than that of captopril.

RESUMO O objetivo deste estudo foi caracterizar os componentes do óleo essencial das folhas de Alpinia zerumbet (OEAz) e suas frações hexânica (FHOEAz), clorofórmica (FCOEAz) e metanólica (FMOEAz), e seu efeito anti-hipertensivo. OEAz foi extraído das folhas por hidrodestilação. Uma alíquota foi submetida à desadsorção seletiva com coluna de gel de sílica e eluída com hexano, clorofórmio e metanol. Os componentes do OEAz e fracções foram analisadas por cromatografia gasosa acoplada à detector de massa e por espectros de ressonância magnética nuclear de hidrogênio. Experimentos de reatividade vascular foram realizados com anéis aórticos isolados de ratos Wistar machos. Efeito anti-hipertensivo foi avaliado em ratos hipertensos submetidos à inibição da síntese de óxido nítrico. A pressão arterial foi medida indiretamente por pletismografia de cauda. FMOEAz mostrou a menor CE50 (150,45 μg/mL), 1,8-cineol (27,81%) e terpinen-4-ol (57,35%) como componentes principais. A administração em dose única por sonda nasogástrica de OEAz, frações e captopril reduziu significativamente a pressão arterial de ratos hipertensos, quando comparados aos animais do grupo controle negativo com água destilada. Em conclusão, a potência da FMOEAz foi maior que a do OEAz e outras frações. O efeito anti-hipertensivo de OEAz e frações foi semelhante, maior do que o controle negativo e menor do que o captopril.

Maternal attitudes towards tooth decay in children aged 12-18 months in Pelotas, Brazil.

AIM: This was to assess mothers' attitudes towards dental caries in children aged 12-18 months. METHODS: This study targeted mothers of children aged 12-18 months. Data about demographic and socioeconomic status were collected by interviews with each mother. In addition, the mother was asked about her attitudes regarding caries in her child's primary teeth. A dental examination of each child was also conducted. Chi-square, bivariate, and multiple logistic regression analyses were performed. RESULTS: A total of 262 mother-child pairs were included, and 18.7 % of the children had dental caries. If a child presented with dental caries in their primary teeth, 93.5 % of the mothers reported that they would take the child to a dentist. Mothers who had only one child and those who had children with dental caries were more likely to report that they did not expect primary dental caries treatment by the dentist. CONCLUSION: Most mothers reported that they would take their children to a dentist when they presented with dental caries. Despite this positive result, educational measures should continue to be emphasised, especially among mothers of children at a higher risk of caries and among first-time mothers.

Is expression of rat breast matrix components influenced by estrogen, progestins and tibolone?

AIM: To study the effects of estrogen therapy, alone or combined with progestogens, and of tibolone on the expression of heparanase (HSPE), extracellular matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), perlecan and proliferating cell nuclear antigen (PCNA) in normal breast tissue. METHODS: Thirty 250-day-old Wistar rats were castrated and 3 weeks later received one of the following treatments by gavage for 5 weeks: (1) estradiol benzoate; (2) estradiol benzoate + medroxyprogesterone acetate; (3) estradiol benzoate + norethisterone acetate; (4) estradiol benzoate + dydrogesterone; (5) tibolone; (6) placebo. Following treatment, the expressions of mRNA for HSPE, MMP-2 and MMP-9 were analyzed by real-time PCR and the protein expressions of HSPE, MMP-2, MMP-9, perlecan and PCNA were quantified by immunohistochemistry. RESULTS: There was a statistically significant difference among the groups for the expression of HSPE mRNA due to high levels in the tibolone group. The groups differed in terms of PCNA, with lower levels found in the tibolone group followed by the estradiol benzoate + dydrogesterone group. A statistically significant positive correlation was observed for PCNA versus perlecan and MMP-9. CONCLUSIONS: There was no difference in the effects of combinations of estradiol and different progestogens on extracellular matrix components, and breast cell proliferation was associated with increases in perlecan and MMP-9.

A comparative chemical and pharmacological study of standardized extracts and vanillic acid from wild and cultivated Amburana cearensis A.C. Smith.

The objectives of this work were to carry out a comparative chemical study and to evaluate the antinociceptive and anti-inflammatory activities of ethanol extracts (EtOHE) and vanilic acid (VA) from cultivated and wild Amburana cearensis A.C. Smith (Fabaceae), an endangered species used in Northeast Brazil for the treatment of asthma. The HPLC analysis of EtOHE, showed that coumarin (CM) and VA were the major constituents from the cultivated plant, while in the extract from the wild plant the major constituents were amburoside A (AMB) and CM. Pharmacological tests were performed with male Swiss mice or male Wistar rats acutely administered with 100-400mg/kg, p.o. of EtOHEs or 12.5-50mg/kg, p.o. of VA. EtOHEs from A. cearensis with 4, 7 or 9 months of cultivation significantly inhibited, from 32 to 64%, both phases of the formalin test in mice. Similar results were observed with the EtOHE from the wild species. VA significantly reduced both phases of the formalin test. This effect was partially reversed by naloxone. EtOHE from cultivated or wild A. cearensis inhibited the carrageenan (Cg)-induced mice paw edema. Furthermore, VA inhibited the paw edema and the leukocyte migration in rat peritoneal cavity induced by Cg. On the other hand, it did not inhibit the edema and the increase of vascular permeability induced by dextran in the rat paw. All together, these results indicate that the EtOHE from cultivated A. cearensis exhibit similar chemical and pharmacological profiles, as related to the wild plant. VA is, at least partially, responsible for these pharmacological effects. Its antinociceptive effect occurs by a mechanism partly dependent upon the opioid system, while the anti-inflammatory action was manifested in inflammatory processes dependent on polymorphonuclear cells and are probably related to the VA inhibition of cytokines as observed by others.

Structure-mutagenicity relationship of kaurenoic acid from Xylopia sericeae (Annonaceae).

Kaurane diterpenes are considered important compounds in the development of new highly effective anticancer chemotherapeutic agents. Genotoxic effects of anticancer drugs in non-tumour cells are of special significance due to the possibility that they induce secondary tumours in cancer patients. In this context, we evaluated the genotoxic and mutagenic potential of the natural diterpenoid kaurenoic acid (KA), i.e. (-)-kaur-16-en-19-oic acid, isolated from Xylopia sericeae St. Hill, using several standard in vitro and in vivo protocols (comet, chromosomal aberration, micronucleus and Saccharomyces cerevisiae assays). Also, an analysis of structure-activity relationships was performed with two natural diterpenoid compounds, 14-hydroxy-kaurane (1) and xylopic acid (2), isolated from X. sericeae, and three semi-synthetic derivatives of KA (3-5). In addition, considering the importance of the exocyclic double bond (C16) moiety as an active pharmacophore of KA cytotoxicity, we also evaluated the hydrogenated derivative of KA, (-)-kauran-19-oic acid (KAH), to determine the role of the exocyclic bond (C16) in the genotoxic activity of KA. In summary, the present study shows that KA is genotoxic and mutagenic in human peripheral blood leukocytes (PBLs), yeast (S. cerevisiae) and mice (bone marrow, liver and kidney) probably due to the generation of DNA double-strand breaks (DSB) and/or inhibition of topoisomerase I. Unlike KA, compounds 1-5 and KAH are completely devoid of genotoxic and mutagenic effects under the experimental conditions used in this study, suggesting that the exocyclic double bond (C16) moiety may be the active pharmacophore of the genetic toxicity of KA.

Studies on the anti-oedematogenic properties of a fraction rich in lonchocarpin and derricin isolated from Lonchocarpus sericeus.

The acute anti-inflammatory properties of a fraction rich in the chalcones lonchocarpin and derricin, from the roots of Lonchocarpus sericeus (HFLS), were studied for the first time, using a paw oedema model induced in rats by various stimuli. Results showed that HFLS (100 and 200 mg kg(-1), i.p.) was ineffective in inhibiting dextran-induced paw oedema. The HFLS (200 mg kg(-1), p.o. or i.p.) also failed to inhibit the bradykinin-induced oedema. In the yeast-elicited oedema, the HFLS (200 mg kg(-1), i.p.) caused inhibitions ranging from 42 to 59% in the first to fourth hours. Orally administered HFLS (200 mg kg(-1)) was active only in the second (27%) and fourth (32%) hours after yeast injection. It was observed that HFLS (50, 100 and 200 mg kg(-1), i.p.) showed inhibitions of 34, 57 and 74%, respectively, in the third hour for the carrageenan-induced oedema. The inhibition was smaller when the HFLS (100 and 200 mg kg(-1)) was administered orally. The effect of the HFLS (20 mg kg(-1), i.p.) in the carrageenan-induced oedema was not modified by the L-NAME, but the association of pentoxifylline and HFLS increased its effect, suggesting an involvement of the PDE enzyme.

Effects of alpha-tocopherol and ternatin antioxidants on morphology and activation of goat preantral follicles in vitro cultured / Efeitos dos antioxidantes alpha-tocoferol e ternatina na morfologia e na ativação de folículos pré-antrais caprinos cultivados in vitro

The effects of α-tocopherol and ternatin on the morphology, activation, and growth of goat preantral follicles in vitro cultured, for one or five days, were evaluated. Ovarian fragments were immediately fixed (non-cultured control) or in vitro cultured for one or five days in Minimum Essential Medium (MEM) with or without α-tocopherol or ternatin supplementation, both at concentrations of 5, 10, or 15µM, corresponding to the following treatments: MEM, TOC5, TOC10, TOC 15, TER5, TER10, and TER15. The percentages of morphologically normal preantral follicles in non-cultured ovarian tissue (control) was 73.2 percent and after five days of culture, there was a decrease on these percentages in all treatments (P<0.05) when compared with non-cultured control. Culture of ovarian cortex for five days increased the percentages of follicular activation in all treatments (P<0.05). Ultrastructural analysis did not confirm the integrity of caprine preantral follicles cultured for five days in medium containing antioxidants. This study demonstrated that α-tocopherol and ternatin can promote follicular activation; however, addition of these antioxidants in the tested concentrations reduced the follicular viability after in vitro culture.

Os efeitos do α-tocoferol e da ternatina sobre morfologia, ativação e crescimento de folículos pré-antrais caprinos cultivados in vitro, por um ou cinco dias, foram avaliados. Os fragmentos ovarianos foram imediatamente fixados (controle não-cultivado) ou cultivados in vitro, por um ou cinco dias, em Meio Essencial Mínimo (MEM) com ou sem suplementação com α-tocoferol ou ternatina nas concentrações de 5, 10 ou 15µM, formando os tratamentos MEM, TOC5, TOC10, TOC 15, TER5, TER10, TER15. O percentual de folículos pré-antrais normais no controle não-cultivado foi de 73,2 por cento, depois de cinco dias de cultivo, houve redução desse percentual em todos os tratamentos, quando comparados com o controle não-cultivado (P<0,05). O cultivo por cinco dias aumentou a ativação folicular em todos os tratamentos (P<0,05). A análise ultra-estrutural não mostrou folículos pré-antrais íntegros após cinco dias de cultivo em meio contendo antioxidantes. Concluiu-se que o α-tocoferol e a ternatina podem promover a ativação folicular, no entanto a adição desses antioxidantes nas concentrações testadas reduziu a viabilidade folicular após o cultivo in vitro.

Relaxant effects of an alkaloid-rich fraction from Aspidosperma ulei root bark on isolated rabbit corpus cavernosum.

We described earlier that an alkaloid-rich fraction (F(3-5)) from Aspidosperma ulei (Markgr) induces penile erection-like behavioral responses in mice. This study verified a possible relaxant effect of this fraction on isolated rabbit corpus cavernosum (RbCC) strips precontracted by phenylephrine (1 microM) or K+ 60 mM. F(3-5) (1-300 microg ml(-1)) relaxed the RbCC strips in a concentration-dependent and reversible manner. The relaxant effect of F(3-5) (100 microg ml(-1)) on phenylephrine contraction was unaffected in the presence of atropine, N-omega-nitro-L-arginine methyl ester or 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one and by preincubation with tetrodotoxin, glibenclamide, apamine and charybdotoxin suggesting that mechanisms other than cholinergic, nitrergic, sGC activation or potassium channel opening are probably involved. However, the phasic component of the contraction induced by K+ 60 mM as well as the maximal contraction elicited by increasing external Ca2+ concentrations in depolarized corpora cavernosa was inhibited by F(3-5). We conclude that F(3-5) relaxes the RbCC smooth muscle, at least in part, through a blockade of calcium influx or its function.

Genotoxic effects of tanshinones from Hyptis martiusii in V79 cell line.

The genotoxic effect of two tanshinones isolated from roots of Hyptis martiussi Benth (Labiatae) was studied using V79 (Chinese hamster lung) cells by the alkaline comet assay and micronucleus test. Tanshinones were incubated with the cells at concentrations of 1, 3, 6 and 12 microg/mL for 3 h. Tanshinones were shown to be quite strongly genotoxic against V79 cells at all tested concentrations. The data obtained provide support to the view that tanshinones has DNA damaging activity in cultured V79 cells under the conditions of the assays.

In vivo growth-inhibition of Sarcoma 180 by piplartine and piperine, two alkaloid amides from Piper.

Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} and piperine {1-5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine} are alkaloid amides isolated from Piper. Both have been reported to show cytotoxic activity towards several tumor cell lines. In the present study, the in vivo antitumor activity of these compounds was evaluated in 60 female Swiss mice (N = 10 per group) transplanted with Sarcoma 180. Histopathological and morphological analyses of the tumor and the organs, including liver, spleen, and kidney, were performed in order to evaluate the toxicological aspects of the treatment with these amides. Administration of piplartine or piperine (50 or 100 mg kg(-1) day(-1) intraperitoneally for 7 days starting 1 day after inoculation) inhibited solid tumor development in mice transplanted with Sarcoma 180 cells. The inhibition rates were 28.7 and 52.3% for piplartine and 55.1 and 56.8% for piperine, after 7 days of treatment, at the lower and higher doses, respectively. The antitumor activity of piplartine was related to inhibition of the tumor proliferation rate, as observed by reduction of Ki67 staining, a nuclear antigen associated with G1, S, G2, and M cell cycle phases, in tumors from treated animals. However, piperine did not inhibit cell proliferation as observed in Ki67 immunohistochemical analysis. Histopathological analysis of liver and kidney showed that both organs were reversibly affected by piplartine and piperine treatment, but in a different way. Piperine was more toxic to the liver, leading to ballooning degeneration of hepatocytes, accompanied by microvesicular steatosis in some areas, than piplartine which, in turn, was more toxic to the kidney, leading to discrete hydropic changes of the proximal tubular and glomerular epithelium and tubular hemorrhage in treated animals.

In vivo growth-inhibition of Sarcoma 180 by piplartine and piperine, two alkaloid amides from Piper

Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} and piperine {1-5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine} are alkaloid amides isolated from Piper. Both have been reported to show cytotoxic activity towards several tumor cell lines. In the present study, the in vivo antitumor activity of these compounds was evaluated in 60 female Swiss mice (N = 10 per group) transplanted with Sarcoma 180. Histopathological and morphological analyses of the tumor and the organs, including liver, spleen, and kidney, were performed in order to evaluate the toxicological aspects of the treatment with these amides. Administration of piplartine or piperine (50 or 100 mg kg-1 day-1 intraperitoneally for 7 days starting 1 day after inoculation) inhibited solid tumor development in mice transplanted with Sarcoma 180 cells. The inhibition rates were 28.7 and 52.3 percent for piplartine and 55.1 and 56.8 percent for piperine, after 7 days of treatment, at the lower and higher doses, respectively. The antitumor activity of piplartine was related to inhibition of the tumor proliferation rate, as observed by reduction of Ki67 staining, a nuclear antigen associated with G1, S, G2, and M cell cycle phases, in tumors from treated animals. However, piperine did not inhibit cell proliferation as observed in Ki67 immunohistochemical analysis. Histopathological analysis of liver and kidney showed that both organs were reversibly affected by piplartine and piperine treatment, but in a different way. Piperine was more toxic to the liver, leading to ballooning degeneration of hepatocytes, accompanied by microvesicular steatosis in some areas, than piplartine which, in turn, was more toxic to the kidney, leading to discrete hydropic changes of the proximal tubular and glomerular epithelium and tubular hemorrhage in treated animals.